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Inorganic Colloidal Nanocrystals

Permanent Link: http://ufdc.ufl.edu/UFE0022614/00001

Material Information

Title: Inorganic Colloidal Nanocrystals Synthesis and Applications
Physical Description: 1 online resource (157 p.)
Language: english
Creator: Wu, Huimeng
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: assembly, dectection, enzyme, gold, iron, nanocrystals, nanomaterial, nanoparticles, oxide, soluble, stability, synthesis, uranium, water
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Nanocrystals (NCs) are very small particles, which contain from a few hundred to thousands of atoms depending on the size of NCs. Because of their special properties compared with the bulk materials, NCs have found many promising applications in areas, such as biomedical diagnosis, catalysis, plasmonics, high-density data storage and solar energy conversion. This dissertation presents studies on the syntheses of metal oxide NCs and hybrid NCs, the surface functionalization of NCs by dual-interaction ligands, and gold-NC-based assay for the detection of beta-galactosidase. Monodisperse colloidal uranium dioxide NCs (UO2 NCs) were synthesized by decomposition of uranyl acetylacetonate. By changing the amount of added surfactant, the sizes of the NCs could vary from 2 ~ 8 nm. Mechanistic studies of the formation of UO2 NCs showed that the condensation product (amide) of oleic acid and oleylamine plays an important role in controlling the particle size. Normally, high-quality NCs are synthesized in organic phase, but most of NC-based bio-applications require water-soluble NCs. To convert these hydrophobic NCs to hydrophilic particles, surface modification is employed. Here dual interaction ligands based on the Tween-derivatives (TDs) were synthesized. Stability tests on TD-capped NCs showed that these dual interaction ligands can significantly increase the stability of NCs compared to single interaction ligands. Further, These TD-capped QDs were further tested as fluorescent labels to detect virus-protein expression in cells. To exploit bio-applications of nanocrystals, gold nanocrystal-based assay to detect enzyme activity was designed. The optical properties of Au-NCs are not only dependent on the particle sizes and shapes, but also the distances between the particles. Here, Lipoic acid-tyramine-beta-galactopyranosyl (LTbeta-gal) was synthesized, as ligands, to cap Au-NCs; and the resultant LTbeta-capped Au-NCs could disperse in water. After the hydrolysis of the ligands with beta-galactosidase, these Au-NCs become to aggregate, which exhibit a red-shift in the absorption spectrum of the Au-NC suspension. The detection of beta-galactosidase was further studies by varying the amounts of beta-galactosidase. Hybrid nanocrystals (HNCs) are attractive candidates for advanced nanomaterials because they contain two or more different nanoscale functionalities, which are expected to possess novel physical and chemical properties. Two kinds of heterodimers (FePt/In2O3 and UO2/In2O3) were prepared using a similar procedure and the synthesized HNCs exhibited different shapes. The studies of high-resolution transmission electron microscopy (HRTEM) indicate that the shapes of these two dimers were controlled by the interfacial structures. The amorphous iron oxide layers on the FePt NC surfaces act as glue to interconnect the FePt with the indium oxide parts and led to a core-seed-shaped heterodimer. Using completely crystalline UO2 NCs as seeds resulted in a peanut-shapd HNC.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Huimeng Wu.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Cao, Yun Wei.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022614:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022614/00001

Material Information

Title: Inorganic Colloidal Nanocrystals Synthesis and Applications
Physical Description: 1 online resource (157 p.)
Language: english
Creator: Wu, Huimeng
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: assembly, dectection, enzyme, gold, iron, nanocrystals, nanomaterial, nanoparticles, oxide, soluble, stability, synthesis, uranium, water
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Nanocrystals (NCs) are very small particles, which contain from a few hundred to thousands of atoms depending on the size of NCs. Because of their special properties compared with the bulk materials, NCs have found many promising applications in areas, such as biomedical diagnosis, catalysis, plasmonics, high-density data storage and solar energy conversion. This dissertation presents studies on the syntheses of metal oxide NCs and hybrid NCs, the surface functionalization of NCs by dual-interaction ligands, and gold-NC-based assay for the detection of beta-galactosidase. Monodisperse colloidal uranium dioxide NCs (UO2 NCs) were synthesized by decomposition of uranyl acetylacetonate. By changing the amount of added surfactant, the sizes of the NCs could vary from 2 ~ 8 nm. Mechanistic studies of the formation of UO2 NCs showed that the condensation product (amide) of oleic acid and oleylamine plays an important role in controlling the particle size. Normally, high-quality NCs are synthesized in organic phase, but most of NC-based bio-applications require water-soluble NCs. To convert these hydrophobic NCs to hydrophilic particles, surface modification is employed. Here dual interaction ligands based on the Tween-derivatives (TDs) were synthesized. Stability tests on TD-capped NCs showed that these dual interaction ligands can significantly increase the stability of NCs compared to single interaction ligands. Further, These TD-capped QDs were further tested as fluorescent labels to detect virus-protein expression in cells. To exploit bio-applications of nanocrystals, gold nanocrystal-based assay to detect enzyme activity was designed. The optical properties of Au-NCs are not only dependent on the particle sizes and shapes, but also the distances between the particles. Here, Lipoic acid-tyramine-beta-galactopyranosyl (LTbeta-gal) was synthesized, as ligands, to cap Au-NCs; and the resultant LTbeta-capped Au-NCs could disperse in water. After the hydrolysis of the ligands with beta-galactosidase, these Au-NCs become to aggregate, which exhibit a red-shift in the absorption spectrum of the Au-NC suspension. The detection of beta-galactosidase was further studies by varying the amounts of beta-galactosidase. Hybrid nanocrystals (HNCs) are attractive candidates for advanced nanomaterials because they contain two or more different nanoscale functionalities, which are expected to possess novel physical and chemical properties. Two kinds of heterodimers (FePt/In2O3 and UO2/In2O3) were prepared using a similar procedure and the synthesized HNCs exhibited different shapes. The studies of high-resolution transmission electron microscopy (HRTEM) indicate that the shapes of these two dimers were controlled by the interfacial structures. The amorphous iron oxide layers on the FePt NC surfaces act as glue to interconnect the FePt with the indium oxide parts and led to a core-seed-shaped heterodimer. Using completely crystalline UO2 NCs as seeds resulted in a peanut-shapd HNC.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Huimeng Wu.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Cao, Yun Wei.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022614:00001


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1 INORGANIC COLLOIDAL NANOCRYSTALS : SYNTHESIS AND BIOAPPLICATIONS By HUIMENG WU A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

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2 2008 Huimeng Wu

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3 To my husband, Xiaoyong Zhao; my Daughter, Ashley Zhao; and my parents.

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4 ACKNOWLEDGMENTS I would first like to thank m y adviser, Dr. Charles Y. Cao for pr oviding interesting and important projects, encouraging me to continue the project when things didnot work so well. I would also like to thank my committee members (Prof. Charles Martin, Prof. Weihong Tan, Prof. Ben Smith, Prof. Paul Holloway and Prof. Ronald Castellano) for helping me apply awards and fellowships. Especially I am grat eful to Prof. Weihong Tan for al lowing me to work in his group before I officially starte d my Ph.D. training here. For the material characterization, I am th ankful to Dr. Kathryn Williams for the thermogravimetry analysis (TGA) and many other instruments and help revising my dissertation. Dr. Williams also helped me apply for the Lockha rt Dissertation Fellowshi p. Also I would like to thank Ms. Kerry Siebein in the Major Analytic al Instrumentation Center (MAIC) for HRTEM measurements. I appreciate valuable help from my colleagues in Prof. Caos group. I especially thank Dr. Jiaqi Zhuang for helpful discussions. Finally, I want to thank my husband for his en couragement and support. I want to deeply and sincerely express my appreciation to my parents for taking care of my daughter and letting me have more time to work on the projects.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES.........................................................................................................................9 ABSTRACT...................................................................................................................................13 CHAP TER 1 INTRODUCTION..................................................................................................................15 1.1 Synthetic Strategies ....................................................................................................... 17 1.2 Properties of NCs ..........................................................................................................22 1.2.1 Optical and Electronic P roperties...................................................................... 23 1.2.2 Magnetic Properties ..........................................................................................27 1.2.3 Therm al Properties............................................................................................ 28 1.2.4 Catalytic Properties ........................................................................................... 29 1.2.5 Mechanical Properties ....................................................................................... 29 1.3 Bio-Applications of NCs ...............................................................................................30 1.3.1 Fluorescent Detection........................................................................................ 30 1.3.2 Colorim etric Detection...................................................................................... 31 1.3.3 Surface Enhanced Ram an Scattering (SERS)................................................... 32 1.3.4 Summ ary of the Present Research..................................................................... 32 2 GENERAL SYNTHESIS AND CHARAC TERI ZATION OF NANOCRYSTALS............. 41 2.1 Synthesis of Nanocrystals.............................................................................................41 2.1.1 Semiconductor Nanocrystals............................................................................. 41 2.1.2 Metal Nanocrystals ........................................................................................... 43 2.1.3 Metal Oxide Nanocrystals .................................................................................45 2.2 Characterization of Nanocrystals ..................................................................................46 2.2.1 Transm ission Electron Microscopy (TEM)...................................................... 46 2.2.2 Powder X-Ray Diffraction (XRD) .................................................................... 48 2.2.3 Dyna mic Light Scattering (DLS)......................................................................49 3 SYNTHESIS OF URANIUM-DIOXIDE NANOCRYSTALS.............................................. 56 3.1 Introduction ................................................................................................................... 56 3.2 Experim ental Section.................................................................................................... 56 3.2.1 Chemicals..........................................................................................................56 3.2.2 Synthesis of Spherical Uranium-oxide Nanocrystals........................................ 57 3.2.3 Synthesis of Octahedral UO2 NCs by a Multiple -injection Method................. 58

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6 3.2.4 The Effect of Additional Mixture of OA and OAm with a Molar Ratio of 1:1 ............................................................................................................ 58 3.2.5 The Effect of W ater........................................................................................... 58 3.2.6 The Am ide Effect.............................................................................................. 59 3.2.6.1 Syntheses of Am ides........................................................................... 59 3.2.7 Characterization of UO2 NCs............................................................................ 60 3.2.7.1 XRD M measuments of UO2 NCs....................................................... 60 3.2.7.2 IR Measurem ents................................................................................61 3.2.7.3 1H-NMR Measurements..................................................................... 61 3.2.7.4 TEM Measurem ents............................................................................ 61 3.3 Results and Discussion .................................................................................................. 61 3.3.1 Synthesis and Characterization of UO2 NCs..................................................... 61 3.3.2 Size Control of UO2 Nanocrystals.................................................................... 62 3.3.3 Shape Control of UO2 Nanocrystals.................................................................63 3.3.3.1 Multip le-injection............................................................................... 63 3.3.3.2 Ligand Effe ct......................................................................................64 3.3.3.3 Self -assembly of UO2 NCs.................................................................64 3.3.3 Mechanistic Study of UO2 NC Formation........................................................65 4 WATER-SOLUBLE NANOCRYSTALS THROUGH DUAL-INTERACTION LIGANDS ...............................................................................................................................78 4.1 Introduction ................................................................................................................... 78 4.2 Experim ental Section.................................................................................................... 80 4.2.1 Chemicals..........................................................................................................80 4.2.2 Synthesis of Tween-derivatives (TDs )..............................................................81 4.2.2.1 Dihydrolipoic Acid-functionalized T weens (TDN-L).........................81 4.2.2.2 Dopa mine-functionalized Tweens (TDN-D).......................................83 4.2.2.3 Carboxyl-group-functionalized TD20-L (TD20-LC)............................86 4.2.3 Gold, Fe3O4 and CdSe/ZnS Nanocrystal Synthesis...........................................87 4.2.4 Preparation of TD-capped W ater-soluble NCs................................................. 88 4.2.5 Antibody-functionalized Cd Se/ZnS Nanocrystals ............................................88 4.2.6 Immunoassay Tests ...........................................................................................89 4.2.7 Stability Tests ....................................................................................................90 4.2.8 Other Measurem ents......................................................................................... 90 4.2.8.1 1H-NMR Measurements..................................................................... 90 4.2.8.2 Determ ination of Fluorescence Quantum Yelds (QY)....................... 90 4.2.8.3 DLS Measurem ents............................................................................ 91 4.2.8.4 TEM Measurem ents............................................................................ 91 4.3 Results and Discussion .................................................................................................. 91 4.4 Conclusion ....................................................................................................................95 5 GOLD NANOCRYSTAL-BASED ASSA Y FOR THE DET ECTION OF GALACTOSIDASE............................................................................................................. 106 5.1 Introduction ................................................................................................................. 106 5.2 Experim ental Section.................................................................................................. 108

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7 5.2.1 Chemicals........................................................................................................ 108 5.2.2 Synthesis of LT-gal.......................................................................................108 5.2.3 Detection of -galactosidase ...........................................................................111 5.2.4 Measurem ents................................................................................................. 112 5.2.4.1 1H-NMR Measurements................................................................... 112 5.2.4.2 UV/Vis Spectra. ................................................................................ 112 5.2.4.3 DLS Measurem ents.......................................................................... 112 5.2.4.4 TEM Measurements............................................................................ 112 5.3 Results and Discussion ................................................................................................112 6 SYNTHESIS OF HYBRID NANOCRYSTALS................................................................. 118 6.1 Introduction ................................................................................................................. 118 6.2 Experim ental Section.................................................................................................. 119 6.2.1 Chemicals........................................................................................................119 6.2.2 Synthesis of FePt Nanocrystals.......................................................................120 6.2.3 Synthesis of UO2 Nanocrystals.......................................................................120 6.2.5 Synthesis of Corn-seed-shaped FePt/In2O3 Hybrid Nanocrystals...................121 6.2.6 Synthesis of Peanut-shaped UO2/In2O3 Hybrid Nanocrystals........................ 121 6.2.7 TEM Measurem ents........................................................................................ 121 6.3 Results and Discussion ................................................................................................122 6.3.1 Synthesis and Characterization of FePt/In2O3 Hybrid Nanocrystals..............122 6.3.2 Synthesis and Characterization of UO2/In2O3 Hybrid Nanocrystals..............124 7 CONCLUSIONS.................................................................................................................. 131 7.1 Summ ary of This Research......................................................................................... 131 7.2 Perspectives ................................................................................................................. 132 APPENDIX A XDR MEASUREMENT OF URANIUMOXIDE NANOCRYSTALS............................... 134 B 1H-NMR SPECTRUM.......................................................................................................... 135 C MS SPECTRUM OF LT-GAL...........................................................................................146 LIST OF REFERENCES.............................................................................................................147 BIOGRAPHICAL SKETCH.......................................................................................................157

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8 LIST OF TABLES Table page 1-1 Electron and hole masses, exciton Bohr radii and band-gap energies for various sem iconductor materials.................................................................................................... 394-1 Stability test of CdSe/ZnS NCs capped with different ligands........................................ 105

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9 LIST OF FIGURES Figure page 1-1 Structure of a typical nanocrystal: the cr ystalline inorganic co re, capped by organic ligands ................................................................................................................................35 1-2 LaMer curve.......................................................................................................................35 1-3 Schematic illustration of the electronic structure............................................................... 36 1-4 Idealized density of states for one band of a se miconductor structure of d, d, d, and d materials.................................................................................................... 36 1-5 The color of CdSe NCs depends on NC sizes.................................................................... 37 1-6 UV/Vis absorption spectra of different size Au NCs: 9 nm 22 nm, 48 nm and 99 nm Au NCs in water................................................................................................................ .38 1-7 The emission wavelengths from representa tive Q D core materials and representative areas of biological interest................................................................................................. 38 1-8 In vivo cancer targeting and im aging of QD-antibody conjugates in living mice............. 39 1-9 The target DNA induces aggregation of oligonucleotide-m odifed Au NCs...................... 39 1-10 Scheme for the SERS detection of DNA fragments.......................................................... 40 2-1 Injection method for synthesis of m onodisperse semiconductor NCs............................... 51 2-2 TEM images of polyhedral metal nanocrystals................................................................. 52 2-3 The electron-optical system of a TEM............................................................................... 53 2-4 The relationship of the spacing ( R ) between diffraction spots and cam era length ( L )......54 2-5 The TEM images of star-shaped Fe3O4 NCs.....................................................................55 3-1 Characterization of UO2 NCs.............................................................................................70 3-2 TEM images of UO2 NCs..................................................................................................71 3-3 Multiple-injection method for preparing octahedral UO2 nanocrystals.............................72 3-4 The particle growth with multiple-in jection of the precursor solutions............................ 72 3-5 A typical TEM image of branched UO2 NCs. The inset is the HRTEM image................ 73 3-6 TEM images of self-assembled UO2 nanocrystals............................................................. 73

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10 3-7 TEM images of assemblies of 9.1 nm octahedral UO2 nanocrystals.................................74 3-8 IR and 1H-NMR spectra of reaction mixture..................................................................... 75 3-9 TEM measurement of the water effect............................................................................... 76 3-10 IR spectra of purified UO2 NCs and OOA ........................................................................76 3-11 TEM measurement of the amide effect.............................................................................. 77 4-1 Schematic illustration of the Tween -derivatives (T Ds) dual interaction........................... 97 4-2 Synthesis of dual-interaction TD ligands........................................................................... 98 4-3 1H-NMR of compound TD20-a...........................................................................................99 4-4 1H-NMR of compound TD20-L..........................................................................................99 4-5 DLS data for TD20-L-functionalized 6.6-nm Au NC s from five parallel thermalstability-test experiments................................................................................................. 100 4-6 TEM images of hydrophobic NCs................................................................................... 100 4-7 Gas-chromatography mass spectrometry (GC-MS)......................................................... 101 4-8 UV-Vis spectra for the stability tests of TD20-L-functionalized Au NCs........................ 102 4-9 Stability tests of TD20-L-functionalized 6.6-nm Au NCs monitored with DLS and UV-Vis spectra.................................................................................................................102 4-10 TEM images of TD20-D-capped Fe3O4 NCs from aqueous solutions at pH 7 and pH 2 for 2h................................................................................................................................103 4-11 TD20-L capped CdSe/ZnS NCs with different sizes in aqueous solution........................ 103 4-12 Stability tests of TDN-L-functionalized 5.6-nm CdSe/ZnS NCs as a function of the fatty-acid chain on these TD ligands............................................................................... 104 4-13 Immunofluorescence tests of NS5A-c ontaining FCA1 ce lls using TD-functionalized CdSe/ZnS core/shell nanocrystal QD..............................................................................104 5-1 galactosidase assay based on Au-NCs. ......................................................................... 116 5-2 Detection of -galactosidase.. ..........................................................................................116 5-3 Kinetics studies of the emzy me-induced Au-NC aggregation. ........................................ 117 6-1 TEM images of FePt seeds and the nanoc rystals extracted from the FePt reaction m ixture before the injection of the indium stock solution............................................... 126

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11 6-2 STEM images of FePt/In2O3 hybrid nanocrystals........................................................... 126 6-3 TEM image of the FePt-Iron oxide core-shell N Cs obtained from the control experiment..................................................................................................................... ...127 6-4 HRTEM studies of an individual FePt/In2O3 hybrid nanocrystal....................................128 6-5 TEM images of UO2/In2O3 hybrid nanocrystals.............................................................. 129 6-6 HRTEM images of an individual UO2/In2O3 hybrid nanocrystal....................................130 A-1 XRD measurement of UO2 Nanocrystals........................................................................134 B-1 1H-NMR of compound TD20-a.........................................................................................135 B-2 1H-NMR of compound TD20-L........................................................................................135 B-3 1H-NMR of compound TD40-a........................................................................................136 B-4 1H-NMR of compound TD40-L........................................................................................136 B-5 1H-NMR of compound TD60-a.........................................................................................137 B-6 1H-NMR of compound TD60-L........................................................................................137 B-7 1H-NMR of compound TD80-a.........................................................................................138 B-8 1H-NMR of compound TD80-L........................................................................................138 B-9 1H-NMR of compound TD20-b........................................................................................139 B-10 1H-NMR of compound TD20-c.........................................................................................139 B-11 1H-NMR of compound TD20-D.......................................................................................140 B-12 1H-NMR of compound TD40-b........................................................................................140 B-13 1H-NMR of compound TD40-c.........................................................................................141 B-14 1H-NMR of compound TD40-D.......................................................................................141 B-15 1H-NMR of compound TD60-b........................................................................................142 B-16 1H-NMR of compound TD60-c.........................................................................................142 B-17 1H-NMR of compound TD60-D.......................................................................................143 B-18 1H-NMR of compound TD80-b........................................................................................143 B-19 1H-NMR of compound TD80-c.........................................................................................144

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12 B-20 1H-NMR of compound TD80-D.......................................................................................144 B-21 1H-NMR of compound TD20-e.........................................................................................145 B-22 1H-NMR of compound TD20-LC.....................................................................................145 C-1 MS spectrum of LT-gal................................................................................................. 146 LIST OF REFERENCES.............................................................................................................147

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13 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy INORGANIC COLLOIDAL NANOCRYSTALS: SYNTHESIS AND BIOAPPLICATIONS By Huimeng Wu December 2008 Chair: Charles Y. Cao Major: Chemistry Nanocrystals (NCs) are very small particle s, which contain from a few hundred to thousands of atoms depending on the size of NCs. Because of their specia l properties compared with the bulk materials, NC s have found many promising app lications in areas, such as biomedical diagnosis, catalysis, plasmonics, high-density data storage and solar energy conversion. This dissertation pres ents studies on the syntheses of metal oxide NCs and hybrid NCs, the surface functionalization of NCs by dualinteraction ligands, and gold-NC-based assay for the detection of galactosidase. Monodisperse colloidal uranium dioxide NCs (UO2 NCs) were synthesized by decomposition of uranyl acetylacetonate. By cha nging the amount of added surfactant, the sizes of the NCs could vary from 2 ~ 8 nm. Mechanistic studies of the formation of UO2 NCs showed that the condensation product (amide) of oleic acid and oleylamine plays an important role in controlling the particle size. Normally, high-quality NCs are synthesized in organic phase, but most of NC-based bioapplications require water-soluble NCs. To convert these hydrophobic NCs to hydrophilic particles, surface modification is employed. Here dual interaction ligands based on the Tweenderivatives (TDs) were synthesized. Stability tests on TD-capped NCs showed that these dual

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14 interaction ligands can significantly increase the st ability of NCs compared to single interaction ligands. Further, These TD-capped QDs were further test ed as fluorescent labe ls to detect virusprotein expression in cells. To exploit bio-applications of nanocrystals, gold nanocrystal-bas ed assay to detect enzyme activity was designed. The optical properties of Au-NCs are not only dependent on the particle sizes and shapes, but also the distances between the particles. Here, Lipoic acidtyraminegalactopyranosyl (LT -gal) was synthesized, as ligands, to cap Au-NCs; and the resultant LT gal-capped Au-NCs could disperse in wate r. After the hydrolysis of the ligands with galactosidase, these Au-NCs become to a ggregate, which exhibit a red-shift in the absorption spectrum of the Au-NC suspension. The detection of galactosidase was further studies by varying the amounts of galactosidase. Hybrid nanocrystals (HNCs) are attractiv e candidates for advanced nanomaterials because they contain two or more different na noscale functionalities, which are expected to possess novel physical and chemical properties. Two kinds of heterodimers (FePt/In2O3 and UO2/In2O3) were prepared using a similar proced ure and the synthesized HNCs exhibited different shapes. The studies of high-resolu tion transmission electron microscopy (HRTEM) indicate that the shapes of these two dimers were controlled by the interfacial structures. The amorphous iron oxide layers on the FePt NC surfaces act as glue to interconnect the FePt with the indium oxide parts and led to a core-seed-sh aped heterodimer. Using completely crystalline UO2 NCs as seeds resulted in a peanut-shapd HNC.

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15 CHAPTER 1 INTRODUCTION Nanoscience and techno logy, which are among the most exciting research areas in modern science, involve studies of matter at th e nanoscale level to discover new properties and applications. Nanocrystals (NCs), which are impor tant building blocks for the development of nanoscience and technology, are ve ry small particles (1 nm~100 nm), which contain from a few hundred atoms to thousands of atoms depending on the particle size. Th e surfaces of NCs are usually capped by organic or inorganic molecules to avoid aggregation and to stabilize NCs in solvents (Figure 1-1). The crystal structures of NCs are the same as those of the corresponding bulk materials, but the nano-sized particles exhibit special properties, including unique optical, magnetic, catalytic, biological and mechan ical properties. For example, semiconductor NCs show sizedependent absorption and emission spectra because of quantum confinement effects.[1,2] Noblemetal NC solutions exhibit different colors, de pending on their sizes and shapes, due to surface plasmon polaritons.[3] Magnetic NCs show size-depe ndent magnetization transitiontemperatures, and Coercivity ( Hc) and the remanence to saturation magnetization ratio ( Mr/ Ms) are also related to particle size. Tetrahexahed ral platinum NCs with high-index facets exhibit enhanced catalytic activity (up to 400%) compar ed to equivalent Pt surface areas for electrooxidation of small organic fuels, such as formic acid and ethanol.[4] CdS semiconductor nanoparticles, enfolded by the chaperonin proteins GroEL and T.th cpn are high thermal and chemical stability in aqueous media and can be readily released from the protein cavities by the action of ATP, analogous to the biolog ical function of the chaperonins.[5] Gold-nanowires exhibit a Young's modulus which is essent ially independent of diameter, whereas the yield strength is largest for the smallest diameter wires, with strengths up to 100 times that of bulk materials.[6]

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16 The investigation of these new properties a nd applications is important for both the understanding of these materials at the funda mental level and the development of novel techniques and applications, in areas such as biological labeling,[2,7-10] solar energy,[11-13] LED development,[14-18] catalyzing.[19,20] and high-density data storage.[21] However, these promising applications can not be fully realized until existing fundamental questions are answered. First, methods must be developed to synt hesize high-quality NCs on a large scale with controlled sizes and shapes. To date, the most successful and widely used method to synthesize nanocrystals relies on rapid precursor injection.[22] Unfortunately, due to rapid injection and reaction kinetics, nanocrystal formation is rather like black magic with the nanocrystal size, size distribution, and shape being ve ry sensitive to subtle differences in the injection process (injection time, speed, etc). Furthermore, it is difficult to scale up the method for nanocrystal synthesis in large quantities. Ther efore, there is a need to devel op easily controllable methods for nanocrystal synthesis. Studies of the mechanisms of nanocryst al nucleation and growth can provide better understanding of how particle size and shape can be controlled. Second, surface engineering of nanocrystals is key to the nanocrystal-based applications. New functionalization methods need to be designed for various applications. Previous research indicates that the properties are very sensit ive to the surfaces of NCs. Modification of nanocrystal surfaces directly impr oves their stability and solubilit y, as well as their chemical and physical properties. Since NCs have exhibited a number of potent ial bioapplications, one goal of surface engineering is to achieve robust and bioc ompatible nanocrystals with special properties. The following portion of this introduction in cludes a brief description of synthesis strategies, size-dependent propert ies and potential applications of NCs, as well as a brief summary of the present research work.

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17 1.1 Synthetic Strategies Based on the reaction m edia, preparation of nanocrystals can be classified as gas phase, liquid phase or solid phase synt heses. Solid-phase syntheses ha ve been employed to alter the crystalline structures of NCs[23] or create hollow structured NCs[24] by annealing NCs at high temperature. Most NCs have been synthesized in either the gas phase or liquid phases. Gas-phase synthesis is a well-known chemical manufacturing technique for ultra-thin coating of particles, such as carbon black and py rogenic silica, using fl ame, plasma, or laser reactors.[25] The products of gas-phase syntheses usually are bare particles with a wide size distribution. Since no ligands stabil ize the particles, these particle s tend to form aggregates in solution. A chemical vapor deposition (CVD) method has been successfully employed for preparation of multiwall or single-wall carbon nanotubes (MWNTs)[26] Recently, gas phase syntheses have been exploited for preparati on of various nanomaterials, such as metal[27,28], metal oxide[29] and semiconductor NCs,[30] and FeSi nanowires.[31] Gas phase synthesis of NCs provides a high throughput method for preparation of nanomaterials, but further investigation need to be performed to improve size distributio n, crystallinity, and surf ace passivation of these nanomaterials. The advances in quantum dot science and t echnology have been made possible by liquidphase synthetic methods, which allow investigati on of the special optical properties of NCs and their potential biomedical applica tions, such as fluorescent labels[2] and cell trackers.[32] Liquid phase synthesis can be performed within structur ed media acting as templates, such as reverse micelles,[33] microemulsions[34,35] and nanoporous membranes[36]. Membrane template synthesis of nanomaterials was developed by Martins group. Depending on th e material and the chemistry of the membrane, the resulting nanomaterial may be either solid (nanowires) or hollow (nanotubes). Martins group has also invest igated applications of nanotube-containing

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18 membranes for separations of DNA, proteins and drugs.[37,38] Template-free syntheses have been performed in both aqueous and organic solvents for preparation of metal,[39] metal oxide[40] and semiconductor NCs,[41] such as Au NCs,[42,43] Ag nanoprisms[44] and CdTe nanowires.[41,45] Aqueous-phase syntheses are usually simple, fa st and relatively inexpensive, but few highquality NCs have been reported by these methods. Recently, great progress has been made in organic-phase synthesis for colloidal inorganic nanocrystals.[46] Numerous monodisperse, sizeand shapecontrollable nanocrystals have been synthesized using organic-phases.[47,48] Although a general approach to the fabrication in a precisely controlled manner is not av ailable yet, it is widely accept ed that the presence of organic surfactants can be key to determin ing the sizes and shapes of the NCs. Since most of the present research has utilized organic-phase syntheses, th ese methods will be descri bed in greater detail. Organic-phase nanocrystal synt hesis involves three important steps: nucleation, growth, and passivation. Synthesis of high-quality colloidal nanocrystals with different sizes and shapes can be achieved by manipulating these three steps. LaMers curve is well-known for describing the formation of precipitates, and it can also be used to understand the formation of NCs from homogeneous, supersaturated media, [49] as shown in Figure 1-2. In a typical synthesis, precursors, ligands and solvents are heated to a certain temperature, at which the precursors react with each other or with the ligands to form active atoms or molecules, called monomers. At the very beginn ing, the reaction medium is saturated with the monomers without the formation of any particles (i.e., nuclei). Wh en the concentration exceeds a critical limit, called the critical limiting super-s aturation, nucleation occu rs. The nuclei start to grow and consume the active monomers, thereby depleting the concentration of monomers. When the concentration of active monomer decr eases, no further nucleation occurs and the

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19 existing particles grow. The growth rate is controlled by both diffusion and reaction kinetics. Finally, the growth of particles is terminated by passivation of the NC surface using organic ligands to coordinate or bind to the nanocrystal surfaces. These ligands prevent agglomeration and fusing of particles, and they make the particles soluble in certain solvents. A prerequisite for achieving monodisperse NC s is decoupling of nuc leation event from the growth step. One method involves shortening th e time period of nucleation in comparison to the growth period. That way, all nuclei will unde rgo a similar growth period, resulting in a narrow size distribution, as show n in Figure 1-2. If the nucleation period is long and concurrent with the growth process, the final partic le size and shape will be poly-disperse. Currently, the most successful and widely-use d NC syntheses have relied on the rapid injection method, first demonstrat ed by Murray and Bawendi in 1993.[22] In this injection method, precursors are swiftly injected into a hot reaction system with rapid stirring. After injection, fast nucleation occurs and this is followed by a rela tively long growth period. Upon nucleation, the concentration of re actant in solution drops below the critical concentration for nucleation, and further material ca n only add to the existing nuclei. Growth rate is controlled by the rate of diffusion of reactant to the particles and/or by the react ion rate. Ultimately, the growth will be balanced by the solubility. Since the nucl eation time is determined by the rates of both injection and diffusion, injection-based synthese s have poor reproducibility and are unsuitable for large scale production, making these methods unsuitable for industrial use. Consequently, non-injection methods are need ed to enable the devel opment of nanotechnologies. In organic phase syntheses, most the formati on of NCs occurs at high-temperature (> 200 oC). In the one-pot method, the precursors are mi xed at room temperature or relatively low temperature before the reaction mixture reaches th e reaction temperature. The heating from room

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20 temperature to a high temperatur e often results in undesired nucle ation concurrent with growth. This factor poses a major challenge in the pr eparation of monodisperse NCs using non-injection methods. To overcome this problem and separate the nucleation and grow th steps, different methods have been tested. Recently, our group ha s reported non-injection syntheses of CdS NCs using nucleation initiators,[50] and CdSe and CdTe NCs by choosing precursors with suitable reactivity.[51] The addition of seed particles to the reac tion mixture is another way to separate the nucleation and growth processes.[52] As described in LaMers curve, when the active monomer concentr ation drops below the critical concentration of supers aturation, nucleation ends and the growth progress of NC can be controlled by the diffusion process. But in act uality, the reaction system is much more complicated than indicated by LaMers model. The growth of NCs can be described by the diffusion of monomer towards the surface of the growing NCs, fo llowed by dissociation rate of the surface capping ligands from the particle su rface, and the subsequent reaction of diffusing particles or atoms assimilated ont o the growing NCs. Thus, any pa rameters which can affect the diffusion rate or the interaction between the li gands and the NC surfaces (e.g. solvents, ligands, reaction temperature, pressure, reaction time and concentration of precursors) all play an important role in dictating the final particle size and shape. The difference of the chemical potential at th e solid/liquid interface al so affects the size distribution of NCs. The chemical potential of a small particle is higher than that of a larger particle. Therefore, the equilibrium solute concentr ation of a small particle is much greater than that for a large particle. The gradients of solute concentration between small particles and large particles result in the mass transport from sma ll particles to large particle, called Ostwald

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21 ripening or the coarsening effect.[53-55] At equilibrium, there is a balance between dissolving and growth of the NCs. The sizes and shapes of NCs can be controlle d by tuning the number of nuclei and the chemical potentials of the components of the reac tion system. This is usually realized by varying the reaction parameters: type s and concentrations of pr ecursors and ligands, reaction temperature, time and pressure, heating and stirring rates. Since the detailed chemical mechanisms of nanocrystal synthesis are not comp letely clear, there are no universal rules which can be applied to all nanocrystal growth. But th ere are some points which are useful. To obtain larger particles, a straightforward way is to increase the reaction time.[56] Based on Gibbs law, a particle in equilibrium with its surroundings should have the minimum surface energy. Normally, the surface energy of spherical NCs is lower th an that of nonspherical particles (e.g. cubes, tetrahedral pyramids and triangular prisms). The surface chemical potentials on the individual facets are different.[57] Thus, for shape control, the surface chemical potential must be considered. To stabilize the NCs, special organic ligands are employed. These or ganic ligands contain the electron-rich capping group, e.g. a phosphi ne, phosphine oxide, amine, carboxylate or thiolate, can coordinate to the el ectron-poor metal ions, such as Cd2+, Zn2+ and Fe3+, or to elemental metals, e.g. Au and Ag. The other end group of the ligand imparts solubility to the NCs. For example, hydrophobic ends, such as al kyl groups, make NCs so luble in low-polarity solvents (e.g. toluene, hexane or chloroform), and hydrophilic ends make NCs water-soluble. The strength of ligand bonding to the nanocrystal s affects the stability of the NCs. Weak bonds between the ligands and the part icle surface will result in unstabl e NCs. Once the NCs lose the ligands on their surfaces, the par ticles will aggregate and precipitate. However, very strong

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22 bonds will block the growth of NCs and result in very tiny particles. In most cases, the interactions between the ligands and the nanocry stal surfaces are not very strong. Therefore, surface modification, also called surface engin eering, by ligand exchange after the initial synthesis is complete allows controlling the solu bility, stability and other properties of NCs. Another way to enhance the stability of NCs is growth of an additional inorganic shell on the core nanocrystal. Passivation by organic cap ping ligands is not perfect. For example, in trioctylphosphine oxide (TOPO) capped CdSe NCs, TOPO preferentially binds to cationic cadmium sites on the surface, leaving anionic se lenium dangling bonds. This, as well as the steric hindrance that the bulky TOPO ligands impose on neighbori ng nanocrystal surface sites, leaves cadmium dangling bonds. These dangling bonds on the NC surface act as trap sites, which provide pathways for non-radiative decay of the nanocrystal excite d states, and result in reduced photoluminescence in semiconductor NCs.[58] Epitaxial growth of an inorganic shell can passivate these dangling bonds and remove trap site s. Thus, the optical pr operties of NCs can be enhanced by addition of an inorganic shell. The first successful preparation of core-shell NCs was synthesis of CdSe/ZnS NCs by Guyot-Sionnest and co-workers.[59] With the addition of the wider bandgap ZnS shell, high emitting NCs were achieved. The quality of core/shell NCs is dependent on the lattice mismatch between the core and shell materials, and usually shell materials with less lattice mi smatch are easier to add. 1.2 Properties of NCs Reducing the size of bulk m aterials to nanoscale dimensions significantly changes the magnitude of chemical and physical properties, including optical, el ectrical and magnetic properties, melting temperature,[1,60] catalytic capabilities and mechanical properties. These special properties come from quantum c onfinement effects a nd surface effects.

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23 1.2.1 Optical and Electronic Properties Figure 1-3 d escribes the changes in the electron ic structure with a decrease of particle size in metals and semiconductors.[1] In both cases, the decrease in crystal size results in discrete energies at the band edges. In a metal, the Fermi level is at the centre of a band, where the energy states do not significantly vary w ith decreasing particle size, and the relevant discreteness of the energy levels is very small. Only for very small particles (tens or hundreds of atoms) at very low temperature (close to absolute zero degree), is this discreteness greater than the thermal energy and measurable. The average spacing in consecu tive energy levels is know as the Kubo gap.[61] In a semiconductor, the Fermi level is between two bands, i.e., in the band gap, which is the spacing between band edges. The di screteness at the band edges resu lts in changes in transition energies as one or more dimensions of the crystals decrease to nano-scale. Figure 1-4 shows the change in the density of states from th e completely unconfined state (3-dimensional bulk material) to the completely confined state (0-dimensional Quantum dots). In the bulk state, the energy levels are nearly continuous. But for the NC structure where the material has been spatially confined in all three directions, the energy levels are restricted to a specific set of completely quantiz ed states. The changes in the en ergy states result in the sizedependent optical and electrical pr operties in semiconductor NCs. One example of quantum confinement in semiconductor NCs is the size-dependent absorption and emission spectra; i. e., the color of NCs depends on their size, as shown in Figure 1-5. For example, by changing the diameter of CdSe NCs from 2.3 nm to 5.5 nm, the energy gap of this material varies from 2.7 eV to 1.9 eV, encompassing almost the entire visible region of the optical spectrum.[62] Semiconductor electronic properties can be de scribed by molecular or bital (MO) theory. Theoretical analysis, based on the effective mass approximation (EMA)[63], shows that the

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24 electronic energy states of NC s strongly depend on the ratio of the nanocrystal radius, R to the exciton Bohr radius, 2*2 Ba= / e where is the exciton reduced mass, is Planck's constant diviced by 2 and is dielectric constant of the crystal. When R >> aB, there is little or no confinement (called infinite hole mass). The Coulomb force domina tes and the exciton acts as a single particle, so that: *** eh mm where em and hm are the effective electron and hole masses, respectively. However, when R ~ aB, a weak confinement occurs. The bandgap shift relative to the bulk band gap is given by:[64] 22 g Ry *2 E = E 2 R (1-1) where* RyE is the bulk exciton binding energy, called the Rydberg energy, *4 Ry 22 e E 2 When R < aB a strong confinement occurs, and the elect ron and hole are decoup led by the dominant quantization effect. The effective reduced mass *is given by: *** eh1/ =1/m+1/m. And the bandgap shift becomes:[64] 22 2 g Ry *2 1.786e E = 0.248E 2R R (1-2) In equation 1-2, the first term represents the qua ntum energy of localization, which increases as the reciprocal of R2 for both electron and hole.[63] The second term represents the Columbic attraction,[63] which is negligible in th e strong confinement regime. The third term is the remnant of the exciton effect.[64] Table 1-1 lists the electron and hole effective masses, exciton Bohr radii (aB) and band-gap energies for various semic onductors. Since the valu e of R is materialdependent, the relative shift of energy can vary with both the size a nd the composition of the material.

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25 Compared with bulk materials, the number of surface atoms in NCs is a large fraction of the total. The surface atoms play a critical role in the surface energy and c ontrol the solubilities, stabilities, and propertie s of the NCs. The surface effect al so influences the electronic energy levels of semiconductor NCs. The explanati on of quantum confinement, based on the EMA model, ignores the atomic detail on the surface. This assumption is only correct when the NCs are capped with a higher bandgap material of su fficient thickness. However, in most cases, dangling bonds, strain defects, and partial passiva tion of the surface have critical effects on the lowest energy states of the nanocrystal. The shift of energy states due to surface effects can be as large as the shift due to confinement. An atomistic tight-binding (TB) theory[65,66] has been proposed to account for these surface effects in semiconductor nanocrystals. R ecently, a realistic TB approach has provided a more accurate description for the bandgap va riation with size, based on the equation: [67] g 21 E= aD+bD+c (1-3) where D is the diameter of the NCs, and a, b and c are material-dependent parameters, which can be evaluated by fitting the variation of the ba ndgap for the different group II-VI semiconductors using equation (1-3).[67] By growing a shell w ith a large bandgap on th e core NC, the surface effect can be reduced. For example, capping the CdS dot with ZnS reduces the effect of the surface on the internal electronic states and op tical properties. Based on the TB model, six monolayers of ZnS are needed to elimin ate the influence of any surface states.[68] Noble metal NC solutions normally display a special color which is absent in the bulk material or the individual atoms. But unlike se miconductor NCs, where the absorption spectrum is due to excitonic transitions from the ground state to excited st ates, the absorption in metal NC solutions is due to the surface plasmon band. When lig ht irradiates a metal nanocrystal, such as a

PAGE 26

26 gold or silver nanocrystal, the electromagne tic field causes oscillation of the conduction electrons. If the collectiv e oscillation of the elec trons has the same frequency as that of the incident photons, localized surface plasma resonance (LSPR) occurs, resulting in unique absorption in the visible or near-infrared regi ons. In semiconductors, there are very few free electrons and the plasmon absorption occurs in the infrared region. The position of the band peak of gold NCs is size-dependent. Figure 1-6 shows the absorption spectra of 9, 22, 48 and 99 nm gold NCs in water.[69] The position of the band peak also depends on the density of electron states on the particle surface, the particle shape, and the surrounding environment, e.g. temperature and solvent.[70] The logarithms of the extinction coefficients of gold NCs are linearly related to the core diameters and are independent of the capping ligands on the particle su rfaces and the solvents used.[71] The aggregation of Au NCs in chloroform results in a red-shift in the absorption spectra. The high-sensitivity of SPR to small environmental changes has been clearly demonstr ated, and applications based on the change of the surface plasmon resonance (SPR) have been widely investigated. Gustay Mie was the first to provide a quantitative description of SPR by solving Maxwells equations with the appropriate bou ndary conditions for spherical particles.[72] In Mies model, the total extinction cross-section, ext, is the sum of the absorption and scattering cross-sections of the NCs over all elec tric and magnetic multipole oscillations.[73] When the particle size is less than the wavelength of the incident light, the scatteri ng part of particles and higher order extinction terms can be neglected, a nd Mies model can be simplified to the dipolar excitation mode:[74] 2232 2 ext 22 126 (2)m mD (1-4)

PAGE 27

27 where D is the diameter of the spherical particle, the wavelength of light, m the dielectric constant of the surrounding medi um (frequency independent), and 1, 2 are the real and imaginary parts of the frequency-dependent dielectr ic constant of the s ubstance (particles). The values of 1 and 2 can be obtained from experiment or theoretical estimation.[75] When 12m ext reaches the maximum. Thus, the SPR of the NCs depends highly on the particle diameter and the dielectric cons tant of the surrounding medium. Mies theory, which was derived for spherica l particles, has been used in evaluating experimental results. Recent advances in the synt hesis of high quality NCs with well defined shapes, such as rods, cubes, triangular prisms, multipods and polyhedra,[76] demonstrate that the shapes of NCs also affects the SPR. Spherical Au NCs exhibit only a single peak, whereas nonspherical gold NCs exhibit multiple scattering peak s in the visible and near-infrared regions due to localized surface plasmons. For example, Au nanorods exhibit two absorption peaks, corresponding to transverse and longitudinal surface plasmons. The transverse band is around 520 nm, whereas the longitudinal band depends on the aspect ratios of the nanorods, and the intensity of these two bands depe nds on the alignment of the rods.[77] In most cases, gold rods are randomly oriented in solution because of Brownian motion. 1.2.2 Magnetic Properties Magnetic NCs, such as cobalt, nickel, iron ox ide, and m ixed NCs, show size-, shapeand composition-dependent properties. Coercivity (Hc) and the remanence to saturation magnetization ratio (Mr/Ms) of magnetic particles have the maximum values when the particles reach the critical sizes, which are around a few to tens of nano meters in diameter, depending on the chemical composition and crystalline structure of the particles.[23,78,79] For the CoNi alloy system, the highest Hc can be achieved when the nanocrystal size is in the range of 20-40 nm.

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28 Domain structure exists in particles larger th an the critical size and is responsible for the decrease of coercivity and remanent magnetizati on. Magnetic studies of these particles have also shown that the magnetic properties are aff ected by the particles surface environment,[80] because defects on the particle surfaces change the satu ration magnetization. The pa rticle shape effect was investigated by comparing spheri cal and cubic cobalt ferrite, CoFe2O4, nanocrystals having the same volume. The results showed that satu ration and remnant magnetization of nanocrystals are determined solely by the size, regardless of the shape (spherical or cubic). However, the shape of the nanocrystals is a dominating factor for the coercivity of na nocrystals due to the effect of surface anisotropy.[81] The transition temperatures are also affect ed by the reduction of particles size. For example, the magnetic transition temperature (cal led the Curie temperature in ferromagnets (Tc) and the Nel temperature (TN) in antiferromagnets) decreases with the decrease in particle size. 1.2.3 Thermal Properties As m entioned above, the transition temperatur e from solid to liquid phase decreases with decreasing size of the NCs. The size-dependent melting temperature of CdS NCs decreases over 55 % when the particle size is less than 15 nm.[60] This special property can be explained by the decrease of the energy barrier for atomic motion. Melting is a process in which the interactions between atoms, ions or molecules are broken thr oughout the structure. Compared to the interior atoms in the NCs, the surface atoms have surface energy, so they require less energy to disrupt the interactions with neighboring atoms and are free to move. The movement of surface atoms causes the surface to be minimized, thus reducing the surface energy. As pa rticle size decreases, the fraction of the surface atoms relative to the total increases. Thus, less energy is required to transform the material from the solid to liquid phase.

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29 1.2.4 Catalytic Properties By decreasing the particle sizes of catalysts, a trem endous increase in the total surface area can be achieved. Thus, nanosized catalysts possess more active sites, which significantly influence the activity of the nanocrystal catalyst For example, gold particles can be used as a catalyst for CO oxidation. [82] The catalytic activity depends on the size of the gold particles, the interactions with the support material, the particle prepar ation method, and the activation procedure. The activity of gold catalysts is a pproximately proportional to the number of lowcoordinated atoms at the co rners of the gold particles.[83] Palladium is the most important catalyst for carbon-carbon bond formation.[84,85] Bawendis group has investigated the catalytic ability of water-dispersible Pd NCs on the Suzuki coupling reaction.[86] When 4-iodotoluene and phenylboronic acid were reacted in the presence of 2 mol % wa ter-dispersed Pd NCs, a 97% yield of the product was obtaine d. In the second recycle reacti on, the yield decreased to 65%, which is comparable to the reactivity of the seco nd recycle reaction in an organic solvent (71%). The surface structures of nanocrystals also a ffect the catalytic ac tivity because of the different bonding abilities on the different crystallographic faces.[4] For example, catalytic hydrogenation of benzene using the (100) surface of platinum yiel ds only cyclohexane, but using the (111) surface, both cyclohexane and cyclohexene are produced.[87] 1.2.5 Mechanical Properties Defect-free silicon spheri cal NCs are four tim es harder than bulk silicon.[88] These special mechanical properties of NCs can also be attributed to surface effects. Recently, a montmorillonite (MTM) / poly(vinyl alcohol) (P VA) nanocomposite was prepared by layer-bylayer assembly. MTM is composed of a 2~1 layered smectite clay mineral with a plated structure. These multilayer nanocomposites are as ha rd as ceramic materials, but the processing temperature is much lower[89] than those materials. A high level of ordering of the nanoscale

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30 building blocks, combined with dense covalent and hydroge n bonding and stiffening of the polymer chains, leads to highly effective load transfer between nanosheets and the polymer.[90] 1.3 Bio-Applications of NCs Optical detection based on NCs is t he most wi dely studied and promising application of NCs. The ultimate goal of optical detection is to enable single-molecule detection in vivo.[91] So far, there are three kind s of applications: semiconductor QDs as labels in fluorescent detection, Au-NC-based colorimetric detect ion, and surface enhanced Raman scattering. Application based on the Frster resonance energy transfer (FRET) or the other properties of NCs, such as use in magnetic separation and detection, sola r cells and light emission diodes (LED)[15], are not covered in this chapter. 1.3.1 Fluorescent Detection Com pared with traditional organic dyes, semiconductor NCs are highly photostable and have tunable, narrow and symmetric emission sp ectra. In addition, multicolour emission can be obtained by a single excitation wavelength. The em ission wavelengths from colloidal QDs made of ZnS, CdS, ZnSe, CdTe and PbSe cover the UV to the infrared region (Figure 1-7), with most bio-applications falling in the visible and near infrared re gions. Thus, semiconductor NCs present very promising applications in biological and biomedical areas. Advances in the synthesis of the NCs make it possible to prepare gram quantities of high quality NCs. By surface modifications, (e.g. ligand exchange[7], or coating with silica or amphiphilic polymers[2,32,93]) the NCs can be made water-so luble. Conjugation with protein, DNA and other biological molecules presents ways to use QDs for biosensors or in diagnosis. In vivo and in vitro tests have been carried out using QDs as fluorescent probes in a variety of biological investigations. For example, QDs ca pped with phospholipid micelles were injected into Xenopus embryos. The studies showed that QD-micelles were stable, cell autonomous, and

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31 slow to photo-bleach, and they had low toxicit y. Nanocrystal fluorescence could be followed to the tadpole stage, allowing lineage-tracing experiments in embryogenesis.[32] Further modification of water-soluble QDs by biological molecules, such as biotin, DNA and antibodies, results in multifunctional QD-conjugates, which can be used as sensors for cancer detection[93,94] Figure 1-8 depicts spectral imaging of QD-an tibody conjugates in living mice harboring tumor cells. QD-antibody conjugates were injected into the tail vein of a control mouse (the left one, no tumor) and a tumor-bearing mouse (the righ t one). The autofluorescence background was separated from the QD signal using spectral mi xing algorithms. The com posite image clearly shows the whole-bodies of bot h mice and the tumor site.[93] The advantages of QDs make it possible to provide both long-term stability and simultaneous detection of multiple signals. But there are still many problems and challenges in the application of QDs; for example, the stability of QDs in water. High quality QDs are usually synthesized in organic phases. However, after transfer to an aqueous phase by ligand exchange, the QDs lose their high stability and al so some of their optical properties.[95] So new methods and/or more robust ligands need to be devel oped. Also non-specific bondi ng and toxicity issues need to be resolved. 1.3.2 Colorimetric Detection Colorim etric sensors based on gold nanocryst al (Au-NC) have become an attractive research area.[96] The bands of SPR peaks of Au-NCs are dependent not only on the size and shape of Au NCs, but also on the distances between the particles. Oligonucleotide-functio nalized gold NCs tend to aggregate in the presence of the complementary target DNA, as shown in Figure 19A and the color of th e solution changes from red to blue (Figure 1-9B). The SPR change of Au-NCs can be measured from the adsorption spectra of Au-NP solutions or it can be observed visually. In addition, th ese Au-NCs demonstrate

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32 high stability and easy surface modification. The mo lar extinction coefficients of Au-NP aqueous solution (~28 M1 cm1 per particle)[97]are much higher than those of most organic dyes. Thus, Au NCs provide promising detection se nsitivities. When bound to metal particles, DNA fragments melt over a much narrower temperatur e range than fragments bound to conventional organic dyes (Figure 1-9C).[3] Based on these properties, Mi rkins group has developed many colorimetric detection methods based on Au NCs, such as detection of DNA mismatch,[3] protein activities,[98,99] special metal ions[100] or other small organic molecules.[101] 1.3.3 Surface Enhanced Raman Scattering (SERS) The surface plasm ons induce a localized electr omagnetic field on the surface of the NCs. The intensity of Raman scattering is proportional to the square of the magnitude of any electromagnetic fields incident on the analyte. When analyte mo lecules are absorbed on metal nanocrystals, the surface provides an extra intens e field and enhances the Raman signal intensity of the analyte by 103-1014, depending on the size and ma terial of the particles.[102] This technology is called surface enhanced Raman sca ttering (SERS), which is a very sensitive diagnostic tool, especially when s ilver-coated particles are used. Figure 1-10 illustrates that SERS of NCs functionalized with Raman-dye-labeled oligonucleotides can be used to detect oligonucleotid e targets. The choice of Raman labels permits multiplexed detection of analytes. The presence of the target is confirmed by silver staining. The SERS method is very selec tive, and the limit of detection (LOD)[10] is several orders of magnitude lower than the LODs of analogous molecular fluorescence based approaches. 1.3.4 Summary of the Present Research This research was untaken to design and improve one-pot syntheses for making highquality metal oxide nanocrystals, to investigat e the methods to synthesize hybride nanocrystals

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33 and to explore surface functionalization of nanocrystals by dual-interaction ligands and nanocrystal-based assay for det ection of enzyme activities. First, syntheses of NCs and their characterization in general will be described in Chapter 2, followed by syntheses of monodisperse colloid al uranium dioxide NC s (Chapter 3). By changing the amount of added surfactant, UO2 NC sizes from 2 to 8 nm can be obtained. Mechanistic studies of the formation of UO2 NCs show that the conden sation product (amide) of oleic acid and oleylamine plays an important role in controlli ng the particle size. Further, octahedral UO2 nanocrystals can be obtained by multiple addition of the precursor solution. Two kinds of heterodimers (FePt/In2O3 and UO2/In2O3) can be prepared using a similar procedure and the synthesized HNCs exhibited different shap es (Chapter 6). High-resolution transmission electron microscopy (HRTEM) studies indicate th at the shapes of these two dimers were controlled by the interfacial structures. The amorphous iorn oxide layers on the FePt NC surfaces act as glue to interconn ect the FePt with the In2O3 parts and led to a core-seed-shaped heterodimer. Using completely crystalline UO2 NCs as seeds results in a peanut-shaped HNC. Second, surface functionalization of NCs by dual interaction liga nds will be described in Chapter 4. Usually, high-quality NCs are synthesized in an organic phase, but most of NC-based bio-applications require water-soluble NCs. To convert the surfaces from hydrophobic to hydrophilic, surface modification of NCs is employe d. For this purpose, dual interaction ligands based on Tween-Derivatives (TDs) were synthesized. Stability tests on these TD-capped NCs show that these dual interaction ligands can signif icantly increase the stability of NCs compared to single interaction ligands. Further, these TD-capped QDs can be boun d with a fluorescent label to monitor virus-prot ein expression in cells.

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34 Third, gold nanocrystal-based assay for the detection of -galactosidase enzyme activity will be described in Chapter 5. The optical properties of Au-NCs are not only dependent on the particle sizes and shapes, but also the distances between the particles. He re, lipoic acid-tyramine-galactopyranosyl (LT-gal) was synthesized, as ligands, to cap Au-NCs; and the resultant LT -gal-capped Au-NCs could disper se in water. After the hydrolysis of the ligands with -galactosidase, these Au-NCs become to aggregate, which exhibit a red-shift in the absorption spectrum of the Au-NC suspension. The detection of -galactosidase was further studies by varying the amounts of -galactosidase. Finally Chapter presents concluding remarks and suggestions for future studies in the synthesis and application of inorganic nanocrystals.

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35 Time Concentration I II III Growth by diffusion or reaction Solubility Critical limiting supersaturation Cs C min C max Ra p id self nucleation Figure 1-1. Structure of a typical nanocrystal: the crystallin e inorganic core, capped by organic ligands Figure 1-2. LaMer curve. In phase I, no nucle ation or growth occurs ; in phase II, rapid nucleation occurs and this is followed by partic le growth in phase II I. (Reprinted with permission from ref 49) Organic ligand Crystalline inorganic core

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36 Figure 1-3. Schematic illustration of the el ectronic structure in A) a metal and B) a semiconductor. The density of states show s quantum size effect in a semiconductor. The HOMO-LUMO separation in the mol ecule becomes the bandgap of the bulk semiconductors. The energy gap of NCs depends strongly on the size of the nanocrystal. (Reprinted with permission from ref 1) Figure 1-4. Idealized density of states for one band of a semiconductor structure of d, d, d, and d materials. In the d case the energy levels are continuous, while in the d or atomic limit the levels are discrete. (Reprinted with permission from ref 1). A B

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37 Figure 1-5. The color of CdSe NCs depends on NC sizes. A) Photo image of CdSe NCs under UV-irradiation; B) absorption spectra (left) and emission spectra (right) of CdSe NCs A Under UV-irradiation B

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38 Figure 1-6. UV/Vis absorption spectra of different size Au NCs: 9 nm, 22 nm, 48 nm and 99 nm Au NCs in water (Reprinted with permission from ref. of 69) Figure 1-7. The emission wavelengths from repres entative QD core materials and representative areas of biological interest (The boxed insert lists materials used for creating magnetic NCs). (Reprinted with permission from ref 92)

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39 Figure 1-8. In vivo cancer targeting and imaging of QDantibody conjugates in living mice. The left mouse is a control (no tumor) and the ri ght one bears a prostate tumor (Reprinted with permission from ref 93) Figure 1-9. The target DNA indu ces aggregation of oligonucleotide-modifed Au NCs. A) the scheme; B) the color change of Au NC solu tions; C) the narrow temperature range of DNA fragments bound to Au NCs, compared to DNA bound to conventional organic dyes. (Reprinted with permission from ref 3). A B C

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40 Figure 1-10. Scheme for the SERS detection of DNA fragments (Repri nted with permission from ref 10) Table 1-1. Electron and hole masses, exciton Bo hr radii and band-gap energies for various semiconductor materials Semiconductor em hm Exciton Bohr Radius () Band-gap Energy (eV) CdS 0.2 0.9 56 2.53 CdSe 0.13 0.8 106 1.74 CdTe 0.11 0.35 150 1.50 GaAs 0.07 0.5 280 1.43 longitudinal 0.98 37 Si Transverse 0.19 0.52 90 1.11 Longitudinal 1.58 50 Ge Transverse 0.08 0.3 200 0.67 PbS 0.1 0.1 400 0.41

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41 CHAPTER 2 GENERAL SYNTHESIS AND CHARAC TERI ZATION OF NANOCRYSTALS Nanocrystals are important building blocks for the development of nanotechnology. The ability to synthesize NCs with controlled size, shape, composition and crystal structure is not only of technological concern, but also of importance for ex ploring novel properties and applications of NCs. In this chapter, the development of NC synthesis is outlined. The instruments, used for characte rization of NCs, mainly incl ude: UV/Vis spectrophotometery, fluorimetery, transmission electron microsc opy (TEM), powder x-ray diffraction (XRD) and dynamic light scattering (DLS). In this chapter, the theory and operation about TEM, XRD and DLS will also be described. 2.1 Synthesis of Nanocrystals A typical nanocrystal syntheti c system contains precursors, capping ligands and solvents, and, som etimes reducing agents (e.g. NaBH4 in gold NP synthesis) or oxidant agents (e.g. trimethylamine N-oxide in In2O3 NC synthesis [103]). The reactions can be performed at room temperature or by heating up 300 oC (or even higher), depending on the precursor reactivity and the boiling temperature of the solvent. The choice of precursors, ligands and solvents varies for different types of NCs. The following section give s a brief review of synthetic methods for three kinds of NCs: semiconductor, metal and metal oxide nanocrystals. 2.1.1 Semiconductor Nanocrystals Spherical semiconductor NCs, also called qu antum dots (QDs), have attracted increasing interesting because of their si ze-dependent optical and electro nic properties and potential bioapplications. The first successful method for c ontrolling the growth of group II-VI and III-V semiconductor NCs was developed by M. L. Steige rwald and L. E. Brus in the mid of 1980s to early 1990s.[104-108] The reaction was carried out by mi xing metaland chalcogen-organic

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42 compounds in organic coordinating solvents and heating to reflux.[107] To obtain highly crystalline NCs with a narrow si ze distribution, M. Bawendi et al. developed the injection method (Figure 2-1) by rapid inj ection of the precur sor solution into pr eheated hot reaction medium: dimethyl cadmium (Cd(CH3)2) (as the cadmium precursor) mixed with trioctytlphosphine (TOP) and trioctytlphosphine oxide (TOPO) (as solvents and also capping ligands.)[22] This method resulted in rapid nucleation and separation of the nucleation and growth stages, as described in chapter 1. Detailed studies of the temporal evolution of NCs, by P. Alivisatos et.al., showed that the monomer concentration plays a key role in controlling the size distribution[109] and also the shapes of nanocrystals.[47] By introducing additional ligands such as hexylphosphonic acid and trioctylphosphine oxide, rod-, arrow-, teardropand tetrapod-sh aped CdSe NCs were obtained.[48] Considering the toxicity and flammability of Cd(CH3)2, cadmium oxide (CdO)[110] and cadmium carboxylates (e.g. cadmium my ristate and stearate)[111] have been successfu lly substituted as Cadmium precursors. Recently, complex nanoheterostructures[112] have been reported to improve the performance of semiconductor NCs. Growth of a shell of a higher band gap material on the core nanocrystal can increase the photoluminescence a nd also the stability of the initial core NCs,[58,59,113], as described in chapter 1. Using CdSe na nocrystals with wurtzite and zinc blende structures as seeds to grow CdS nanorods, CdSe/CdS na norods and nanotetrapods were obtained.[114] Both of these structures showed excelle nt luminescent properties (QY > 80% and > 50% for nanorods and nanotetrapods, respectively) b ecause of efficient energy transfer from the CdS arms into the emitting CdSe core.[114]

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43 2.1.2 Metal Nanocrystals Metal (e.g. Au, Ag, Pt, Cu, Fe and Co) NCs provide promising applications in the areas of optoelectronics, catalysis, data storage and bio-sensors. Among them, gold co lloids (Au NCs) are among the most stable and fascinating meta l NCs, due to their special surface plasma resonance (SPR) properties and applications. Various methods have been investigated to prepare Au NCs. Convenient synthesis methods incl ude the two-phase Brust-Schiffrin method[115] and chemical reduction by trisodium citrate. In the Brust-Schiffrin method, HAu(III)Cl4 is used as a gold precursor. It is dissolved in toluene in the presence of tetraoctylammonium bromide (TOAB), as a phase-transfer reagent, and NaBH4 as the reducing agent. Thiol ligands (e.g. dodecanethiol) are used to stabilize these Au NCs. The size of Au NCs can be adjusted from 1.5 nm to 5.2 nm by changing thiol/gold mole ratios However, this is a two-phase synthesis an d the evaporation of toluene by heating can cause Au NCs aggregation. Also the si ze distribution of Au NCs made by this method is broad. To improve the quality of colloidal Au NCs, single-phase syntheses have been developed. Citrate reduction of HAuCl4 in boiling aqueous solution (als o called Frens's synthesis) is another popular method for preparing relatively large and water-soluble Au NCs (10 nm). Trisodium citrate acts as both a reducing agent and a capping ligand to form a citrate ion layer on the particle surface to st abilize the Au NCs. Gold NCs in the size range from 20 to 40 nm can be obtained by simply varying the solution pH with fixed concentrations of HAuCl4 and citrate.[116] Other precursors, such as gold(III) chloride (AuCl3), silver(I) acetate(Ag(CH3COO)), anhydrous, copper(II) acetate (Cu(CH3COO)2), or platinum(IV) chloride (PtCl4) were used as precursors for synthesis of different metal NCs.[117] Toluene was the solvent with decyldimethylammonium bromide (DDAB) as a surfactant. Tetrabutyl ammonium borohydride

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44 (TBAB) or hydrazine was used as the reducing agen t, and fatty acids or aliphatic amines were added as ligands. Although this strategy can provide nearly monodisperse Au NCs ranging from 1-15 nm in diameter, the challenge remains of obtaining monodisperse NCs on a large scale. Using gold(I) chloro(triphenylphosphine) (AuPPh3Cl)[118] as a gold precursor instead of AuCl3, gram quantities of nearly monodi sperse Au NCs (< 10 nm in diameter) can be synthesized by a single step. Recently, increased emphasis has been placed on the synt hesis of nonspheri cal metal NCs because of their shape-dependent chemical and physical properties. C. J. Murphy developed a seed-mediated growth method to prepare gold na norods with various aspect ratios at room temperature.[52,119,120] Gold NCs in the 3~4-nm range were prepared by addition of a strong reducing agent (NaBH4) into an aqueous solution of HAuCl4 and cetyltrimethylammonium bromide (CTAB). The seed solution was then mixed with the growth solution, composed of HAuCl4, a weak reducing agent (ascorbic acid), surf actant and silver nitrate. The amount of silver nitrate added determined the aspect ratio of the Au nanorods.[52] Y N. Xia[121,122] and P.D. Yang [87,123-125] developed a polyol process to prepare polyhedral Au, Ag, Pt nanocrystals by employing the poly(vinyl pyrrolidone) (PVP) as a surface-capping agent. A metal salt is dissolved in a polyol liquid, whic h acts as both the reducing agent and solvent, and the mixture is heated to near-reflux temperatures. Figure 2-2 shows TEM images of polyhedral metal nanocrystals. Different shapes of nanocrystal s can be obtained by adjusting the metal salt concentration, the amount of PVP, the choice of polyols and the reaction time. Polyhedral metal nanocrystals are almost entirely bound by the fcc {100} and {111} planes. The similarity between these noble metals suggests a general nucleation and growth mechanism, in which

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45 stabilization of the {111} facets relative to the {100} facets can be used to tune final nanocrystal shape.[87] Cobalt, iron and iron platinum nanocrystals ar e also very important because of their special magnetic properties. Sizeand shape-co ntrollable Co NCs have been prepared by P. Alivisatos et.al., using rapid injection of organometallic r eagents (cobalt pentacarbonyl Co(CO)5) into a hot coordinating solvent.[39] C.B. Murray and S. H. Sun have reported a series of studies about the synthesis of Fe and FePt NCs.[21,126-128] 2.1.3 Metal Oxide Nanocrystals Nanocrystals of m any metal oxides, including ZnO, SnO2, In3O4, Fe3O4, FeO, NiO, MnO and CoO, are important building blocks for mate rials because of their special properties, e.g. catalyst capability and magnetic properties. The ability to control the size, shape, composition, crystal structure, and surface properties facilitate their promising application. The metal oxide NCs can be synthesized by both non-aqueous and aqueous processes.[129] Normally, synthesis performed in organic systems provides better control over particle size, size distribution, shape, crystal structure and com position, because of the slower reaction rates compared to aqueous solutions. The choice of su itable precursor compou nds is very important. The precursors employed in preparing high-quality metal oxide NCs are organometallic compounds. e.g. iron (III) acetylacetonate (Fe(acac)3)[130], iron (III) acetate, iron pentacarbonyl (Fe(CO)5)[131] and iron (III) oleate,[56,132] which decompose in organic solvents to yield highly uniform and crystalline iron oxide NCs. The lig ands on the surface of me tal oxide NCs normally are alkyl carboxylic acids or alkyl amines. Sometim es, the ligands also serve as the solvent. The common solvents are alcohols, 1-octadecene (ODE), dioctyl ether (DOE), diphenyl ether (DPE) and toluene. Adjusting the reflux temperature of reaction mixture by varying the solvent allows the synthesis of metal oxide NCs with different sizes,[56] and the reaction temperature also

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46 controls their shapes. Because of the different surface energy of each crystal faces, the final geometry of metal oxide NCs is determined by balancing the relative growth rates among the crystallographic faces.[133] 2.2 Characterization of Nanocrystals The characterization of NCs includes the identification of the crystal morphology, the crysta lline structure and the composition of the part icles, and also the measurement of optical or other properties. This chapter will focus on the mechanisms of transmission electron microscopy (TEM), powder x-ray diffraction (XRD), dynami c light scattering (DLS), which are three important techniques used in this research, 2.2.1 Transmission Electron Microscopy (TEM) Transm ission Electron Microsc opy (TEM) provides a direct measure of the morphology of NCs, including the size, the si ze distribution and the shape of th e particles, as well as their crystal lattice spacing. From the image, the sizes of the particles can be determined by comparison to the scale bar; an d the size distribution is ev aluated from the statistical measurement of hundreds of particles. The resolution of a TEM is de fined by the Rayleigh criterion: 0.61 sin (2-1) where is the wavelength of the radiation, is the refractive index of the viewing medium and is the semi-angle of collection of the magnifyi ng lens. For TEM, the refractive index and semiangle are fixed; thus, the reso lution is mainly dependent on th e wavelength of the radiation source. The electron wavelength is relate d to the electron energy, described as: 1/21.22 ~ E (2-2)

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47 For example, for a 100 keV electron, the wa velength is 0.004 nm. Thus, the higher energy electrons have a smaller wave length and a higher resolution. Figure 2-3 illustrates the basic electron-opti cal system of a TEM. An electron beam passes from the gun through the specimen to the screen. The parts above the specimen (not shown in Figure 2-3) belong to illumination system and the parts below the specimen constitute the imaging system. There are two major modes in the TEM measurement, which are controlled by adjusting the imaging system lens. If the back focus plane of objective lens acts as the object plane for the intermediate lens, then the diffracti on pattern will be obtained on the screen. If the image plane of the objective lens acts as the obje ct plane of the intermed iate lens, an image will be projected onto the screen. The diffraction pattern obtained from the whole area of the specimen, as depicted in Figure 2-3A, is normally buckled, and the direct b eam is so intense that it damages the viewing screen. Therefore, selective area diffraction is performed by inserting an aperture above the specimen to reduce the intensity of the pattern fall ing on the screen and al so to select a special area of the specimen to contribute to the diffracti on pattern. From the diffraction pattern, the dspacing can be calculated by the following equation: R dL (2-3) where R is the distance between the diffracted beam and the direction beam at a certain camera length ( L ), d the lattice spacing, and the wavelength of electron b eam. Figure 2-4 illustrates the relationship between the spacing ( R ) of diffraction spots and camera length ( L ). Since the wavelength of the electron beam is determined by the electron energy, Rd is constant for a given camera length. To calibrate the length, a standa rd sample with known crystal spacing, such as polycrystalline NiO or Au, is measured. And R is obtained from the diffraction pattern. Once R

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48 has been evaluated, the elect ron beam wavelength and known d of the standard can be used to calculate L High Resolution Transmission Electron Microscopy (HRTEM) is an imaging mode, which provides a tool to image crystallographic st ructure of a sample at an atomic scale. The image is formed by the interference of the di ffracted beams with the direct beam (phase contrast). To obtain HRTEM, the point resolution of the microscope should be sufficiently high and also the orientation of the specimen should be along a zone axis. From the image of HRTEM, the d-spacing can be measured from the fringes. Figure 2-5 shows an example of TEM measurement of Star-shaped Fe3O4 NCs. A typical low resolution TEM image is shown in Figure 2-5A. From this image, the shap e and size of the sample (Fe3O4 NCs) can be estimated. Figure 25B is a HRTEM image and the insert is the di ffraction pattern; both s how the crystal morphology of the nanostars. The lattice fringes in HRTE M have an interplanar distance of 0.19 nm, corresponding to the lattice sp acing of {400} planes in Fe3O4 nanopartilces. Since the TEM images are obtained by the direct electron beam, the sample must be made sufficiently thin to allow electron transparency. All the TEM specimens for this research were prepared by evaporating one drop of nanocry stal solution on carbon-coated copper grids. The TEM micrographs were obt ained using Jeol 2000cx and Je ol 2010F transmission electron microscopes operating at 200 kV. 2.2. 2 Powder X-Ray Diffraction (XRD) X-ray diffraction (XRD) provides information a bout the identity of the sample and the sizes of the particles. The diffrac tion peak positions of NCs are the same as the lattice reflections of the bulk materials, but the width of a diffraction peak is dependent on the sizes of the particles. From the size br oadening, the domain size ( D ) of the NCs can be calculated by the Debye-Scherrer equation:

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49 1.2 cos D (2-2) where is the observed diffraction angle, is the full-width at half-maximum of the peak, and is the wavelength of the radiation (CuK =1.54 ). The average particle size is obtained by averaging D for different peaks. This equati on is valid for spherical particles and more complicated equations are required for other particle shapes. 2.2.3 Dynamic Light Scattering (DLS) Both TEM and DLS are employed for the m easurement of nanocrystal size. TEM is based on the scattering and diffraction of electr ons when the electron beam passes through the sample, which must be conductive or semi-conductive material and in the solid state. Thus, only the electron-dense inorganic core can be observe d by TEM, and the organic ligands can not been seen. In dynamic light scattering (DLS), organic ligands also affect the diffusion of the particles, and are included in the measured radius. Thus, by using both TEM and DLS, the hydrodynamic size, nanocrystal shape, thickness of capping liga nds on the particle surf ace, and the aggregation format in the solution can be determined. DLS is based on the fluctuations of scattered light intensity by particles. When the particles are dispersed in a solution at a very low concentration, they move randomly due to the Brownian motion. When light passes through the solution, it is sca ttered by the particles. The intensity of the scattered light varies b ecause the different phases undergo constructive and destructive interference. As the particles move over distan ces equal to the wavelength of the light, the phases of the s cattered waves and the intensity variation (or speckle) are dramatically changed. The fluctuations of in tensity of the scattere d light passing through a small pinhole (smaller than the size of the speck le) are related to the diffusion time, which

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50 depends on the particle size and shape, because small particles move faster than large particles. Particles with a broad size distribution give broadened DLS peaks, but even in perfectly monodisperse particle solutions, e ffects such as interparticle interactions, orientation dynamics of asymmetric particles, and conformational dynamics or deformations of flexible particles lead to a much more co mplicated correlation functions. These effects are usually insignificant for scattering by particles smaller than the wavelength of the light but become important, and are often overw helming, for larger particles.

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51 Figure 2-1. Injection method for synthe sis of monodisperse semiconductor NCs Heating mantle

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52 Figure 2-2. TEM images of polyhedral metal na nocrystals. Cubes, cuboc tahedra, and octahedra have been obtained for silver (A-C, scale bar = 100 nm), gold (D-F, scale bar=1 m), and platinum (G-I, scale bar = 2 nm). (Reprinted with permission from ref 87) E F D H I G B C A

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53 Figure 2-3. The electron-optical system of a TEM. A) in diffraction model, in which the diffraction pattern I is projected onto the vi ewing screen; B) in the image model. An image is projected onto the viewing screen. In each case, the intermediate lens selects either the back focal plane or the image plane of the objective lens as its object. (Reprinted with permission from ref 134) A B

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54 Figure 2-4. The relationship of the spacing ( R ) between diffraction spots and camera length ( L ) (Reprinted with permission from ref 134)

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55 Figure 2-5. The TEM images of star-shaped Fe3O4 NCs. A) The low resolution TEM image; B) high resolution TEM image. The diffr action pattern of star-shaped Fe3O4 NCs is shown in the insert. A B

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56 CHAPTER 3 SYNTHESIS OF URANIUM-DIOXIDE NANOCRYSTALS 3.1 Introduction This chapter describes the one-pot synthe sis of high-quality uranium dioxide (UO2) nanocrystals in an organic phase. Uranium oxide s are important materials for technological applications. Enriched uranium dioxide is the ma jor component of the fuel materials for nuclear reactors.[135] Depleted uranium oxides can be used for radiation shielding,[135] and they are also highly efficient and stable catalysts for the destruction of chlorine-containing organic pollutants at moderate temperatures.[136,137] In addition, uranium dioxide is a material with a high seebeck coefficient,[138] which could be important in thermopower applications. Because nanomaterials can exhibit solution processability, as well as size-depe ndent physical and chemical properties,[139] the ability to synthesize high-quality, co lloidal uranium-oxide NCs would create a new opportunity to facilitate uranium-oxide-based applications. However, there has been little previous research on the synthesis of collo idal uranium-oxide NCs of high quality.[140] Herein, we report a successful organicphase synthesis for producing monodisperse uranium-dioxide NCs. Also, because of the high contrast of UO2 NCs in TEM imaging, it can be used as a model system to study the mechanism of the nanocrystal formation. 3.2 Experimental Section 3.2.1 Chemicals Uranyl(VI) acetylacetonate (UAA, 99%) was purchased from STREM Chemicals. 1octadecene (ODE, 90%), oleic aci d (OA, 90%), oleylamine (OAm, 70%), dioctylamine (98%), N-methyl-N-hexadecyl amine, oleamide (OAP, 99%), hexadecane-1,2-diol (98%), 4dimethylaminopyridine (DMAP, 99%), p-toluenesulfonic acid monohydr ate (TA, 98%), and 1,3-

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57 diisopropyl carbodiimide (DIPC, 99%) were purchased from Aldrich. All the other reagents were purchased from Fisher Scientific International Inc. 4-( N,N -dimethylamino)pyridinium-4-toluenesulf onate (DPTS) was prepared by mixing THF solutions of 4-dimethylam inopyridine (2 M, 50 mL) and p-toluenesulfonic acid monohydrate (2 M, 50 mL) at room temperature with stirring. The resulting precipitate was filtered and dried under vacuum.[141] 3.2.2 Synthesis of Spherical Uranium-oxide Nanocrystals The uranium-oxide NCs were synthesized by thermal decomposition of uranyl acetylacetonate (UAA) in a solu tion of oleic acid (OA), oley lamine (OAm) and octadecene (ODE). In a typical synthesis, UAA (186 mg, 0.4 mmol) was dissolved in a solution of OA (1.0 g) and ODE (1.0 g) at 150 oC. After the solution was cooled to room temperature, OAm (1.0 g) was added, and then the resulting mixture was degassed under vacuum (~20 mtorr) at 100 C for 10 min. Under Ar flow, the reaction solution was he ated to 295 C over about 8 min, aged at the same temperature for 5 min, and subsequently cooled to room temperature. The NCs were precipitated from the reaction solution by a dding acetone and further purified by adding a mixture of hexane and acetone (1:4). The black nanocrystal precipitate was easily redispersed in non-polar organic solvents such as hexane or to luene. The typical reaction yield was about 78%. The reaction yield (Y ) of UO2 nanocrystal synthesis was determined by / YPR where P is the mole number of UO2 in the total nanocrystal products and R is the mole number of UAA. The moles of UO2 in the total nanocrystal products ( P ) were calculated using formula WQ P M W where W is the total weight of nanocrystal product, Q is the weight percentage of UO2 in the NCs, and MW is the formula weight of UO2. Q was determined by thermogravimetric analysis (TGA) by measuring the weight change due to loss of organic ligands on the nanocrystal surface.

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58 To monitor this reaction, aliquots were ta ken from the reaction mixture at different temperatures during heating from room temperat ure to the refluxing temp erature. These aliquots were diluted with toluene and then directly dropped onto TEM grids without any purification. 3.2.3 Synthesis of Octahedral UO2 NCs by a Multiple-injection Method Seed uranium dioxide NCs (5.4 nm) were s ynthesized as above, purified once by addition of acetone and dried under vacuum. Seed UO2 NCs (17 mg) was mixed with OA (0.75 g), OAm (0.75 g), and ODE (5 g), and heated to 300 oC. The precursor solution (growth solution) was premade by dissolving UAA (186 mg, 0.4 mmol) in a mixture of OA (0.92 g) and ODE (8.0g) at 150 oC, and the solution was stored at room temperature. At 300 oC, 1 mL of the precursor solution was injected into the seed solution ever y 10 min. Prior to each injection, a 0.2 mL of aliquot was removed from the reaction solutio n for monitoring the growth. The NCs were precipitated from the aliquot by adding acetone, and the black NCs were re-dispersed in hexane, and then directly dropped to TEM grids. 3.2.4 The Effect of Additional Mixture of OA and OAm with a Molar Ratio of 1:1 To explore the effect of additional OA and OAm, a series of syntheses were carried out, in which various amounts of a 1:1 mole ra tio OA/OAm mixture were added to the UO2 synthesis solution (UAA, 0.4 mmol; OA, 1.0g; and OAm, 1.0g). These synthe tic experiments were carried out according to the procedure described in section 3.2.2. 3.2.5 The Effect of Water To examine the effect of water, a synthesi s was carried out according to the protocol described in section 3.2.2. In addition, water (1.0 mL) was slowly (over a 10 min period) added to the reaction solu tion after the temperature reached 200 oC during the heating stage. After the water was added, the reacti on temperature was about 260 oC, and subsequently it was continuously heated to 295 oC.

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593.2.6 The Amide Effect N O N O NH2 O H N O Tertiary(3o)amide Primary(1o)amide:OAP Secondary(2o)amide:OOA I:OAT1II:OAT2 Scheme 3-1. The molecular structures of three types of amides. 3.2.6.1 Syntheses of Amides Synthesis of N-( cis -9-octadecenyl)-oleamide (OOA). Two synthetic routes were used for synthesis of OOA. Route 1: OA (3.1 g, 0. 011 mol), OAm (2.7 g, 0.010 mol), and DPTS (3.7 g, 0.013 mol) were added to a flask with CH2Cl2 (25 mL) at room temperature under stirring. After 10 min, 1,3-diisopropyl cabodiimide (1.9 g, 0.015 mol) was added, and the reaction solution was further stirred for 5 hours. Then the reaction solution was diluted with CH2Cl2 (30 mL) and extracted with water (50 mL) three tim es to remove DPTS. The crude product was purified by flash chromatography. Yield: 70%. R oute 2: OA (2.8 g, 0.01mol) and OAm (2.7g, 0.01mol) were heated to 300 oC and kept at that te mperature for 1 hr. Yield: 100 %. These two

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60 methods both led to the same product (OOA). 1H-NMR (300-MHz, CDCl3): (ppm) 0.89 (t, J=6.6 Hz, 6H), 1.27 (m, 42H), 1.49 (m, J=6.6Hz, 2H), 1.63 (m, 2H), 2.01(m, 8H), 2.15 (t, J=7.5Hz, 2H), 3.24 (m, 2H), 5.35 (m, 4H). Synthesis of other amides. OA was mixed with other amines (dioctylamine, N -methylN -hexadecyl amine in a 1:1 molar ratio) and the mixture was heated to 300oC and kept at that temperature for 1 hr. The products were named N,N -dioctyloleamide (OAT1, Scheme 3-1 ) and N -hexadecyl-N-methyloleamide (OAT2 Scheme 3-1). 1H-NMR of OAT1 (300-MHz, CDCl3): (ppm) 0.89 (9H), 1.27 (m, 42H), 1.52 (m, 2H), 1.63 (m, 4H), 2.01(m, 4H), 2.24 (m, 2H), 2. 76 (t, 2H), 3.24 (m, 2H), 5.35 (m, 2H). 1H-NMR of OAT2 (300-MHz, CDCl3): (ppm) 0.87 (7H), 1.27 (m, 46H), 1.51 (m, 2H), 1.62 (m, 2H), 2.01(m, 4H), 2.28 (m, 2H), 2. 93 (d, 3H), 3.30 (m, 2H), 5.35 (m, 2H). The OOA effect. To explore the effect of adding OOA to the reaction mixture, we carried out a series of synthe ses in which various amounts of OOA were added to the system described in section 3.2.2. Other amide effects. To explore the effects of di fferent amides, syntheses were performed as in section 3.2.2, but 4g of one of the amides, shown in Scheme 3-1 (OAT1, OAT2 or OAP) was added to the reaction system. 3.2.7 Characterization of UO2 NCs 3.2.7.1 XRD Measurements of UO2 NCs Powder X-ray diffraction patterns were measured on a Philips PW 3720 X-ray diffractometer with Cu-K radiation. Approximately 20 mg of NCs was dispersed in 0.5 ml of toluene. Then the solution was deposited onto a low-scattering quartz plate, and the toluene was evaporated under air overnight. The Bragg diffractions of the ur anium-oxide NCs can be indexed

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61 to nearly all of those of the st andard bulk face-centered cubic UO2, which are quite distinguishable from the Br agg diffractions of bulk UO3 or U3O8 structures (Appendix A). 3.2.7.2 IR Measurements IR spectra were obtained using a Perkin-Elmer 1600 FT-IR spectrometer. The specimens were prepared by directly loading aliquots (0 .1 mL) of a hot reaction mixture onto a NaCl window. 3.2.7.3 1H-NMR Measurements 1H-NMR spectra were recorded using a Va rian Mercury NMR Spectrometer (300 MHz). The samples were prepared by adding aliquots (~0.02 mL) of reaction mi xtures to deuterated chloroform (CDCl3, ~0.8 mL). 1H-NMR measurements were used to examin e the condensation reaction of OA and OAm in a modified UO2 synthesis (section 3.2.2) The aliquots we re taken from the reaction system and mixed with CDCl3. The protons close to th e amide functional group (CH2-NH-CO-) were used as probes to monitor the condensation reaction. 3.2.7.4 TEM Measurements TEM measurements were performed on a JE OL 200CX operated at 200 kV. To prepare the specimens, a particle solution (10 L) was dropped onto a 200-mesh copper grid and dried overnight at ambient conditions. 3.3 Results and Discussion 3.3.1 Synthesis and Characterization of UO2 NCs Spherical UO2 NCs were characterized by TEM and XRD. The TEM image (Figure 3-1A) shows that the uranium-oxide NCs are nearly monodisperse, spherical part icles with a diameter of 5.4 nm and RSD of 3 %. The XRD pattern shown in Figure 3-1B in dicates that the NCs consist of uranium dioxide. The XRD pattern of the nanocrystal sample exhibits the highly

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62 crystalline peaks that can be indexed to nearly all the Bragg reflections corresponding to the standard and phase pure face-centered c ubic (fcc, rock salt) structure of UO2 ( Fm3m a = 0.5468 nm). These Bragg reflections are qui te distinguishable from those of the typical crystal structures of other uranium oxides such as U3O8 and UO3 (Appendix A). Moreover, this structural assignment is consistent with the high-resolu tion TEM (HRTEM) image (Figure 3-1, inset), which shows the characteristic cr oss-fringe pattern of the fcc-cr ystal structure viewed along the <011> zone axis. The ordered distance of 0. 32 nm shown in the high-resolution image, corresponding to the lattice spaci ng of the {111} faces in the fcc UO2, is in good agreement with the result from the XRD measurement. 3.3.2 Size Control of UO2 Nanocrystals In the synthesis of UO2 NCs, two kinds of ligands were employed: oleic acid (OA) and oleylamine (OAm). To understand the functions of OA and OAm in the formation process, we systematically investigated how the molar rati o between OA and OAm affects the size of final NCs (Figure 3-2AC and E). A series of synthesis experiments was car ried out with nine different OA/OAm mole ratios, but with the same total solv ent amount (OA + OAm = 4.0 g), ODE (1.0 g), and with constant amount of UAA (0.4 mmol). With increasing OA/OAm mole ratio, the size of the final NCs increased, a nd a maximum size was obtained when the ratio was 1:1. Then the size of final NCs decreased with a further increase of the molar ratio, shown in Figure 3-2E. However, the reacti on yield was not significantly ch anged with the increase of the OA/OAm ratio. Taken together, these results indi cate that a minimum number of stable nuclei were formed at the OA/OAm mole ratio of 1:1, and the increase of either OA or OAm led to an increase in the number of nuclei, and a decrease in final nanocrystal size.

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63 Moreover, the size of NCs from the synthesis with OA/OAm ratio of 1:1 was larger than that of the final particles from the original sy nthesis (6.2 nm vs. 5.4 nm). The only difference between these two syntheses is the total amount of OA plus OAm (4.0 g vs. 2.0 g). This result may indicate that increasing the am ount of 1:1 OA: OAm mixture can lead to larger final NCs. To examine the generality of this conc lusion, we carried out five further UO2 NC syntheses with various total amounts of the 1:1 OA/OAm mixture. Indeed, the si ze of the final NCs increased with the increasing total OA/OAm amount, as shown in Figure 3-2D. The largest diameter was 8 nm, corresponding to OA+OAm =18 g, as shown in Figure 3-2F. Further increasing the amount of the 1:1 OA/OAm mixture resulted in the decrease of particle size. Figure 3-2F also shows that increasing the amount of ODE solvent (OA+OAm constant at 2.0 g) decreases the NC size. 3.3.3 Shape Control of UO2 Nanocrystals 3.3.3.1 Multiple-injection Multiple-injection of the precursor solution (called growth solution) into the hot seed UO2 NCs mixture was employed to synthesize octahedral UO2 NCs. Figure 3-3A describes the process of multiple injection, and Figure 3-3B-D are TEM images of spherical seed UO2 NCs, multishaped UO2 NCs, and octahedral UO2 NCs, respectively. After multiple-injection of the growth solution, UO2 NCs gradually changed from 5.4 nm spherical NCs to 9.1 nm octahedra. Since the volume of growth solution was the same for each injection, it was expected that the total particle volume would increase linearly with injection number. Figure 3-4 is a plot of volume (relative to the volume of 5.4 nm seed particles) versus the injection number. For the first three injections, the relative volume of the partic les increased linearly. Ho wever, after the fourth injection, the particle volume leveled off and the remaining injections resulted in only slow growth of particles. This can be explai ned through the formation of additional UO2 nuclei after the fifth injection. According to the LaMer model (see Figure 1-2), nucleation occurs only when

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64 the active monomer concentration exceeds a critical limit (super-saturation). In the presence of seed NCs, the injected precursor solution resulted in growth on the seed NCs for the first several injections. However, with the growth of NCs, the reaction mixture gradually changed from a clear solution to a cloudy mixture, due to the decreased solubility of UO2 NCs with increasing particle size. Thus, the growth rate of NCs decreased, and the precursor concentration began to accumulate. Eventually, the cri tical concentration was exceeded and more nuclei formed. Also, a high concentration of active monomer is key to formation of octahedral UO2 NCs. Non-spherical particles usually have a higher surface energy than spherical ones.[123] Therefore, making non-spherical particles requires growth co nditions with a high chemical potential, which can be achieved by increasing the per-pa rticle concentration of precursors.[51] 3.3.3.2 Ligand Effect The functional groups of the ligands play a significant role in the NC synthesis. For example, when hexadecane-1,2-diol (HDD) was used instead of OAm, branched NCs of UO2 were obtained. Figure 3-5 shows the TEM image of the branched NCs of UO2 and the insert HRTEM image shows that these particles are highly crystalline with a fringe distance of 0.32 nm, corresponding to the lattice spaci ng of the {111} planes in fcc UO2. 3.3.3.3 Self-assembly of UO2 NCs As described above, high -quality monodisperse UO2 NCs can be obtained by organicphase syntheses, which are easily adjusted for gr am-scale synthesis. Since the size distribution of UO2 NCs is very narrow, these NCs easily fo rm self-assembled ordered monolayers (2dimensional assembly) and multilayer 3-dimensional assemblies. Figure 3-6A shows the doublelayer self-assembled pattern formed by monodisperse 5.4 nm spherical UO2 NCs; Figure 3-6B shows monolayer self-assemble pattern formed by two sizes of spherical UO2 NCs. The NC systems with hexagonal arrangements were created by simple self-assembly on hydrophobic

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65 surfaces, formed by deposition of a hexane dispersion of the UO2 NCs on an amorphous carboncoated copper grid under toluene vapor, followe d by evaporation of the solvent. By controlling the concentration of UO2 NC solution, 2-dimensional and 3-dimensional assemblies can be obtained. 3.3.3 Mechanistic Study of UO2 NC Formation The formation of uranium dioxide from a ur anyl precursor indicates that reduction of U(VI) to U(IV) is included in the nanocrystal synthesis. In this system, oleylamine may act as the reducing agent, with the reaction yield of a typical nanocrystal synthesis is about 78%. In addition, the synthesis is hi ghly reproducible. Among replicat e experiments, the typical deviations of experimental data (in terms of final nanocrystal si ze and size distribution) are less than 3%. Such a highly reproduc ible synthesis allows a detailed mechanistic analysis of nanocrystal formation by comparing experiment al data obtained using different reaction conditions. In the growth process, oleic acid and ol eylamine ratios and total amounts significantly affect the particle size, as shown Figure 3-2 previously. The size of UO2 NCs obtained from 1:1 OA: OAm mixtures are larger than those obtained from other rati os. Increasing the total solvent amount (OA + OAm mixture) at th e 1:1 ratio can lead to furthe r size increase, as shown in Figure 3-2F, with maximum size of 8 nm, obtained when the total amount was 18 g. Thus, a fundamental question is raised as to how the amount of the OA/OAm mixt ure affects the final particle size. IR analyses (Figure 3-8A) show that UO2-nanocrystal formation is accompanied by the formation of N -(cis -9-octadecenyl)-oleamide (O OA), due to the condens ation reaction of OA and OAm. This result suggests that the OA +OAm condensation r eaction may affect UO2 nanocrystal synthesis. 1H-NMR analyses (Figure 3-8B) further in dicate that the condensation reaction was

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66 nearly complete before the nucleation of UO2 NCs. Therefore, the pr oducts of the condensation reaction may play significant roles in controlling the formation of UO2 NCs. The two major products of the condensation reaction are water and OOA. To test the effect of added water, we carried out UO2 synthesis while adding water during the heating stage. Figure 3-9A shows the TEM image of UO2 NCs with addition of wate r, and Figure 3-9B is a typical TEM image of 5.4 nm UO2 NCs without the addition of water. It is obvious that water does not significantly affect the size of the fina l products, thus indicatin g that OOA is important in the formation of UO2 NCs. To examine the effect of OOA in more deta il, we first synthesi zed OOA according to a literature method. Then, six UO2-synthesis experiments were car ried out based on the original synthesis (UAA, 0.4 mmol; OA, 1.0g; OAm, 1.0g and ODE, 1.0 g) but with various amounts of OOA added. The results from these experiments s how that the sizes of final particles increase with the amount of additional OOA (Figure 3-2F), and that the NCs reach their maximum size of 7.8 nm when the additional OOA is 18 g. Signifi cantly, the effect of OOA almost perfectly matches that of the 1:1 OA/OAm mixture (up-tria ngles in Figure 3-2F). These results provide unambiguous evidence that it is OOAnot the mixture of OA and OAmthat plays the major role in controlling the formation of UO2 NCs. Furthermore, to determine whether the amide functional group or the hydrocarbon ch ain on OOA generates the effect on UO2-nanocrystal formation, we carried out five syntheses based on the conditions used in the original synthesis but with different amounts of octadecene (ODE). The results show that additional ODE leads to the opposite effect on UO2 nanocrystal formation: the size of the final products decreases as the amount of ODE increases (Figure 3-2F). Therefore, it is likely that the amide functional group on OOA generates the major effect on the formation of UO2 NCs.

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67 Interestingly, IR analysis of purified UO2 NCs shows conclusive evidence that OOA is not on the nanocrystal surface, but that the NCs are passivated by OA through a chelating bidentate interaction (Figure 3-10) In the IR spectrum of the UO2 NCs, the peak at 1531 cm-1 comes from the asymmetric vibration band of C=O, and the peak at 1456 cm-1is the symmetric vibration band of C=O. The wavenumber separa tion between the asymmetric and symmetric bands is 75 cm-1. Such a small wavenumber separation i ndicates that oleate anions are bonded to the UO2 nanocrystals through chelat ing bidentate interaction.[143,144] Moreover, the IR spectrum of UO2 nanocrystals is quite disti nguishable from the IR spectrum of OOA. This result clearly shows that OOA is not on the surface of UO2 nanocrystals. These results indicate that the effect of OOA on the nanocrystal synthesis is likely achieved via tuning the reactivity of the interm ediate states (or active monomers) in the formation of UO2 particles. The product yields for these reactions are similar and independent of the final particle size. Thus, the size of NCs is controlled by the number of nuclei formed, with particle size decreasing as the num ber of nuclei increases. This i ndicates that the amide affects the growth of NCs by blocking nucleation via an interaction between th e amide and the surface of NCs. To check the effect of the amide functional group on the activity of the intermediate states in the formation of UO2 particles, four types of amid es were studied. The molecular structures of these amides are shown in Sche me 3-1: oleamide (OAP) is a primary amide; N -(cis 9-octadecenyl)-oleamide (OOA) is secondary amide, N,N -dioctyloleamide (OAT1) and N hexadecylN -methyloleamide (OAT2) are both tertiary amides, differing in the lengths of the carbon chains on the amide nitrogen. In OAT1 there are two C-8 chains; and in OAT2, one carbon chain is C-1 and the other is C-15. Figure 3-11 shows the TEM images of UO2 NCs,

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68 obtained by the addition of th e different amides. The UO2 NCs generated from OOA system have 6.5 nm diameters with RS D around 5%. The diameters of UO2 NCs from the OAT1 and OAT2 systems are 5.7 nm with RSD around 5% a nd 5.1 nm with RSD around 8%, respectively. The OAP system produced UO2 NCs with a very broad size range of 1-4.5 nm. Since the reaction mixtures are the same, and the only difference is the type of additional amide, the particle size must be affected by the amide. The intermediate states of the formation of UO2 NC are free UO2 units and very small UO2 clusters, both having an interaction with amides. As the concentration of amide group in th e reaction mixture increases, the amide-UO2 interaction becomes more favorable, resulting in fewer nuclei being formed. Thus, increasing the amount of OOA results in large UO2 NCs. The strength of the interaction between amide functional groups with the intermediate states al so affects the nucleation. Scheme 3-2 illustrates the hydrogen-bonding between amides and UO2. The tertiary amides: OAT1 and OAT2 have weaker interaction with UO2 than the secondary amide OOA. Therefore, more nuclei are generated in the OAT1 and OAT2 systems and smaller UO2 NCs are obtained. The stereohindrance effect in OAT2 is weaker than that in OAT1. Thus the interaction of OAT1 with the intermediate stage is a lit tle stronger than that of OAT2, and fewer nuclei are formed in OAT1, compared to OAT2 system. Scheme 3-2. Schematic illustration of the interaction between amides and UO2 molecules strong weak too strong 2o amide 3o amide 1o amide

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69 The interaction between the primary amide OAP and the intermediate states is so strong that the growth of nuclei is blocked. Thus sm aller NCs with a very broad size distribution are obtained. In this case, the pr oduct yield of the reaction was only 46% compared to 78% in a typical synthesis. In conclusion, we have developed an or ganic-phase synthesis for making high-quality, colloidal UO2 NCs. By multi-addition of the uranium precursor solution, octahedral UO2 NCs were obtained. Second, we have mapped out the functions of the solvents (OA, OAm, and ODE) in the synthesis, and have found that OOA a product of the condensation of OA and OAm can substantially affect the formation of UO2 NCs. Importantly, these results provide fundamental insight into the mechanism of UO2 nanocrystal synthesis. In addition, because a mixture of OA and OAm has been widely used in preparation a variety of high quality metal or metal-oxide NCs,[21,145] the results herein should also be important for understanding the detailed mechanisms of those syntheses.

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70 Figure 3-1. Char acterization of UO2 NCs. A) A TEM image of UO2 NCs made in the typical synthesis; B) XRD pattern of the UO2 nanocrystals. The standard diffraction peak positions and relative intensities of bulk cubic UO2 are indicated. The inset shows a HRTEM image (6.5 nm 6.5 nm) of the nanocrystal sample. (Reprinted with permission from ref 142) B A

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71 Figure 3-2. TEM images of UO2 NCs A), B) and C) from synthe sis with OA/OAm ratio of 1:3, 1:1, and 3:1, respectively. D) A TEM im age of the NCs from a synthesis with UAA (0.4 mmol), ODE (1.0 g), OA (10 g) and OAm (10 g). The scale bar is 10 nm.[142] Plots of nanocrystal diameter E) as a f unction of OA/OAm molar ratio from various syntheses; F) as a function of the amount of additional solvents a dded to the original reaction mixture: up-triangles for the mixture of OA and OAm at a molar ratio of 1:1, down-triangles for OOA, and black squares for ODE. C B A F E D

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72 Figure 3-3. Multiple-injection me thod for preparing octahedral UO2 nanocrystals. A) The scheme; B) spherical seed UO2 NCs (diameter, D = 5.4 nm); C) multishaped UO2 NCs after the 1mL injection of the growth solution four times (edge length of octahedra, a = 8.3 nm); D) octahedral UO2 NCs after the 1mL injection of the growth solution seven times ( a = 9.1 nm). Figure 3-4. The particle growth with multiple-injection of the precursor solutions. Dashed line represents linear growth rate, a nd spheres are experimental data. D C A B

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73 Figure 3-5. A typical TEM image of branched UO2 NCs. The inset is the HRTEM image Figure 3-6. TEM images of self-assembled UO2 nanocrystals (A) monodisperse UO2 NCs 5.4 nm in diameter; (B) two sizes of UO2 NCs, 6.0 nm and 2.0 nm. B A

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74 Figure 3-7. TEM images of asse mblies of 9.1 nm octahedral UO2 nanocrystals. (A) monolayer assembly; (B) and (C) 3d self-assembled pa tterns with a projection direction of <110> and <111>, respectively; (D) a large area assembly with projection direction <111>. B A C D

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75 C Figure 3-8. IR and 1H-NMR spectra of reaction mixture. A) IR spectra of a reaction mixture (in violet) and a supernatant solution (i n orange) from a typical 5.4-nm-UO2-nanocrystal synthesis after the reacti on solution was aged at 295 oC for 5 min; OOA (in green); OA (in red); and OAm (in blue). B) 1H-NMR spectra (2.5-3.5 ppm region) of a reaction mixture (i) at 25 oC, (ii) at 200 oC for 20 min, and (iii) at 295 oC for 5 min. C) the equation of the condensation reaction of OA and OAm. Protons on the carbon close to the nitrogen, are starred on th e OAm and OOA structures and corresponding NMR spectra. (Reprinted with permission from ref 142). Su p ernatant B A

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76 Figure 3-9. TEM measurement of the water effect A) The particles made in the synthesis with water added; and B) in a typical 5.4-nm-UO2 synthesis. (Reprinted with permission from ref 142). Figure 3-10. IR spectra of purified UO2 NCs (top) and OOA (bottom). (Reprinted with permission from ref 142) A B

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77 Figure 3-11. TEM measurement of the amide effect. TEM images of UO2 NCs, synthesized by addition of A) OOA, B) OAT1, C) OAT2 and D) OAP. A B C D

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78 CHAPTER 4 WATER-SOLUBLE NANOCRYSTALS TH ROUGH DUAL-INTERACTION LIGANDS 4.1 Introduction Because of their unique size-dependent opt ical, electronic, magnetic, and chemical properties, inorganic NCs are beco ming powerful tools in biological and medical ap plications for sensing, labeling, optical imaging, magnetic resonance imaging (MRI), cell separation, and treatment of disease.[146-148] These applications, however, require NCs that are soluble and stable in aqueous solutions. This creates a need for furt her engineering of nanocrystal coatings, because high-quality NCs are often synthesized in or ganic phases and stabilized with hydrophobic ligands.[46] To date, two major methods have been deve loped to modify the coatings of hydrophobic NCs using organic ligands. The first approach is based on coordinate bon ding. Functional groups (such as thiol,[7] dithiol,[95] phosphine[20] and dopamine[149]) are used to link hydrophilic groups directly onto the surface of hydrophobic NCs by re placing the original hydrophobic ligands. The second approach uses hydrophobic van der Waal s interactions, through which the hydrophobic tails of amphiphilic ligands interact with ( but do not replace) the hydrophobic ligands on the NCs,[150] leading to the formation of nanocrystal-micelles. Many types of water-soluble NCs made by these two approaches suffer low stab ility and/or high non-sp ecific binding with nontarget biomolecules. Water-soluble NCs coated with PEGylated amphiphilic polymers have very high stability and low nonspecific-adsorption levels,[95,150] but the PEGylated polymer shells often produce large hydrodynamic diameters (HDs), on the order of 30-40 nm, which can limit the use of these NCs in applications such as in vivo cell imaging. Herein, we describe an alternative nanocrystal-sur face-engineering approach that uses a new class of ligands (here called dual-interaction ligands) to pr oduce water-soluble NCs. The dualinteraction ligands are based

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79 on Tween-derivatives (TDs), as shown in Figure 4-1. These dual-interact ion ligands can bind to the hydrophobic NCs through both c oordinate bonding to the inorganic crystal and hydrophobic van der Waals interactions with the non-pol ar tails of the orga nic surface ligands. The dual-interaction ligands are synthesized through simple modifications of Tweens, polyethylene glycol (PEG) sorbit an fatty-acid esters, which all have 20 ethylene-glycol units distributed among their four branches (Figure 42). Depending on the length of their fatty-acid tail, these sorbitan fatty-acid esters are commercially named Tween 20, Tween 40, Tween 60 and Tween 80, as shown in Figure 4-2. Because of their low toxicity, these Tween compounds are often used as food additives. More importantly, Tween compounds are wide ly used as protein stabilizing and blocking agents to minimize non-specific binding in immunoassays such as western blotting and ELISA (enzyme-linked i mmunosorbent assay). These properties make Tween compounds unique for coating water-soluble NCs for use in biomedical applications. However, the Tween compounds cannot be used di rectly to stabilize hydrophobic NCs in water because the hydrophobic van der Waals interactions between the Tween fatty-acid tails (R group in Figure 4-2) and the hydrophobic surface ligands are relatively weak. To overcome this difficulty, we have introduced a coordinating functional group via one of the OH-groups. The resulting Tween derivatives (TDs) were expected to have affinity to the surface of hydrophobic NCs through both coordinate bonding and hydrophobic van der Waals interactions. Two types of coordinating groups were us ed to functionalize Tween compounds for surface engineering of different types of NCs, as shown in Figure 4-1B. For noble-metal particles and semiconductor QDs, a dithiol coordinating group was introduced into Tween compounds via two steps (Figure 4-2A): (1) attachment of a lipoi c-acid group via a mild esterification reaction; and (2) reduction of the product with sodium borohydride to conve rt the lipoic-acid group into a

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80 dihydrolipoic-acid moiety. Th e final products are named TDN-L. For NCs of transition-metal oxides such as Fe3O4, a dopamine group was used to modify Tween compounds via a succinicacid cross-linker in a two-step synthesis (Figure 4-2B): (1) incorporati on of a succinic-acid group by an esterification reaction with succinic anhydride, and (2) c oupling of a dopamine group with the succinic-acid cross-linker through an EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride)-mediated amide-formation reaction. The reaction sequence yields final products named TDN-D. Moreover, an additional carboxyl group can be attached onto these TDN-L compounds, for example, succinic-acid-functionalized TD20-L (TD20-LC, shown in Figure 4-2C). The additional carboxyl group allo ws protein attachment through a mild EDC-mediated coupling reaction to prepare NC-antibody conjugates. The typi cal yields of these reactions are higher than 80%. The structures of the resulting TD ligands were confirmed using 1H-NMR, but the exact positions of the attached functional groups in the TD ligands are not identified. In this study, water-soluble gold and Fe3O4 NCs and CdSe/ZnS quantum dots (QDs) were prepared using dual-interaction ligands with re latively small hydrodynamic diameters (HDs), i.e., less than 20 nm. These NCs exhibit extraordinary stab ility over in a wide range of pH (e.g. 1-14), salt concentration, and thermal treatment (at 100 oC). In addition, these NCs can be further functionalized with antibodies for monito ring virus-protein e xpression in cells. 4.2 Experimental Section 4.2.1 Chemicals Butylamine (99%), dimethylaminopyridine (DMAP, 99%), dopamine hydrochloride, hexamethyl disilathiane ((TMS)2S), lipoic acid ( 99%), 1-methyl-2-pyrrolidinone (NPA, 99%), N,N -diisopropyl carbodiimide (DIPC, 99% ), iron (III) chloride (FeCl3H2O, 98%), 1octadecene (ODE, 90%), octadecylamine (ODA, 97%), oleic acid (OA, 90%), p-toluenesulfonic acid monohydrate (98%), rhodamine 6G (99%), tributylphosphine (TBP, 97%),

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81 trioctylphosphine oxide (TOPO, 99%), 1,3-diisopropyl cabodiimid e (DIPC, 99%), polyethylene glycol sorbitan monolaurate (T ween-20), polyethylene glycol sorbitan monopalmitate (Tween40), polyethylene glycol sorbitan monostearate (Tween-60) and polyethylene glycol sorbitan monooleate (Tween-80), 1-ethyl3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), N -hydroxysuccinimide (NHS) were purchased fro m Aldrich. Cadmium oxide (CdO, 99.998%), selenium (Se, 99.99%), dodecyl trimethylammoni um bromide (DTAB, 97%) were purchased from Alfa Aesar. Sodium oleate (95%) was purchased from TCI. Nanopure water (18.2 M cm) was prepared by a Barnstead Nanopure Diamond syst em. All the other reagents were purchased from Fisher Scientific International Inc. 4-( N,N -dimethylamino)pyridinium-4-toluenesulf onate (DPTS) was prepared by mixing THF solutions of DMAP (2 M, 50 mL) and p-toluenesulfonic acid monoh ydrate (2 M, 50 mL) at room temperature with stirring. The resulting precipitate was filtered and dried under vacuum. 4.2.2 Synthesis of Tween-derivatives (TDs ) 4.2.2.1 Dihydrolipoic Acid-functionalized Tweens (TDN-L) The synthetic route to dihydrolipoi c acid-functionalized Tweens (TDN-L) is shown in scheme 4-1. Scheme 4-1. Synthetic route to dihy drolipoic acid-functionalized Tweens (TDN-L). Lipoic acid

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82TD20-a: [5-(1,2-Dithiolan-3-yl)-1-oxopenty l]polyethylene glycol sorbitan monolaurate. Tween-20 (4.91 g, 4.0 mmol), lipoic acid (0.83 g, 4.0 mmol) and DPTS (1.37 g, 4.4 mmol) were mixed in CH2Cl2 (20 mL) and stirred for several minutes at room temperature. Then, DIPC (0.63 mL, 4.4 mmol) was added to the mixture. After being stirred at room temperature overnight, the reaction mixture was washed with water (30 mL) four times. The organic phase was dried over magnesium sulfate (MgSO4), filtered and concentrated. The crude product was purified by column chromatography on silica gel (eluents: et hyl acetate/hexane 9:1 and chloroform/methanol 9:10). Yield: 88%. 1H-NMR (300 MHz, CDCl3, Figure 4-3): (ppm) a 0.88 (t, 3H), b 1.25 (m, 16H), c 1.47 (m, 2H), d 1.63 (m, 6H), e 1.90 (m, 1H), f 2.33 (m, 4H), g 2.45 (m,1H), h 3.13 (m, 3H), i 3.63 (m, 82H), j 4.21 (m, 4H), k 4.56 (m, 2H ). TD20-L: (6,8-Dimercapto-1-oxoocty)polyethyl ene glycol sorbitan monolaurate. TD20-a (4.96 g, 3.5 mmol) was dissolved in a mixt ure of EtOH/water (50 mL, 1:4). Then NaBH4 (0.23 g, 6.0 mmol) was slowly added. The reaction mixture was stirred for 2 h until the solution became colorless. Then, the solution was diluted with water (50 mL) a nd extracted with CHCl3 (50 mL) five times. The combined organic phase was dried over MgSO4 and filtered. The solvent was removed under reduced pressure to gi ve a white oily product. Yield: 82%. 1H-NMR (300 MHz, CDCl3, Figure 4-4): a 0.88 (t, 3H), b 1.24 (m, 16H), c 1.47 (m, 2H), d 1.63 (m, 6H), e 1.90 (m, 1H), f 2.33 (m, 4H), g 2.92 (m,1H), h 2.70 (m, 3H), i 3.63 (m, 82H), j 4.21 (m, 4H), k 4.56 (m, 2H). TD40-L: (6,8-Dimercapto-1-oxoocty)polyethyl ene glycol sorbitan monopalmitate was synthesized using conditions similar to those for the synthesis of TD20-L. 1H-NMR (300 MHz, CDCl3,): (ppm) 0.88 (t, 3H), 1.24 (m, 24H), 1.47 (m, 2H), 1.63 (m, 6H), 1.90 (m, 1H), 2.33 (m, 4H), 2.92 (m,1H), 2.70 (m, 3H), 3.63 (m, 82H), 4.21 (m, 4H), 4.56 (m, 2H). 13C-NMR (100 MHz,

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83 CDCl3): (ppm) 174.06, 173.70, 72.78, 70.79, 70.56, 69.43, 63.67, 63.56, 61.95, 42.98, 39.52, 38.95, 34.43, 34.21, 32.13, 29.90, 29.87, 29.69, 29.57, 29.50, 29.36, 26.72, 25.13, 24.73, 22.90, 22.50, 14.34. TD60-L: (6,8-Dimercapto-1-oxoocty)polyethyl ene glycol sorbitan monostearate was synthesized using conditions similar to those for the synthesis of TD20-L. 1H-NMR (300 MHz, CDCl3,): (ppm) 0.88 (t, 3H), 1.24 (m, 28H), 1.47 (m, 2H), 1.63 (m, 6H), 1.90 (m, 1H), 2.33 (m, 4H), 2.70 (m, 3H), 2.92 (m,1H), 3.63 (m, 82H), 4.21 (m, 4H), 4.56 (m, 2H). 13C-NMR (100 MHz, CDCl3): (ppm) 174.06, 173.70, 86.49, 86.21, 82.78, 79.59, 72.78, 71.24, 70.79, 70.56, 69.43, 63.68, 63.56, 61.95, 42.98, 39.52, 38.95, 34.43, 34.21, 32.13, 29.90, 29.87, 29.69, 29.57, 29.50, 29.36, 26.72, 25.13, 24.73, 22.90, 22.50, 14.34. TD80-L: (6,8-Dimercapto-1-oxoocty)polyethyl ene glycol sorbitan monooleate was synthesized using conditions similar to those for the synthesis of TD20-L. 1H-NMR (300 MHz, CDCl3,): (ppm) 0.88 (t, 3H), 1.24 (m, 20H), 1.47 (m, 2H), 1.63 (m, 6H), 1.90 (m, 1H), 2.33 (m, 4H), 2.70 (m, 3H), 2.92 (m,1H), 3.63 (m, 82H), 4.21 (m, 4H), 4.56 (m, 2H), 5.34(t, 2H). 4.2.2.2 Dopamine-functionalized Tweens (TDN-D) Scheme 4-2. Synthetic route to dopamine-functionalized Tweens (TDN-D) Succunic anhydride Et3N

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84 The synthetic route to dopamine-functionalized Tweens (TDN-D) is shown in scheme 42. TD20-b: (3-Carboxy-1-oxopropyl)polyethylene glycol sorbitan monolaurate. A mixture of Tween-20 (4.91 g, 4.0 mmol), succini c anhydride (0.41 g, 4.0 mmol) and DMAP (18 mg, 0.15 mmol) in 20 ml of dry acetonitrile was refluxed overnight with stirring. The solution was cooled to room temperature and the solv ent was evaporated under reduced pressure. The oily residue was dissolved in CHCl3 (100 mL) and washed with HC l (1 N, 40 mL) three times and then water (60 mL) three times. The organic phase was dried over MgSO4 and filtered. The yellow oily product was obtained after re moval of the solvent. Yield: 93%. 1H-NMR (300 MHz, CDCl3): (ppm) 0.88 (t, 3H), 1.25 (m, 16H), 1.60 (m, 2H), 2.32 (t, 2H), 2.63 (s, 4H), 3.63 (m, 82H), 4.23 (m, 4H), 4.57 (m, 2H). TD20-c: [4-[(2,5-Dioxo-1-pyrrolidinyl)oxy]1,4-dioxobutyl]polyethylene glycol sorbitan monolaurate. TD20-b (4.85 g, 3.7 mmol) and N -hydroxysuccinimide (NHS, 0.43 g, 3.7 mmol) were mixed in CH2Cl2 (20 mL). Then EDC (0.71 g, 3.7 mmol) was added. After being stirred at room temperature overnight, the reaction mixture was diluted with CH2Cl2 (30 mL).[ S2] This solution was washed with HCl (0.1 N, 60 mL) twice and then br ine (60 mL) twice. The organic phase was dried over MgSO4, filtered and concentrated. The crude product was purified by column chromatography on silica gel (e luents: ethyl acetate/hexane 9:1 and chloroform/methanol 9:1) to give a yellow oily product. Yield: 89%. 1H-NMR (300 MHz, CDCl3): (ppm) 0.88 (t, 3H), 1.25 (m, 16H), 1.60 (m, 2H), 2.32 (t, 2H), 2.84 (s, 4H), 2.78(t, 2H), 2.97 (t, 2H), 3.63 (m, 82H), 4.26 (m, 4H), 4.57 (m, 2H). TD20-D: [[[2-(3,4-Dihydroxyphenyl)ethyl]amino]carbonyl]polyethylene glycol sorbitan monolaurate. Dopamine hydrochloride (0.63 g, 3.3 mmol) and triethylamine (0.33 g,

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85 3.3 mmol) were mixed in 1ml of pyridine. Then the mixture was added to a solution of TD20-c (4.63 g, 3.3 mmol) in pyridine (10 mL). After being stirred for 2 h, the pyridine and solvent were removed under reduced pressure. The oily residue was dissolved in CH2Cl2 (20 mL) and the insoluble solid was removed by filtration. The filtered solution was washed with water (30 mL) three times. The organic phase was dried over MgSO4 and filtered. A dark yellow oily product was obtained after evaporation of the solvent. Yield: 90 %. 1H-NMR (300 MHz, CDCl3): (ppm) 0.88 (t, 3H), b 1.25 (m, 16H), 1.62 (m, 2H), 2.33 (m, 2H), 2.43 (t, 2H), 2.84 (m, 4H), 3.435 (t, 2H), 3.64 (m, 84H), 4.21 (m, 4H), 4.56 (m, 2H), 6.55 (d, 1H), 6.70 (s, 1H) and 6.79 (d, 1H). TD40-D: [[[2-(3,4-Dyhydroxyphenyl)ethyl]am ino]carbonyl]polyethylene glycol sorbitan monopalmitate was synthesized using conditions sim ilar to those for the synthesis of TD20-D. 1H-NMR (300 MHz, CDCl3): (ppm) 0.88 (t, 3H), 1.25 (m, 24H), 1.62 (m, 2H), 2.33 (m, 2H), 2.43 (t, 2H), 2.84 (m, 4H), 3.43 (t, 4H), 3.64 (m, 84H), 4.21 (m, 4H), 4.56 (m, 2H), 6.55 (d, 1H), 6.70 (s, 1H) and 6.79 (d, 1H). TD60-D: [[[2-(3,4-Dyhydroxyphenyl)ethyl]am ino]carbonyl]polyethylene glycol sorbitan monostearate was synthesized using conditions simila r to those for the synthesis of TD20-D. 1H-NMR (300 MHz, CDCl3): (ppm) 0.88 (t, 3H), 1.25 (m, 26H), (m, 2H), 2.33 (m, 2H), 2.43 (t, 2H), 2.84 (m, 4H), 3.43 (t, 4H), 3.64 (m, 84H), 4.21 (m, 4H), 4.56 (m, 2H), 6.55 (d, 1H), 6.70 (s, 1H) and 6.79 (d, 1H). TD80-D: [[[2-(3,4-Dyhydroxyphenyl)ethyl]am ino]carbonyl]polyethylene glycol sorbitan monostearate. was synthesized using conditions simila r to those for the synthesis of TD20-D. 1H-NMR (300 MHz, CDCl3): (ppm) 0.88 (t, 3H),1.25 (m, 20H), 1.62 (m, 2H), 1.99

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86 (m, 4H), 2.33 (m, 2H), 2.43 (t, 2H), 2.84 (m, 4H), 3.43(t, 4H), 3.64 (m, 84H), 4.21 (m, 4H), 4.56 (m, 2H), 5.34 (t, 2H), 6.55 (d, 1H), 6.70 (s, 1H) and 6.79 (d, 1H). 4.2.2.3 Carboxyl-group-functionalized TD20-L (TD20-LC) The synthetic route to ca rboxyl-group-functionalized TD20-L (TD20-LC) is shown in scheme 4-3. Scheme 4-3. Synthetic route to carboxyl-group-functionalized TD20-L (TD20-LC) TD20-e: -[5-(1,2-Dithiolan3-yl)-1-oxopentyl]-(3-carboxy-1-oxopropyl) polyethylene glycol sorbitan monolaurate. A mixture of TD20-a (4.96 g, 3.5 mmol), succinic anhydride (0.36 g, 3.5 mmol) and DMAP (18 mg, 0.15 mmol) in dry ace tonitrile (20 mL) was refluxed overnight with stirring. The solution was cooled to room temperature, and the solvent was evaporated under reduced pressure. Th e oily residue was dissolved in CHCl3 (10 mL) and washed with HCl solution (1 N, 40 mL) twice an d with water (50 mL) twice. The organic phase was dried over MgSO4 and filtered. The yellow oily product was obtained after evaporation of the solvent. Yield: 98%. 1H-NMR (300 MHz, CDCl3): (ppm) 0.88 (t, 3H), 1.24 (m, 16H), 1.46 (m, 2H), 1.63 (m, 6H), 1.90 (m, 1H), 2.32 (m, 4H), 2.45 (m,1H), 2.65 (m, 4H), 3.13 (m, 3H), 3.63 (m, 82H), 4.21 (m, 6H), 4.56 (m, 2H). TD20-LC:-[5-(1,2-Dithiolan-3-yl)-1-oxopentyl]-(6,8-dimercapto-1-oxooctyl)polyethylene glycol sorbitan monolaurate. TD20-e (5.14 g, 3.4 mmol) in NaHCO3 aqueous solution (0.25 M, 100 mL) was cooled with an ice bath for 5 minutes. Then NaBH4 (0.53 g, 14

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87 mmol) was added slowly. The reaction mixture wa s stirred for 2 h until the reaction mixture turned colorless. Then, HCl (6 N, 10 mL) was added to quench the reaction. The mixture was extracted with CHCl3 (50 mL) five times. The combined organic solution was dried over MgSO4 and filtered. A white oily product was obtained af ter evaporation of the solvent. Yield: 92%. 1HNMR (300 MHz, CDCl3): (ppm) 0.88 (t, 3H), 1.24 (m, 16H), 1.47 (m, 2H), 1.63 (m, 6H), 1.90 (m, 1H), 2.33 (m, 4H), 2.92 (m,1H), 2.65 (m, 4H ), 2.70 (m, 3H), 3.63 (m, 82H), 4.21 (m, 6H), 4.56 (m, 2H). 4.2.3 Gold, Fe3O4 and CdSe/ZnS Nanocrystal Synthesis 1-Dodecanethiol-capped 6.6-nm gold NCs were synthesized according to the literature procedure.[ S4 ] In a typical synthesis, AuCl3 (0.068 g) was dissolved in a DTAB solution (0.185 g of DTAB in 20 ml of toluene) with ultrasonication to form a dark orange solution. Then a freshly prepared aqueous solution of NaBH4 (75 mol) was added dropwise to the solution with vigorous stirring. After 20 minutes, 1-dodecanethi ol (1.6 mL) was added and the stirring was continued for 10 minutes. The NCs were precipita ted by adding ethanol, and the solid was redispersed in toluene (20 mL) in the presence of 1-dodecanethiol (1.6 mL) and refluxed for 30 minutes under nitrogen. The NCs were precipitated from the reaction solu tion with ethanol (30 mL), isolated by centrifugation, and re-dispersed in CHCl3. The resulting NCs have a diameter of 6.6 nm with a standard deviation of 7.0 %. Oleic-acid-capped 5.8-nm Fe3O4 NCs were synthesized accordi ng to the literature method.[S5] Oleylamine-capped 5.6-nm CdSe/ZnS core/shell NCs. CdSe/ZnS core/shell NCs were prepared by a two-step procedure consisting of synthesis of CdSe core NCs[152] and growth of ZnS layers. The syntheses were conducte d according to the literature method.[153]

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884.2.4 Preparation of TD-capped Water-soluble NCs Hydrophobic NCs (i.e., Au, Fe3O4 or CdSe/ZnS) (5 M) and TDs (e.g., TDN-L, TDN-D or TD20-LC) (10 mol) were mixed in CHCl3 (5 mL). The solution was stirred at room temperature for 10 minutes. Then triethylamin e (0.05 mL) was added to the mi xture. The resulting mixture was stirred further for 10 minutes. After evaporation of the solvent, the NCs were re-dispersed in water. The nanocrystal solution was filtered through a 0.22m MCE syringe filter (Fisher Scientific). The excess of TD ligands was re moved by spin filtration (Millipore, 10K NMWL, 10000g, 10 min) four times. The resulting NCs were re-dispersed in water (18 ) for further studies. 4.2.5 Antibody-functionalized CdSe/ZnS Nanocrystals Hydrophilic CdSe/ZnS QDs with a diameter of 5.6 nm (RSD ~ 8.0 %) and capped with a mixture of compound TD20-L and TD20-LC (5:1 molar ratio) were pr epared as described above. CdSe/ZnS QDs (0.20 nmol) were dissolved in 2-( N -morpholino)ethanesulfonic-acid buffer solution (MES buffer, 0.1 M, 150 L, pH = 6.0). An aqueous solution of 1-ethyl-3-(3dimethylaminopropopyl)carbodiimide (EDC, 5.0 mg/mL, 50 L) and an aqueous solution of Nhydroxysulfosuccinimide (Sulfo-NHS, 5.0 mg/mL, 50 L) were added to the QD-buffer solution. The mixture solution was incubated for 1 h at room temperature with gentle shaking. Then, 2-mercaptoethanol (1.0 L) was added to the reaction mi xture to quench the EDC. The excess reducing agent and inactiv ated cross-linker were removed by filtering through a NAP-5 column using phosphate buffered saline (PBS buf fer, pH = 7.4) as the elution buffer. The collected QD solution was concentrated to 50 L by spin filtration (10K NMWL, Millipore, 10000g, 10 min) and re-dissolved in PBS buffer solution (250 L, pH 7.4). NS5A-specific mouse monoclonal antibody (100 g) was added to the QD soluti on, and the resulting mixture was incubated for 2hr at room temperature. Hydroxylamine (0.5 L) was added to quench the

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89 reaction. The mixture was transferred to a spin filter (100K NMWL, Millipore, 10000g, 10 min) and concentrated to 50 L. Then PBS buffer solution (200 L, pH 7.4) was added and the mixture was spun again. The washing step was re peated 2 times to remove thoroughly the free antibodies. Finally, the purified antibody-functionalized QDs (0.15 nm ol) were re-dispersed in a PBS buffer solution (450 L, pH 7.4). Sodium azide was added to the solution of antibodyconjugated QDs (with a concentration of 0.01 % w/v) as a preservative. 4.2.6 Immunoassay Tests FCA1 HCV replicon cells were grown on glass coverslips for 24 h, and the cells were fixed in an ethanol solution with 5% acetic acid at -20 oC overnight. The fixed cells were washed with PBS (pH 7.4) at room temperature tw ice (5 minutes each time), and the cells were blocked by 1:50 normal goat serum for 30 min at room temperature. Then the cells were incubated with NS5A-specific-antibody functionalized QDs (50 nM, 0.20 mL) at room temperature for 1 h. After the cells were washed with PBS (pH 7.4) 3 times (5 minutes each), the nuclei of the cells were counterstained with DAPI (4 ,6-diamidino-2-phenylindole,Vector Laboratories Inc, Burlingame, CA) as an intern al reference, and the extra DAPI was removed with PBS (pH 7.4). Finally, the FCA1 cells were examined under a fluorescence microscope (Olympus BX51, Olympus Imaging America Inc, Center Valley, PA). In a control test, TD-cappe d QDs (with the ratio of TD80-L: TD20-LC = 5:1), which were not functionalized with NS5A-specific anti body, were used for staining FCA1 cells. Histograms of fluorescent images show the m ean pixel intensities. The intensity ratio of blue channel (from DAPI) and red channel (from QDs) was calcu lated for each cell, and more than 300 cells were analyzed.

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904.2.7 Stability Tests Each stability test was repeated more than five times using the same batch of TD-capped NCs. The sample standard deviation (S) and relative standard devi ation (RSD) were calculated by the following equations, respectively: N 2 i i=11 (x-x) N-1 S=; and RSD = S / x where xi is the result of each experiment and x is the average value. For example, five sets of dynamic light scatte ring (DLS) data (Figur e 4-5) were obtained from five parallel stability-test experime nts. The nanocrystal hydrodynamic size in each experiment is Figure 4-5A) 16.3 nm, B) 17.8 nm C) 17.1 nm, D) 17.3 nm and E) 17.0 nm. So the average size of these five experiments was 17.1 nm 0.54 nm and RSD = 3.2%, as calculated by the above equations. 4.2.8 Other Measurements 4.2.8.1 1H-NMR Measurements 1H-NMR spectra were recorded using a Va rian Mercury NMR Spectrometer (300 MHz). The samples were prepared by adding aliquots of products (10 mg) to deuterated chloroform (CDCl3, ~ 0.6 mL). 4.2.8.2 Determination of Fluorescence Quantum Yelds (QY) Fluorescence spectra were measured using a Fluorolog-3 spectrof luorometer (Horiba Jobin Yvon, Irvine, CA). Room-temperature fluorescence QY of the CdSe/ZnS core/shell QDs was determined using the literature method.[58] LD690 (63% QY) was used as reference and the excitation wavelength was 500 nm.

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914.2.8.3 DLS Measurements The nanocrystal aqueous solutions were first filtered through a 0.22m MCE syringe filter (Fisher Scientific). The hydrodynamic sizes of NC s were obtained using a Brookhaven Instrum ents dynamic light scatte ring instrument (DLS) (Corpor ation, Holtsville, NY) at 25 oC. 4.2.8.4 TEM Measurements TEM measurements were performed on a JE OL 200CX operated at 200 kV. To prepare the specimens, a particle solution (10 L) was dropped onto a 200-mesh copper grid, and dried overnight at ambient conditions. 4.3 Results and Discussion To examine the function of these TD liga nds, three types of hydrophobic NCs were used: gold NCs (6.6 nm in diameter with RSD 7.0 %), Fe3O4 (5.8 nm in diameter with of 6.0%), and CdSe/ZnS QDs (5.6 nm in diameter with RSD of 8.0%) (Figure 4-6A-C). In the first set of experiments, TD20-L (or TD20-D) was used to functionalize the gold and CdSe/ZnS (or Fe3O4) NCs, respectively. The ligand-exchange reactions were performed in chloroform for 20 min. After evaporation of the chloro form, the resulting hydrophilic NCs are highly soluble in water, with a transfer yield of nearly 100%. The extra liga nds in the hydrophilic-nanocrystal solutions were removed by spin filtration four times. TEM measurements show that the TD-func tionalized hydrophilic NCs exhibit nearly identical size and shape, as compared to their hydrophobic counterpa rts (Figure 4-6D-F). Dynamic light scattering (DLS) measurements show that th e HDs of these NCs are 17.1 nm for the TD20-L-functionalized Au particles, 16.3 nm for the TD20-D-functionalized Fe3O4 NCs, and15.9 nm for the TD20-L-functionalized CdSe/ZnSe QDs (Figure 4-6G-I). After subtracting the respective core sizes from th e HDs, we obtain a nearly identic al shell thickness of about 5.2 nm for all three types of NCs. The shell thickness is very close to the average length of these TD

PAGE 92

92 ligands (~4.9 nm). This result shows that only one monolayer of TD lig ands is functionalized onto the nanocrystal surface. In addition, gas-chromatography-mass spectrometry (GC-MS) measurements show that these TD ligands do not totally remove the original hydrophobic ligands of these NCs. In a sample of TD20-D-functionalized Fe3O4 NCs, oleic acid (the original ligand) was unambiguously identified by GC-MS (Figure 4-7). Taken together with the results above, this partial ligand exchange of suggests that TD ligands indeed functionalize hyd rophobic NCs through both coordina te bonding, as well as via hydrophobic van der Waals interactions between the fatty-acid chain in th e TD ligands and the hydrophobic ligands on the NCs. This nanocrystal f unctionalization with dua l-interaction ligands is further consistent with the results from the following nanocrystal-stability measurements. The stability of the hydrophilic NCs was i nvestigated as a f unction of pH, salt concentration and time of a thermal treatment at 100 oC. For the TD20-L-functionalized gold NCs, the stability tests were monitored using both LDS and UV-Vis absorption spectroscopy, as shown in Figure 4-8. In boiling water (pH 6.5) for 4 h, these gold NCs do not exhibit significant change in their hydrodynamic diameters (HD), as m easured using LDS, or in the position of their absorption peak, as measured by ab sorption spectrosc opy (Figure 4-9A). The results from pHstability tests (Figure 4-9B) show that these hydr ophilic gold NCs are stable from pH 2 to 13 for more than one week. At pH 1, the HD of these particles is slightly decreased, but without a change in the position of absorption maxima for more than two hours. At pH 14, the particles show small changes in both HD and absorption-p eak position, but the na nocrystal solution is stable for more than three days at this conditi on. In addition, the gold NCs are stable almost indefinitely in NaCl solutions with concentra tions up to 5 M (Figure 4-9C). Altogether, these results show that TD20-L-functionalized gold NCs exhibit ex traordinary stability in various

PAGE 93

93 extreme conditions. Such stability is even highe r than that of gold NCs heavily functionalized with alkylthiol-capped oligonucle otides, which have been used in commercial biomedical diagnosis because of their high stability in high-concentration salt solutions. The TD20-D-functionalized Fe3O4 NCs also exhibit excellent stability in these tests. These Fe3O4 NCs are stable in a solution of boiling wa ter (pH 6.5) for 3 h (Figure 4-9D). pHstability tests show that these Fe3O4 NCs are stable from pH 3 to 14 for more than one week. At pH 2, the HD of these Fe3O4 NCs slightly increases (Fig ure 4-9E). Surprisingly, TEM measurements show that after a 2hr treatment at pH 2 the NCs exhibit no measurable change in size and shape, as compared with the NCs in the control experiment at pH 7 (Figure 4-10). Moreover, the TD20-D-modified Fe3O4 NCs are stable nearly in NaCl solutions with concentrations up to 2 M. In a 4-M NaCl solution, these Fe3O4 NCs are stable for more than 4 h (Figure 4-9F). These results show that TD20-D-functionalized Fe3O4 NCs exhibit a much higher stability than Fe3O4 NCs functionalized with PEGylated-dop amine ligands, which attach onto the nanocrystal surface through coordinate bonding only.[154] Therefore, the excelle nt stability of TDfunctionalized hydrophilic NCs may be attributed to the ability of the TD ligands to bind to the nanocrystal surface via both coordi nate bonding and hydrophobic interactions. Figure 4-11 shows four di fferent colors of TD20-L capped CdSe/ZnS NCs in aqueous solution. These NCs are very bright under a UV-lamp. The absorption spectra and emission spectra of these NCs are shown in Figure 4-11C and D. To compare the stability of TL20-Lcapped NCs with the other ligands, the same Cd Se/ZnS NCs were used, but the ligands on the NC surfaces were different. Table 4-1 lists the resu lts of transfer efficiency of ligand exchange, hydrodynamic size measured by DLS, and the stability test of these NCs. It clearly shows that TD20-L functionalized CdSe/ZnS NCs are relatively small but stable NCs.

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94 To further explore the effect of the hydrophobi c van der Waals intera ctions between TD ligands and the nanocrystal hydrophobi c coating, the stability of TDN-L-functionalized CdSe/ZnS QDs was studied as a functi on of the fatty-acid chain in the TDN-L ligands (N = 20, 40, 60 or 80). In these experiments, 5.6-nm oleylamine-coated CdSe/ZnS particles were transferred into the aqueous phase through surfac e functionalization with the four types of TDNL ligands. The resulting TD20-L-modified CdSe/ZnS NCs were st able from pH 3.5 to 11 (Figure 4-12A). Such stability is higher than that of CdSe/ZnS particles functionalized with PEGylatedlipoic-acid ligands, which lack hydrophobic in teractions with th e nanocrystal surface.[95] When the length of the fatty-acid chain was increased to C16 and C18, TDN-L-modified CdSe/ZnS NCs (N = 40, 60, or 80) are stable in a wi der pH range, from 1 to 14, according to DLS measurements (Figure 4-12A). Such the fatty-acidchain-dependent stability is also observed in the stability tests with NaCl solutions. TD20-L-modified particles are stable in NaCl solutions only up to 0.6 M, while TD40-L-modified particles are stable in a 2-M NaCl solution. With a further increase of fatty-acid length to C18, TD80-L(or TD60-L)-functionalized CdSe/ZnS NCs are stable in a nearly saturated NaCl soluti on (Figure 4-12B). Taken together, these results demonstrate that the stability of NCs indeed de pends on the length of the fatty-acid chains on the TD ligands. This chain-length-dependent stability sugge sts that the van der Waals interactions between the fatty-acid chains and the oleylamine coating play a significant role in stabilizing NCs in aqueous solutions. The longer the fatt y-acid chain, the great er the van der Waals interactions with the oleylamine coating. In addition, the van der Waals interactions should create a hydrophobic shell on the nanocrystal surface. Such a hydrophobic shell can provide additional protection for the hydr ophilic NCs by preventing hydrophilic reagents (such as H+)

PAGE 95

95 from reacting with the nanocrystal surface. Inde ed, we have found that the fluorescence quantum yield of TD80-L-functionalized CdSe/ZnS QDs is maintain ed at about 50% for more than three months in aqueous solutions between pHs 3.5 a nd 12.5. Furthermore, the fluorescence brightness of these NCs does not significantly change for 2 hours in an aqueous solution of pH 2 (Figure 412C). To demonstrate the suitability of using these water-soluble NCs for biomedical diagnosis, TD-modified CdSe/ZnS QDs were used as fluor escence labels to monitor the expression of a HCV (Hepatitis C virus) protein (NS5A) inside FCA1 cells. In these experiments, 5.6-nm oleylamine-capped CdSe/ZnS QDs were fi rst functionalized w ith a mixture of TD80-L and TD20-LC (5:1). Anti-HCV NS5A monoclonal an tibodies were then attached onto the resulting QDs through an EDC coupling reaction. In a control test, the QDs without NS5Aspecific antibodies showed very low non-speci fic adsorption onto NS5A-containing FCA1-cell substrates (Figure 4-13A). In contrast, the an tibody-modified QDs exhi bit a very high specific affinity to such substrates (Figure 4-13B). Significantly, the fluorescence intensity from QDs labels is more than 75 times stronger than that in the control test (Figure 4-13C). 4.4 Conclusion Dual-interaction ligands were successfu lly used to convert hydrophobic gold, Fe3O4 and CdSe/ZnS NCs into hydrophilic NCs. A series of dual-interaction ligands was synthesized through simple modifications of Tween compoun ds. The hydrophilic NCs functionalized with these TD ligands exhibit high stab ility in aqueous solutions with a wide range of pH and salt concentration, and under thermal treatment at 100 oC. Such extraordinary nanocrystal stability is attributed to the new type of surface functi onalization through both coordinate bonding and hydrophobic van der Waals interactions. In additi on, the TD-functionalized QDs are excellent fluorescent labels for detecti ng the HCV-NS5A expression in FCA1 cells. Moreover, the new

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96 surface-functionalization approach can be readily generalized for NCs with other compositions. Finally, because of their excellent stability these TD-functionalized NCs should play an important role in a variety of nanocry stal-based biomedical applications.

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97 Figure 4-1. Schematic illustration of the Tween -derivatives (TDs) dual interaction. A) The molecular structure of Tween-derivatives. B) The dual interactions: coordinate bonding to the inorganic crystal and hydrophobi c van der Waals interaction with the non-polar tails of the organi c surface ligands. Dithiol gr oup coordinates with Au and semiconductor QDs and diphenol group has stro ng interaction with metal oxide NCs. (Reprinted with permission from ref 151).

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98 Figure 4-2. Synthesis of dualinteraction TD ligands: A) lipoic-acid-functionalized TDs; B) dopamine-functionalized TDs, and C) carboxyl-group-functionalized TD20-L. In this study, water-soluble gold, Fe3O4 NCs and CdSe/ZnS quantum dots (QDs) functionalized with dual interaction ligands were prepared with relatively small hydrodynamic diameters (e.g., less than 20 nm). These NCs exhibit extraordinary stability over in a wide range of pH (e .g., 1-14), salt concentration, and thermal treatment (at 100 oC). In addition, these NCs can be further functionalized with antibodies for monitoring virus-protein expression in cells. (Reprinted with permission from ref 151). A B C

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99 Figure 4-3. 1H-NMR of compound TD20-a Figure 4-4. 1H-NMR of compound TD20-L

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100 Figure 4-5. DLS data for TD20-L-functionalized 6.6-nm Au NC s from five parallel thermalstability-test experiments (at 100 oC and pH 6.5 for 120 min). (Reprinted with permission from ref 151). Figure 4-6. TEM images of hydrophobic NCs: A) 6.6-nm Au, B) 5.8-nm Fe3O4 and C) 5.6-nm CdSe/ZnS core/shell NCs prior to f unctionalization; and their hydrophilic counterparts functionalized with TD20 ligands: D) Au NCs/TD20-L, E) Fe3O4 NCs / TD20-D and F) CdSe/ZnS QDs/TD20-L. Dynamic light scattering spectra of these hydrophilic NCs: G) Au NCs/TD20-L, (h) Fe3O4 NCs/TD20-D and I) CdSe/ZnS QDs / TD20-L. (Reprinted with permission from ref 151) B C D E A B C A E F D H I G

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101 Figure 4-7. Gas-chromatography mass spectrometry (GCMS): A) a gas chromatogram of a solution with the ligands from TD20-D-functionalized Fe2O3 NCs; and B) a mass spectrum of the compound with a retention time of 10.81 min. This spectrum is nearly identical to the standard mass spectrum of oleic acid. The sample was prepared as follows: TD20-D-functionalized Fe2O3 NCs (~15 mg) were dissolved by HCl (10 M, 1 mL), and then water and excess HCl were evaporated from the solution using a rotary evaporator. The resulting yellow, oily resi due was dissolved in methanol (1 mL) for the GC-MS measurements. (Reprinted with permission from ref 151). B A

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102 Figure 4-8. UV-Vis spectra for the stability tests of TD20-L-functionalized Au NCs: A) thermal stability test at 100 oC as a function of time: Black: 0 h, Red: 1 h, Green: 2 h, Blue: 3 h and Magenta: 4 h; B) stabil ity as a function of pH: Black: pH 1; Red: pH 2, Green: pH 3, Blue: pH 7, Cyan: pH 12, Magenta: pH 13 and Orange: pH 14; C) stability as a function of NaCl concentration. Black: 0 M, Red: 1 M, Green: 2 M, Blue: 3.5 M and Orange: 5 M. (Reprinted w ith permission from ref 151). Figure 4-9. Stability tests of TD20-L-functionalized 6.6-nm Au NCs monitored with DLS (black) and UV-Vis spectra (Red): A) Thermal-stability test at 100 oC; B) pH-stability test; and C) stability as a function of NaCl concentration. D), E) and F) are the respective stability tests for TD20-D-functionalized 5.8-nm Fe3O4 NCs. (Reprinted with permission from ref 151). B C A B A C E D F

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103 Figure 4-10. TEM images of TD20-D-capped Fe3O4 NCs from aqueous solutions at A) pH 7 and B) pH 2 for 2h(Reprinted w ith permission from ref 151). Figure 4-11. TD20-L capped CdSe/ZnS NCs with different sizes in aqueous solution. A) Photo image and B) fluorescence image, C) absorption spectra and D) emission spectra. A B A B C D

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104 Figure 4-12. Stability tests of TDN-L-functionalized 5.6-nm CdSe/ZnS NCs as a function of the fatty-acid chain on these TD lig ands A) pH and B) NaCl: TD20-L (blue square); TD40L (red dot); TD60-L (green triangle up); and TD80-L (black triangle down). C) Fluorescence images of TD80-L-functionalized 5.6-nm Cd Se/ZnS NCs as a function of pH, which was indicated by pH-test paper. Figure 4-13. Immunofluores cence tests of NS5A-containi ng FCA1 cells using TDfunctionalized CdSe/ZnS core/shell nanocryst al QDs: A) without and B) with NS5Aspecific antibodies. The nuclei of these FCA1 cells were counterstained with DAPI (4',6-diamidino-2-phenylindole, exhibiting blue fluorescence) as an internal reference. The images were taken under an Olympus fluorescence microscope. C) Fluorescenceintensity radios of blue channel (from DAPI) and red channel (from QDs) were calculated on the basis of the fluorescence images taken from the immunostaining tests. A: control test, and B: test with antibody-functiona lized QDs. (Reprinted with permission from ref 151). A B C A B C

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105 Table 4-1. Stability test of CdSe/Z nS NCs capped with different ligands Ligands T a (%) DLS (nm)b pH [NaCl] TD20-L 100 ~18 3.5~11 0.7M DHLA-PEG2000[95] 100 ~29 5.5~11 0.7M PEG2000-PMAO (30:1)[150] 65 ~28 3.5~12 0.7M PEG5000-PMAO (30:1)[150] 100 ~35 3.5~13 1.0M Cysteine 100 ~9 2~13 c 0.3 Modified PAA[155] 70 ~14 4.5~13 0.4 a: transfer efficiency of ligand exchange; b: at pH~6. 5; c: Cysteine-capped CdSe/ZnS NCs are not stable at pH around 5.

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106 CHAPTER 5 GOLD NANOCRYSTAL-BASED ASSA Y FOR THE DETECTION OF -GALACTOSIDASE 5.1 Introduction Because of its high selectivity and stability, -galactosidase is a commonly used reporter gene marker in gene expression studies.[156,157] The current method for detecting -galactosidase involves the use of fluorom etry based upon organic dyes.[158-160] After incubation with the enzyme, a weakly fluorescent substrate is c onverted into a highly fluorescent substrate. However, most of organic dyes are toxic and can not be used to study ge ne expression in living systems. Recently, a magnetic resonance imaging (MRI) contrast agent was designed to identify -galactosidase,[161-163] in which the access of water to a chelated paramagnetic ion (Gd3+) is blocked by the galactopyranosyl ring. In the pres ence of galactosidase, the galactopyranosyl ring can be cleaved by -galactosidase, resulting in a change in the T1.[163] However, this method has low sample throughput, and it requires multi-step and low-yield synthesis, and complicated instrumentation. Therefore, a need exists for de veloping simple and easy assays for the detection of -galactosidase. Chromophoric colorimetric methods have attr acted attention due to their easy readout (even by the naked eye). Compared with fluorometric met hods, colorimetric detections are not hindered by background from either autofl uorescence of analytes or fluorescence created during sample preparation. However, colorimetric methods normally suffer from low sensitivity and high limits of detection (LOD) ~1M).[164] Recently, Au NP-based colorimetric sensors have become an attractive research area because of the special optical and electronic properties of Au NCs, in particular surface plasmon resonance (SPR).[165] The positions of SPR peaks of Au-NCs are dependent not only on the size and shape of Au NCs, but also on the distances between the particles. The SPR change of AuNCs can be measured from the adsorption spectrum of a Au-NP solution, and it can even be

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107 observed by the naked eye. In addition, thes e Au-NCs possess high stability and easy surface modification. The molar extinction coeffici ent of Au-NCs aqueous solutions (~28 M1 cm1 per particle), which is extremely high compared to those of most organic dyes, provides a promising detection sensitivity. Therefore, Au-NCs have been applied for a wide variety of assays, such as detection of proteins,[98] metal ions[166] or other small organic molecules.[164] However, assays for -galactosidase activity based on Au-N Cs have not been developed. Therefore, we have designe d a new type of Au NP-based biosensor to detect -galactosidase, based on the distance-dependent SPR of Au-NCs. Figure 5-1 shows the molecular design of the sensor and the general principle for detection of -galactosidase. Galactopyranos yl functionalized ligands, LT-gal are used to cap Au-NCs and stabilize the NCs in aqueous solution. The galactopyranosyl ring is cleaved from the ligand after the addition of -galactosidase, resulting in the Au-NP aggregation, and the color of the Au-NP solution changes from red to purple. Scheme 5-1. Synthetic route of LT-gal ligands. HO NH2 S S HO O HO N H S S O DIPC/DPTS 1y.85% Ac2O,py O HO OH OH OH OH O AcO OAc OAc OAc OAc O AcO OAc OAc OAc Br HBr 2y.98% 3y.93% N H S S O O AcO OAc OAc OAc O N H S S O O HO OH OH OH O CsCO3/DMF NaOMe/MeOH 4y.48% 1 5y.43%(LT -gal) (LT)

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1085.2 Experimental Section 5.2.1 Chemicals. N,N -Diisopropylcarbodiimide (DIPC, 99% ), iron (III) chloride (FeCl3H2O, 98%), 1octadecene (ODE, 90%), octadecylamine (ODA, 97%), p-toluenesulfonic acid monohydrate (98%), ()-lipoic acid (99%), tyramine (99%), D-(+)-galactose (99%), acetic anhydride (99%), hydrobromic acid solution (33% in acetic acid), cesium carbonate (99.9%), triphenylphosphinegold( I) chloride (AuPPh3Cl, 99.9+%), tert-butylamine borane (TBAB, 97%), 1-dodecanethiol (97%), -galactosidase from E.Coli (molecular weight 540,000, EC 3.2.1.23) and -glucosidase from almonds (EC 3.2.1.21) we re purchased from Aldrich. Nanopure water (18.2 M cm) was prepared by a Barnstead Nanopure Diamond system. All the other reagents were purchased from Fisher Scientific International Inc. 4-(N,N-Dimethylamino)pyridinium-4-toluenesulf onate (DPTS) was prepared by mixing THF solutions of DMAP (2 M, 50 mL) and p-toluenesulfonic acid monoh ydrate (2 M, 50 mL) at room temperature with stirring. The resul ting precipitate was filtered and dried under vacuum.[142] 5.2.2 Synthesis of LT-gal The synthetic route to prepare LT-gal is shown in Scheme 5-1. Lipoic acid and tyramine react in the presence of DIPC and DPTS to form 5-(1,2-dithiolan-3-yl)-N-(4hydroxyphenethyl)pentanamide (compound 1). Galactose is first acetylated and the acetyl-group at the anomeric carbon is replaced with bromide to form 1--bromo-2,3,4,6-tetraacetyl-Dgalactose (compound 3). Compound 3 condenses with 1 which is deacetylat ed via reaction with sodium methoxide (NaOMe).

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109Preparation of 5-(1,2-dithiolan-3-yl )-N-(4-hydroxyphenethyl)pentanamide (compound 1, LT). Tyramine (3.3 g, 24.0 mmol), lipoic acid (2.6 g, 12.8 mmol), and DPTS (4.7 g, 15.1mmol) were mixed in pyridine (30 mL ) and stirred for several minutes at room temperature. Then, DIPC (2.3 mL 15.1 mmol) was added to the mi xture. After being stirred at room temperature overnight, the reaction mixture was filtered and the solvent was removed under reduced pressure. The oily residue was dissolved in CHCl3 (100 mL) and washed with water (60 mL) three times. The organi c phase was collected, dried over MgSO4, filtered and concentrated. The crude product was purified by column chromatography on silica gel (ethyl acetate/hexane 7:3) to give a yellow powder. Yield: 85%. 1H-NMR (300 MHz, CDCl3): 6.807.02 (dd, 2H), 3.50 (m, 2H), 3.07 (m, 3H), 2.76 (t, 2H), 2.43 (m, 1H), 2.12 (t, 2H), 1.90 (m, 1H), 1.64 (m, 1H), 1.40 (m, 2H). Preparation of pentaacetyl-D-galactose (compound 2). D-galactose (3.00 g, 16.7 mmol) was dissolved in dr y pyridine (33 mL) at 0 oC under Ar flow. Then acetic anhydride (31.5 mL, 333 mmol) was added slowly. The reaction mixture was stirred at 0oC for 1hr before a catalytic amount of DMAP (200 mg, 1.67 mmol) was added. Then the reaction mixture was stirred at room temperature for 6 h. The resul ting clear yellow solution was poured into rapidly stirring ice water (500 mL) and ex tracted with ethyl acetate (75 mL) two times. The combined organic phase was dried over MgSO4 and filtered. The solvent was removed under reduced pressure to give a white oily product. Yield: 98 %. 1H-NMR (300 MHz, CDCl3): 6.35 (s, 1H), 5.46-5.47 (m, 1H), 5.29-5.31 (m, 2H), 4.29-4.33 (m, 1H), 4.02-4.10 (m, 2H), 1.96-2.12 (m, 15H). Preparation of 1--bromo-2,3,4,6-tetraacetyl-D-galactose (compound 3). Compound 2 (5.77 g, 14.7 mmol) was dissolved in a HBr ( 14.7 mmol) acetic acid solution (20 mL). After 3 h, the reaction mixture was evaporated and coevaporated with dry toluene under reduced

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110 pressure to give a brown oily product. Yield: 93%. 1H-NMR (300 MHz, CDCl3): 6.66 (d, 1H), 5.45-5.47 (m, 1H), 5.36 (m, 1H), 5.00 (m, 1H), 4.41-4.46 (m, 1H), 4.05-4.10 (m, 2H), 1.95-2.09 (m, 12H). Preparation of 2-(4-(2-(5-(1,2-dithiol an-3-yl)pentanamido)ethyl)phenoxy2,3,4,6tetraacetyl-D-galactose (compound 4). Compound 1 (1.00 g, 3.07 mm ol) was dissolved in dry N,N-dimethylformamide (5 mL) and cesium carbonate (4.00 g, 12.28 mmol) was added to the solution at 0 oC. Compound 3 (5.03 g, 12.28 mmol) in dry N,N-dimethylformamide (20 mL) was added dropwise under an Ar flow, and the reaction solution was stirred at room temperature for 4 h. Then the inorganic precipitate was removed by filtration and the filtrate was concentrated under reduced pressure. The residue was diluted with water and extr acted with ethyl acetate, and the combined organic extracts were dried over MgSO4 and filtered. After removing the organic solvent by evaporation, the crude product was purified by column chro matography on silica gel (ethyl acetate/hexane 1:1 to 9:1) to give a yellow powder. Yield: 48%. 1H-NMR (300 MHz, DMF): 6.94-7.12 (m, 2H), 5.42 (m, 2H), 5.12 (m, 1H ), 5.01 (d, 1H), 4.22-4.04 (m, 2H), 3.45 (m, 2H), 3.11 (m, 3H), 2.76 (t, 2H), 2.43 (m, 1H), 2.02-2.16 (m, 12H), 1.95 (m, 1H), 1.68 (m, 1H), 1.42 (m, 2H). Preparation of LT -gal (compound 5). Compound 4 (0.97 g, 1.47 mmol) was dissolved in methanol (20 mL). Sodium methoxide (0.02 g, 0.37 mmol) was added at 0 oC and the solution was stirred at room temperature for 2 h. The r eaction mixture was neutrali zed with Amberlite IR120 plus (H+). The Amberlite was removed by filtration, and the filtrate was evaporated. The remaining organic solid was purified by column chromatography silica gel (chloroform/methanol 9:1) to give a yello w powder. Yield: 43%. 1H-NMR (300 MHz, CDCl3): ): 6.92-7.08 (dd, 2H), 5.12 (d, 1H), 4.72-4.80 (dd, 2H), 4.60 (t, 1H), 4. 42 (d, 1H), 3.45 (m, 2H), 3.11 (m, 3H), 2.60 (t,

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111 2H), 2.40 (m, 1H), 2.01(t, 2H ), 1.80(m, 1H), 1.60 (m, 1H), 1.42 (m, 1H), 1.28 (m, 2H). [M+H]+=488.18 (Appendix C). Preparation and 8.0 nm gold nanocrystals. Gold NCs were synthesized according to the literature procedure.[118] In a typical synthesis, AuPPh3Cl (0.124 g, 0.25 mmol) and dodecanethiol (0.125 mL, 0.50 mmol) were dissol ved in benzene (20 mL) to form a clear colorless solution. Then tert-butylamine borane (TBAB, 0.22 g, 2.5 mmol) was added. The resulting solution was stirred at 100 oC via silicon oil bath for 1 h. The NCs were precipitated from the reaction solution with ethanol (30 mL), isolated by centrifugation and re-dispersed in CHCl3. The resulting NCs have a diameter of 8.0 nm with a standard deviation of 7.0 %. Preparation of LT -gal-capped water-soluble NCs. Hydrophobic 8.0 nm Au NCs (5 mol) in CHCl3 (2 mL) and LT-gal (10 mol) in ethanol (2 mL) were mixed. Then triethylamine (0.05 mL) was added and the result ing mixture was stirred overnight. Then Au NCs were precipitated and the solvent was discarded. These NCs were washed with chloroform twice and re-dispersed in water. The excess of LT-gal ligands was removed by spin filtration (Millipore, 10K NMWL, 10000g, 10 min) four times. The resulting NCs were re-dispersed in aqueous buffer solution (PBS, pH 7.4, 25 oC) for further studies. Using the same method, AuNCs capped by a mixture of ligands (LT-gal and LT with different weig ht ratios: 4:1, 2:1, 4:3, 1:1, and 3: 4) were also prepared. 5.2.3 Detection of -galactosidase Solutions of PBS buffer (pH 7.4, 1. 25 mL), a magnesium chloride (MgCl2, 30 mM, 0.05 mL) were prepared in separa te tubes, Varying amount of -galactosidase were added and the mixtures were equilibrated to 37 oC. Then the LT-gal capped Au-NP solution was added to each mixture. The peak positions and absorbances at 523 nm were monitored. The intensities were

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112 measured at the wavelength of 523 nm. A bla nk PBS buffer solution and a glucosidase solution were used for control tests. 5.2.4 Measurements 5.2.4.1 1H-NMR Measurements 1H-NMR spectra were recorded using a Va rian Mercury NMR Spectrometer (300 MHz). The samples were prepared by adding aliquots of products (10 mg) to CDCl3. 5.2.4.2 UV/Vis Spectra UV/Vis spectra of Au-NCs were meas ured using UV-1700 PharmaSpec UV-Visible spectrophotometer (Shimadzu, MD). 5.2.4.3 DLS Measurements The nanocrystal aqueous solutions were filtered through a 0.22m MCE syringe filter (Fisher Scientific). The hydrodynamic si zes of NCs were obtained using Brookhaven Instr. Corp. dynam ic light scattering instrume ntal (Holtsville, NY) at 25 oC. 5.2.4.4 TEM Measurements TEM measurements were performed on a JEOL 200CX operated at 200 kV. The specimens were prepared as described in prev ious chapters. 5.3 Results and Discussion The galactopyranosyl-ring functionalized ligand (LT-gal) was synthesized by simple organic reactions, as described above. To stabilize Au-NCs, a dithiol coordination group was incorporated into the ligand. The ligand-excha nge reaction was performe d to make the Au-NCs water-soluble with a transfer yield of nearly 100 %. These functionalized Au-NCs are stable in the aqueous buffer solution (PBS, pH 7.4, 25 oC) for more than five months. The UV/Vis absorption spectrum of the Au-NCs in the PBS buffer solution shows a sharp peak at 523 nm, which indicates that these functionalized Au-NCs are well dispersed in the buffer solution. The

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113 TEM images show a monolayer of the functiona lized Au-NCs, indicating that no aggregation occurs. To demonstrate the suitability of using thes e Au-NCs (8.0 nm in diameter) for enzyme detection, a solution of functionalized Au NCs (PBS, pH 7.4) was added to enzyme galactosidase buffer solution (PBS, pH 7. 4) with various concentration at 37 oC, and UV/Vis measurements were performed to monitor the aggr egation of Au NCs. In control experiments, the Au-NC buffer solution was added to a blank PBS buffer solution (without any enzyme) and a -glucosidase buffer solution (PBS, pH 7.4) at 37 oC. The control solutions showed a very small red-shift ( 3 nm) and a tiny decrease in absorban ce after being mixed with the Au NC suspension for 6 hr. In contrast, Figure 52 shows that the mixture of Au NCs and galactosidase exhibits a 21 nm red-shift and a dram atic decrease in absorbance within 1 hr after the addition of Au-NC solution. The red-shift absorbance of the SPR (from 523 nm to 544 nm) and the decrease of the absorbance at 523 nm are due to the aggregation of Au-NCs. The change is easily observed even by the naked eye. Interestingly, when the pure LT-gal-capped Au-NCs were used as the probes to detect galactosidase, no shift was observed in the UV/ Vis absorption of the Au-NC suspension. We attribute this to the possibility that -galactosidase cannot reach the active site of galactopyranosy ring when LT-gal is the only ligand bound to the surface of Au-NCs. Introducing another short ligand, LT, to the surfaces of Au-NCs separates the LT-gal sufficiently for the active-site on the LT-gal, which allows to interact with -galactosidase. Since the pure LT capped Au-NCs are unable to disperse in the aqueous solution, changing the ratios between LT-gal and LT affects the solubility of corresponding Au-NCs and causes different sensitivities of these Au-NCs.

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114 To test the effect of the use of two ligands on the sensitivity of these assays, the Au-NCs capped with LT and LT-gal at different ratios were pr epared. The kinetics of Au-NP aggregations was studied using the same conditions for these different Au-NCs. As the indication of Au-NP aggregation, the absorbance at 523 nm was measured every 15 minutes after mixing the enzyme solution with the Au-NP suspension, as shown in Figure 5-3A. The aggregation of Au-NCs is faster when the surface of Au-NCs is covered by a higher percent of LT. However, the higher percent of LT capped on the Au NCs results in a decrease in the stability of the AuNCs. The highest percentage of LT on the Au-NCs is about 57% (3:4 molar ratio LT-gal: LT). Further increase of the amount of LT on the Au -NP surface induces Au-NP aggregation in the absence of enzyme. The detection of enzyme was furthe r studied for Au-NCs capped with LT-gal and LT of at a ratio 3: 4. Different concentrations of -galactosidase (0, 5, 10, 20, 40, 60, 80, 140, 200, 250 and 300 units/ml) were tested, as shown in Figure 5-3B. A linear relationship between the enzyme concentration and the wavelength shift of Au-NCs suspensions is observed when the concentration of enzyme is less than 100 units/m L. However, the wavelength shift reaches the maximum when the concentration of -galactosidase is aroun d 140 units/mL. If the concentration of -galactosidase is further increased, th e absorption shift begins to drop. One possible reason is that the excess -galactosidase in the solution can attach to the surface of AuNCs and facilitate the solubility of Au-NCs in the aqueous solution.[12] In summary, we have dem onstrated a new design of -galactosidase assay based on AuNCs aggregation. The enzyme was detected by the red-shifted absorbance of the SPR and the decrease of the absorbance of Au-NP solutions. The sensitivity of the Au-NP based assay was tuned by varying the ratios of dual ligands, LT-gal and LT. The design is useful not only for the

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115 detection of -galactosidase, but also for the detection of other enzymes via rational design of the ligands on Au-NCs. For example, a phosphate gr oup functionalized ligand can be used for the detection of phosphatase.

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116 Figure 5-1. -galactosidase assay based on Au-NCs .A) The molecular structure of LT-gal. Galactopyranosyl ring is the hydrophilic part in LT-gal. After interacting with galactosidase, the galactopyra nosyl ring is cleaved from LT-gal, which induces the Au-NCs to aggregate. B) The color of Au-NP aqueous solution changes from red to purple due to the aggregation of Au-NCs. Figure 5-2. Detection of -galactosidase. A) UV/Vis spectra of LT-gal capped Au-NC solutions at 0 min (black line), 15 min (re d line), 30 min (green), 60 min (blue), 100 min (cyan), 120 min (purple) and 240 min (orange) af ter the addition of the galactosidase buffer solution; B) the red-shift of the peak () and the decrease of the absorbance () of Au-NP solutions. B A B A

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117 B A Figure 5-3. Kinetics studies of the emzyme-induced Au-NC a ggregation. A) Au-NCs were capped with LT-gal and LT at different molar ra tios. The spectra were measured every 15 min after the addition of Au-NCs su spension. B) The absorption shifts of Au-NC solution at different concentrations of -galactosidase, measured at 30 min after the addition of Au-NCs suspen sion. Au-NCs were capped with LT-gal and LT at the molar ratio of 3:4

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118 CHAPTER 6 SYNTHESIS OF HYBRID NANOCRYSTALS 6.1 Introduction Compared to single-component inorganic nanomaterials, hybr id nanocrystals (HNCs) are attractive candidates for advanced nanomaterials because they contain two or more different nanoscale functionalities. By tuning the compositi ons and tailoring the topological domain for each composition, these nanocomposites can exhibit novel physical and chemical properties that will be essential for future te chnological applications. For ex ample, the core/shell structure provides a protective shell on th e core material and significa ntly improves stability. The difference in surface chemistry of heterodime r-structured nanocrystals allows different functional molecules to attach to the heterodimer, which is very useful in biomedical applications. In general, the topologies of hybrid nanocryst als are controlled by both kinetic processes and thermodynamic factors during the synthesis.[167] It is believed that interfacial structures play an important role in the final crystallographic stru ctures of HNCs. Core/shell HNCs can be formed when the two components have simila r lattice constants and when the synthesis parameters allow the interfacial energy to be kept low. So far, core/shell structured HNCs have been prepared for various semiconductors (such as CdSe, CdS, CdTe, ZnS, ZnSe, InAs) in which the outer shell of a higher band gap material in creases the photostabil ity and enhances the photoluminescence (PL) quantum yield of the core[58,59,113,168]. When the materials possess limited miscibility or a large interfacial energy, heterodimer structured nanocrystals can be formed, in which the two materials are phase segreg ated into two separate particle domains, and the limited junction areas allow the minimization of interfacial energy. H eterodimer formation has been exploited to grow FePt/CdS[169], FePt/CdSe[170], FePt/Fe3P4 [167], Fe2O3/group II-IV sulfide,[171] Fe3O4/Ag [172] Fe3O4/Au[173], and CoPt3/Au[174] HNCs.

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119 The influence of the lattice mismatch on th e formation of HNCs has been explained by the coincidence site lattice (CSL) theory[175]. For specific orientations of crystal lattices, there are specific lattice points of one lattice which coincide with points of the other lattice.[176] Therefore, on a crystal substrate, with an exposal facet ha ving a certain crystallographic orientation, a second material with another type of lattice te nds to grow, and its rela tive orientation produces the best fit of the lattice points of the two structur es at the interfacial plane. This match occurs at regular intervals along two directions of the inte rfacial plane to define the two-dimensional cell that describes the interface. The better the fit be tween lattice points of th e two structures and the shorter the repetition intervals, the lower the interfacial energy will be.[171] So far, the synthesis parameters of he terostructured NCs have not been fully characterized out, and CSL theory cannot always explain the relationships between two domains of HNCs. The development of HNCs with contro lled structures and in terfacial interactions requires further studies of the growth of HNCs. In this chapter, two kinds of heterostructured dimers (FePt/In2O3 and UO2/In2O3) were synthesized using a seed-growth procedure and different topologies were observed. The interf acial structure was investigated carefully by HRTEM and an amorphous layer on the seed na nocrystals played an important role in the formation of the heterodimers. 6.2 Experimental Section 6.2.1 Chemicals Platinum (II) acetylacetonate (Pt(acac)2, 99%) and uranyl(VI) acetylacetonate (UAA, 99%) were purchased from STREM Ch emicals. Iron pentacarbonyl (Fe(CO)5, 98%), indium (III) acetate (99.99%), trimethylamine N-oxide (TMNO, 98%), 1-octade cene (ODE, 90%), oleic acid (OA, 90%), oleylamine (OAm, 70%), were purchased from Aldrich. All the other reagents were purchased from Fisher Scientific International Inc.

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1206.2.2 Synthesis of FePt Nanocrystals Modified literature procedures were used to prepare 6.7 nm FePt NCs. In a typical synthesis, Pt(acac)2 (100 mg 0.25 mmol) was mixed with 7.5 ml of ODE, and the mixture was degassed under vacuum at 60oC for 10 min. Then the mixture was heated to 130 C under Ar flow, resulting in a clear yellow soluti on, which indicated that the Pt(acac)2 salt was completely dissolved in ODE. Subsequently, Fe(CO)5 (95 mg, 0.5 mmol) dissolved in 785 mg (2.5 mmol) of OA, was swiftly injected into th e reaction mixture. After 5 min, 955 mg (2.5 mmol) of OAm was added to the reaction mixture. Th en, the reaction mixture was heat ed to 200 C at a rate of approximately 10 C/min, aged at this temperature for 1hr, and then cooled to room temperature. The NCs were precipitated from the reacti on mixture by adding acetone. The black NCs were further washed twice with a mixture of hexane and acetone (1:4) to remove excess surfactant and unreacted precursors. Finally they were fully diss olved in non-polar solvents, such as hexane, toluene or chloroform. 6.2.3 Synthesis of UO2 Nanocrystals The method described in section 3.2. 3 was used to prepare 6.2 nm UO2 nanocrystals. 6.2.4 Preparation of Indium Stock Solution Indium acetate (165 mg, 0.4 mmol), oleylamine (0.55 mL), and oleic acid (0.6 mL) were mixed with ODE (7 mL) in a th ree-neck flask. The mixture was degassed under vacuum at 110 C for 30 min to form a clear light-green solution. Trimethylamine N-oxide (TMNO, 161 mg, 1.45 mmol) was added to the vigorously stirred hot mixture under Ar. Then, the reaction mixture was further degassed at 120 C for 1hr. The result ant yellow indium stock solution, was stored at room temperature under Ar protection.

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1216.2.5 Synthesis of Corn-seed-shaped FePt/In2O3 Hybrid Nanocrystals. After heating at 200 C for 1 h, the reacti on mixture for the synthesis of FePt NCs (section 6.2.2) was further heated to 300 C at a rate of approximately 7 C/min under Ar flow. Indium stock solution was then injected into the FePt NCs solution. After heating at 300 oC for 30min, the mixture was cooled to room temp erature. The hybrid nanoc rystals (HNCs) were precipitated from the reaction solution by addi ng acetone and were furt her purified by adding a mixture of hexane and acetone (1:4). The black nanocrystal precipitate was easily redispersed in non-polar organic solvents such as hexane or toluene. A small aliquot of the FePt r eaction mixture was extracted via a syringe before injection of the indium stock solution at 300 oC. The aliquot was suddenly cooled, and the NCs were purified as described above. A control experiment was also performed by heating the FePt reaction mixture to 300oC for 30 min without injection of the indium stock solution. 6.2.6 Synthesis of Peanut-shaped UO2/In2O3 Hybrid Nanocrystals. After heating at 300 C for 5min, the reaction mixture of synthesis of UO2 NCs (section 6.2.3) was cooled to room temperature and mi xed with 8 ml of ODE. The mixture was then heated to 300 C at a rate of approximately 15 C/min under Ar flow. Indium stock solution was then injected into the UO2 NCs solution. After heating to 300 oC for 30min, the mixture was cooled to room temperature. Th e hybrid nanocrystals (HNCs) were precipitated from the reaction solution by adding acetone and further purif ied as described above (section 6.2.5). 6.2.7 TEM Measurements TEM measurements were performed on a JE OL 200CX operated at 200 kV. To prepare the specimens, a particle solution (10 L) was dropped onto a 200-mesh copper grid, and dried overnight at ambient conditions.

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1226.3 Results and Discussion 6.3.1 Synthesis and Characterization of FePt/In2O3 Hybrid Nanocrystals Cubic FePt nanocrystals with an average si ze of 6.7 nm (Figure 6-1A) were obtained after heating the reaction mixture at 200 oC for 1 hr. Following the formation of the FePt NCs, further heating of the reaction mixture to 300 oC induced the additional growth of thin ironlayers, which were further oxidized under air and converted to iron oxide layers. Figure 6-1B shows the core/shell-structured nanocrystals. Since the image contrast of Iron oxide is lower than that of FePt, the light thin shells come fr om Iron oxide layers and the dark cores are FePt nanocrystals. The shells of Iron oxide on the cores are not evenly dispersed, resulting in incomplete coverage of FePt cores. Figure 61C shows corn-seed-shaped hybrid nanocrystals, which were obtained after the in jection of indium stock solution and subsequent heating. Figure 6-1D illustrates the formation of the corn-seed-shaped dimer. In this system, the FePt-Iron oxide NCs act as seeds for the growth of indium oxide NCs and prevent the homogenous nucleation of indium oxide. Hetero geneous deposition of indium oxide on certain surfaces of FePt-Iron oxide NCs leads to the formation of corn-seed-shaped dimers. Since only about half of the FePt-Iron oxide NC surface is covered by In2O3, the HNCs have corn-seedshaped structures. According to the coincidence site lattice theory, the lattice mismatch at the heterointerface in these dimers should be small, but this was not observed in further experiments described below. To further characterize these hybrid nanoc rystals, scanning transmission electron microscopy (STEM) was performed, to obtain mo re structural and compositional information about these nanocrystals. The contrast in dark-f ield STEM (DF-STEM, Figure 6-2A) is strongly dependent on atomic number, whereas bright-fie ld STEM (BF-STEM, Figure 6-2B) is in some ways complementary to the dark-field contrast image. Thus, FePt cores show as bright areas in

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123 DF-STEM and dark areas in BF-S TEM. Conversely Iron oxide or In2O3 areas are dark in DFSTEM and bright in BF-STEM. However, the contrasts of Iron oxide or In2O3 are similar, so the domains of Iron oxide or In2O3 cannot be determined in these images. Since the composition of the large part of the dimer is not clear, there is a possibility that it could be Iron oxide To eliminate this possi bility, a control experiment was performed by heating the FePt reaction mixture to 300 oC for 30 min without injection of the indium precursors. The resultant nanocrystals were observed by TE M, as shown in Figure 6-3. The Iron oxide shells are thicker than those in Figure 6-1B due to the extra aging tim e, but they are significantly smaller than the dimers obtained by the injectio n of the indium stock solution (Figure 6-1C). Thus, the further growth achieved in Figure 6-2B is most likely caused by the heterogeneous growth of In2O3 on the surfaces of the FePt-Iron oxide NCs. High resolution TEM (HRTEM) studies provid ed more detail information about the crystal structures and orientations of these dimers. Figure 6-4 shows the HRTEM images of these individual dimers and different orienta tions of the FePt nanocrystals and the In2O3 nanocrystals are observed. The spacing of lattice fringes in Figure 6-4A are 1.95 and 2.70 corresponding to the {200} lattice planes for FePt and {123} lattice planes for In2O3, the lattice mismatch of respectively. The mismatch associated with th is interface is about 28%. Such a huge lattice mismatch has not been commonly obse rved in other types of dimers,[171,173]and are not predicted by the coincidence site lattice theory. These HR TEM images also clearl y show that there are amorphous areas between the interfaces of the FePt and In2O3 NCs, as marked by red arrows. (No amorphous area is observed in Figure 6-4D, because the FePt part is sitting on the surface of the In2O3 NC and the interface is hidden. Therefore, it can be inferred that the amorphous layers

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124 on the FePt nanocrystals acts as a glue and cause corn-seed-shaped dimers. Since there are no preferred heterointerface conf igurations, the growth of In2O3 can have different orientations. 6.3.2 Synthesis and Characterization of UO2/In2O3 Hybrid Nanocrystals As a comparison, another type of dimer was synthesized: UO2/In2O3 dimer. The synthesis of UO2 nanocrystals has been described in de tail in chapter 3. The synthesized UO2 nanocrystals are completely single-crystalline and stable in air, and have high contrast on TEM images. Thus, the UO2 nanocrystal is a good candidate for the study of dimer formation. As described in section 6.2.6, the procedure for making UO2/In2O3 dimers was the same as that for FePt/In2O3 dimers, but 6.2 nm UO2 NCs were used as seeds (Figure 6-5A). The injection of the indium stock solution also led to the formation of dimers. However, the shapes of the UO2/In2O3 dimers are different compared to the FePt/In2O3 dimers. The TEM image (Figure 6-5B) shows that UO2/In2O3 dimers are peanut-shaped. This shape was further confirmed by DF-STEM and BFSTEM (Figure 6-5C and D). In this system, UO2 show as bright areas in DF-STEM and dark areas in BF-STEM, whereas In2O3 is dark in DF-STEM and bright in BF-STEM. Figure 6-5E illustrates the formation of the peanut-shaped dimers. HRTEM measurement was also performed in the investigations of the UO2/In2O3 dimers. Only two types of heterointerf ace configurations were observed in this system. Figure 6-6A shows the most popular configuration. The fringe distances in Figure 6-5A are 2.73 and 2.53 corresponding to the {200} lat tice planes for FePt and th e {400} lattice planes for In2O3 respectively. The calculated lattice mismatch, asso ciated with this interface, is about 7%, which in the range of common dimers. In the other t ype of heterointerface c onfiguration, shown in Figure 6-6B, the lattice mismatch at the interface is also about 7 %. The images clearly demonstrate a high crystallinity across the entire heterostructure. In conclusion, two kinds of heterostructured dimers, FePt/In2O3 and UO2/In2O3, were synthesized using a similar procedure.

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125 The injection of the same indium stock solution into the different seed solutions led to dimers with different shapes. The structures of the interfaces in these two systems were carefully investigated by HRTEM. The corn-seed-shaped FePt/In2O3 dimers cannot be explained by lattice mismatch, as proposed by the CSL theory. The amorphous Iron oxide layers on the FePt seed surfaces are believed to act as glu e in the heterostructure. The UO2/In2O3 dimers were synthesized as a comparison. In this system no amorphous layers were observed on the UO2 seed surfaces. This result agrees with the CS L theory because of the small lattice mismatch (~7%). The resultant dimers have peanut-shaped structures and fewer interfacial areas compared to the corn-seed-heterodimers. Further studies ar e currently underway to gain a deeper insight into the mechanism of dimer formation.

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126 Figure 6-1. TEM images of A) FePt seeds, B) the nanocrystals extracted from the FePt reaction mixture before the injection of the indium stock solution. A thin layer of iron oxide was formed after the reaction mixture was heated to 300 oC; C) FePt/In2O3 hybrid nanocrystals, obtained from the reaction mixt ure after the injection of the indium stock solution and heating at 300 oC for 30 min. (d) Schematic illustration of the formation of the corn-seed-shaped dimer. Figure 6-2. STEM images of FePt/In2O3 hybrid nanocrystals A) dark-f ield STEM and B) brightfield STEM A B B C A D

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127 Figure 6-3. TEM image of the FePt-Iron oxide core-shell NCs obtained from the control experiment, in which the reaction mixture was heated to 300 oC and aged at that temperature for 30 min without the inject ion of the indium stock solution.

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128 Figure 6-4. HRTEM studies of an individual FePt/In2O3 hybrid nanocrystal. Red arrows indicate areas of amorphous Iron oxide layers. The scale bar is 5 nm. A B C D

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129 Figure 6-5. TEM images of A) 6.2 nm UO2 seeds, B) UO2/In2O3 hybrid nanocrystals; C) darkfield STEM image of UO2/In2O3 hybrid nanocrystals and D) bright-field STEM image of UO2/In2O3 hybrid nanocrystals. E) Schematic illustration of the formation of the peanut-shaped hybrid nanocrystals. A B C D E

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130 Figure 6-6. HRTEM images of an individual UO2/In2O3 hybrid nanocrystal The scale bar is 5 nm. A B

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131 CHAPTER 7 CONCLUSIONS 7.1 Summary of This Research A broad range of nanocrystals were synthesized a nd studied in this research. First of all, a one-pot synthesis for making high-quality mo nodisperse uranium-dioxide nanocrystals was developed. The size of UO2 NCs could be controlled by adjus ting the amount of ligands, and the shape of the particles was varied by tuning the precursor concentr ation per particle. Studies of the mechanism for controlling the nanocrystal fo rmation demonstrated that the amide, the condensation product of oleic aci d and oleylamine substantially affects the formation of UO2 nanocrystals. Since oleic acid and oleylamine are widely used in synthesizing a variety of highquality metal and metal-oxide nanocrystals, our results are very important for understanding the detailed mechanisms of these syntheses. Second, a new concept for converting hydr ophobic NCs (e.g., Au, Iron oxide and CdSe/ZnS quantum dots) into hydrophilic NCs through dual-interaction ligands was demonstrated. A series of dual-interaction lig ands, Tween derivatives (TDs), have been synthesized. The hydrophilic nanocrystals (Au and Fe3O4NCs and CdSe/ZnS core/shell QDs) functionalized with these TDs exhibit high stab ility in aqueous solutions. Moreover, the TDfunctionalized quantum dots were applied as fluorescence labels for monitoring the expression of a HCV (Hepatitis C virus) protein (NS5A) in FCA1 cells. Because of their excellent stability, these TD-functionalized nanoc rystals can play an important role in a variety of nanocrystal-based biomedical applications. Third, a gold nanocrystal-based assay to detect enzyme activity was designed taking advantage of the aggregation of the NCs induced by changes of ligands on the particle surface. A natural enzyme, -galactosidase was chosen as the model system. As the proof-of-concept

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132 experiment, a ligand functionalized with -D-galactopyranoside was synthesized. Upon hydrolysis of the ligands by -galactosidase, the gold NCs begin to aggregate, which induces a red-shift of the absorption maximum. Fourth, more complicated hybrid nanocrystals were synthesized. Hybrid nanocrystals are interesting because of the combin ation of two or more different materials in one nanocrystal in separated phases. FePt/In2O3 and UO2/In2O3 were prepared by a seed-growth method. The studies of the formation of FePt/In2O3 HNCs indicate that the amorphous iorn-oxide layer on the seed particle surface acts as glue to interconnect the two components, and controls the final topologies of heterostructured NCs. 7.2 Perspectives Nanocrystals have demonstrated many potential applications, especia lly in optoelectronic and biomedical areas. The development of na nocrystal synthesis allows preparation of monodisperse, shapeand size-controllable nano crystals. In addition to the challenge of synthesizing high-quality nanocrystals, surface engineering and the controlled assembly of nanocrystal are also very important for the development of nanocrystal-based devices. For possible future directions, some researches are suggested in two areas: synthesis and surface engineering. Synthesis. The development of new nanostructure d materials and the investigation of new properties of nanomaterials w ill still be hot research areas. Th e concern about the toxicity of nanocrystals, such as CdSe/ZnS QDs, require s exploration of the biological effects of nanocrystals and eventually the development of lo w and non-toxic materials. And of course, to provide enough materials for testi ng, methods to prepare high-quality nanocrystals in large scale are needed. Hybrid nanocrystals are a new generation of NCs, whic h are expected to have a wide

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133 range of applications in the future. Understanding of the crystal growth of these systems is crucial for future development. Surface engineering. New functionalization methods are needed to be make nanocrystals suitable for various applications. The challenges existing in nanoscience, such as how to improve the stability and the performan ce of nanocrystals, how to realize selective surface modification, how to conjugate with biol ogical molecules without losing their biological activities, and how to realize nanocrystal assemb lies to build nanomachines are all related to the surface engineering of nanocrystals.[177,178]

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134 APPENDIX A XRD MEASUREMENT OF URANIUMOXIDE NANOCRYSTALS Figure A-1. XRD measurem ent of UO2 Nanocrystals. A) The standa rd diffraction peak positions and relative intensities are indicated for the bulk and pure-phased face-centered cubic (fcc) UO2; (B) the XRD spectrum for synthesi zed NCs; (C) for orthorhombic U3O8 and (D) for primitive cubic UO3 The Bragg diffractions of the uranium oxide NCs can be indexed to nearly all of those of the standard bulk face-centered cubic UO2. These Bragg diffractions are quiet distinguishable from those Bragg diffractions of bulk UO3 or U3O8 structures. A B C D

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135 APPENDIX B 1H-NMR SPECTRUM Figure B-1. 1H-NMR of compound TD20-a Figure B-2. 1H-NMR of compound TD20-L

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136 Figure B-3. 1H-NMR of compound TD40-a Figure B-4. 1H-NMR of compound TD40-L

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137 Figure B-5. 1H-NMR of compound TD60-a Figure B-6. 1H-NMR of compound TD60-L

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138 Figure B-7. 1H-NMR of compound TD80-a Figure B-8. 1H-NMR of compound TD80-L

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139 Figure B-9. 1H-NMR of compound TD20-b Figure B-10. 1H-NMR of compound TD20-c

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140 Figure B-11. 1H-NMR of compound TD20-D Figure B-12. 1H-NMR of compound TD40-b

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141 Figure B-13. 1H-NMR of compound TD40-c Figure B-14. 1H-NMR of compound TD40-D

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142 Figure B-15. 1H-NMR of compound TD60-b Figure B-16. 1H-NMR of compound TD60-c

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143 Figure B-17. 1H-NMR of compound TD60-D Figure B-18. 1H-NMR of compound TD80-b

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144 Figure B-19. 1H-NMR of compound TD80-c Figure B-20. 1H-NMR of compound TD80-D

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145 Figure B-21. 1H-NMR of compound TD20-e Figure B-22. 1H-NMR of compound TD20-LC

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146 APPENDIX C MS SPECTRUM OF LT-GAL A B Figure C-1. MS spectrum of LT-gal. (A) The MS spectrum and (B) the moleucular weight of LT-gal. N H S S O O HO OH OH OH O ChemicalFormula:C22H33NO7S2ExactMass:487.17 MolecularWeight:487.63 m/z:487.17(100.0%),488.17(26.0%),489. 17(11.0%),489.18(2.9%),490.17(2.3%) ElementalAnalysis:C,54.19;H,6.82;N,2.87;O,22.97;S,13.15

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157 BIOGRAPHICAL SKETCH Hui meng Wu received her Bachelor of Science in Macromolecular Science and Engineering from Fudan University in 1999. Sh e worked under the direction of Prof. Yuliang Yang doing Living Free Radical Poly merization of Styrene. Then in 2003, she got her master degree in physical chemistry, from Institute of Chemistry, Chinese academy of Sciences, under the direction of Prof. Yanlin Song and Prof. Le i Jiang doing ultrahigh density data storage by scanning tunneling microscopy (STM). In August, 2004, she moved to Gainesvill e, Florida and began her Ph.D. study in chemistry department at the University of Florida. She spent four years of her graduate research working with Prof. Yunwei Cao to complete her Ph.D. study. Her resear ch area in Prof. Caos group includes the synthesis of high-quality NCs, surface modification of NCs and also exploring the potential bio-application of NCs.