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Preparation of Hydrophilic Nanocrystals Using Lipoic Acid Based Synthetic Ligands

Permanent Link: http://ufdc.ufl.edu/UFE0022745/00001

Material Information

Title: Preparation of Hydrophilic Nanocrystals Using Lipoic Acid Based Synthetic Ligands
Physical Description: 1 online resource (54 p.)
Language: english
Creator: Yang, Shuo
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Over the past several decades, nanocrystals have generated a tremendous amount of interest. They are employed in biological and medical studies for sensing, labeling, optical imaging, and drug delivery. High-quality colloidal nanocrystals are normally synthesized in organic phase with the presence of strongly coordinating hydrophobic organic ligands. However, using nanocrystals in biological system means that these nanomaterials have to be well dispersed and stable in aqueous solution. To meet the criteria, surface functionalization is needed to tailor the solubility and biocompatibility of nanocrystals and preserve their desired properties. To prepare water-soluble nanocrystals, there are generally two approaches. The first one is ligand exchange via coordinate bonding and the other one makes use of hydrophobic van der Waals interaction between amphiphilic polymers and original surface ligands. Considering both advantages and disadvantages of the above two approaches, a new class of dual-interaction ligand was developed by using Tween20 and lipoic acid. DHLA-Tween20 ligand modified nanocrystals are more stable than the other water-soluble nanoparticles functionalized with either ligand exchange or polymer encapsulation approach. By having both coordinate bonding and hydrophobic molecular interaction, this new ligand renders modified hydrophilic nanocrystals superior stability and keeps the particle size relatively small. This new surface modification approach can be used to nanocrystal with different compositions and will help develop more biomedical applications using nanomaterials. Another lipoic acid based ligand was synthesized for gold nanoparticle functionalization. It is expected to develop a platform for simple and rapid phosphatase detection by incorporating phosphate group into synthetic ligand structure. Although the stability is relatively low, the hydrophilic gold nanoparticles were obtained through surface modification using the phosphate group-containing ligand. Further work is needed to produce more stable water-soluble Au nanoparticles and test their efficiency in phosphatase detection assay.
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 Shuo Yang.
Thesis: Thesis (M.S.)--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: UFE0022745:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022745/00001

Material Information

Title: Preparation of Hydrophilic Nanocrystals Using Lipoic Acid Based Synthetic Ligands
Physical Description: 1 online resource (54 p.)
Language: english
Creator: Yang, Shuo
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Over the past several decades, nanocrystals have generated a tremendous amount of interest. They are employed in biological and medical studies for sensing, labeling, optical imaging, and drug delivery. High-quality colloidal nanocrystals are normally synthesized in organic phase with the presence of strongly coordinating hydrophobic organic ligands. However, using nanocrystals in biological system means that these nanomaterials have to be well dispersed and stable in aqueous solution. To meet the criteria, surface functionalization is needed to tailor the solubility and biocompatibility of nanocrystals and preserve their desired properties. To prepare water-soluble nanocrystals, there are generally two approaches. The first one is ligand exchange via coordinate bonding and the other one makes use of hydrophobic van der Waals interaction between amphiphilic polymers and original surface ligands. Considering both advantages and disadvantages of the above two approaches, a new class of dual-interaction ligand was developed by using Tween20 and lipoic acid. DHLA-Tween20 ligand modified nanocrystals are more stable than the other water-soluble nanoparticles functionalized with either ligand exchange or polymer encapsulation approach. By having both coordinate bonding and hydrophobic molecular interaction, this new ligand renders modified hydrophilic nanocrystals superior stability and keeps the particle size relatively small. This new surface modification approach can be used to nanocrystal with different compositions and will help develop more biomedical applications using nanomaterials. Another lipoic acid based ligand was synthesized for gold nanoparticle functionalization. It is expected to develop a platform for simple and rapid phosphatase detection by incorporating phosphate group into synthetic ligand structure. Although the stability is relatively low, the hydrophilic gold nanoparticles were obtained through surface modification using the phosphate group-containing ligand. Further work is needed to produce more stable water-soluble Au nanoparticles and test their efficiency in phosphatase detection assay.
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 Shuo Yang.
Thesis: Thesis (M.S.)--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: UFE0022745:00001


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1 PREPARATION OF HYDROPHILIC NANOCRYSTALS US ING LIPOIC ACID BASED SYNTHETIC LIGANDS By SHUO YANG A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2008

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2 2008 Shuo Yang

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3 To my wife and my parents

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4 ACKNOWLEDGMENTS First, I would like to thank m y wife and my parents. It is their unconditional love and supports that helped me get to where I am today. I want to thank my advisor, Dr. Y. Charles Cao, for his guidance and support. I appreciate all of his help and suggestions on my research an d my life. I also want to thank Huimeng Wu for all the help in the lab and Dr. Jiaqi Zhuang for the helpful discussions. Additionally, I want to say thank you to all the Cao group me mbers for their help and friendship. Finally, I would like to expre ss my appreciation to Dr. Ben Smith for helping me through my difficult times.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4LIST OF FIGURES.........................................................................................................................7ABSTRACT.....................................................................................................................................9 CHAP TER 1 PREPARATION OF HYDROP HILIC NANOCRYSTALS .................................................. 111.1Introduction................................................................................................................... 111.2General Methods of Nanocrystal Surface Modification............................................... 111.2.1Ligand Exchange Method.................................................................................121.2.2Polymer and Silica Encapsulation..................................................................... 151.3Biomedical Applications of Water-Soluble Nanocrystals............................................ 171.3.1Cellular Labeling............................................................................................... 171.3.2In vivo and Deep Tissue Imaging..................................................................... 182 PREPARATION OF HYDROPHILIC NA NOC RYSTALS USING DHLA-TWEEN20 LIGAND......................................................................................................................... ........192.1Introduction................................................................................................................... 192.2 Experimental Section.................................................................................................... 212.2.1Material.............................................................................................................212.2.2Instrumentation................................................................................................. 212.2.3Experimental Procedures.................................................................................. 222.2.3.1Synthesis of 1-dodecanethiol -capped gold nanocrystals....................222.2.3.2Synthesis of dihydrolipoic acid (DHLA) functionalized Tween20.... 232.2.3.3Preparation of water-soluble gol d nanoparticles and CdSe/ZnS quantum dots....................................................................................... 242.3 Results and Discussion.................................................................................................. 242.3.1Ligand Synthesis...............................................................................................242.3.2Preparation of Water-soluble Au Nanoparticles and CdSe/ZnS QDs............... 252.3.3Stability............................................................................................................. 292.4Conclusion....................................................................................................................333 PREPARATION OF HYDROPHILI C GOL D NANOPARTICLES USING PHOSPHATE BASED SYNTHETIC LIGAND.................................................................... 353.1Introduction................................................................................................................... 353.2Experimental Section.................................................................................................... 373.2.1Material and Instrumentation............................................................................373.2.2Experiments...................................................................................................... 37

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6 3.3Results and Discussion.................................................................................................. 403.4Conclusion....................................................................................................................464 SUMMARY AND FUTURE WORK.................................................................................... 474.1Summary.......................................................................................................................474.2Future Work..................................................................................................................48LIST OF REFERENCES...............................................................................................................50BIOGRAPHICAL SKETCH.........................................................................................................54

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7 LIST OF FIGURES Figure page 1-1 Typical experiment set-up of the injection-based m ethod................................................. 12 1-2 Quantum dot surface modification strategies.................................................................... 13 1-3 Schematic of a mercaptoacetic acid capped CdSe/ZnS QD coupled to a protein............. 14 1-4 Schematic of single-QD encapsulation in a phospholipids micelle................................... 15 1-5 Schematic illustration of amphiphilic polym er coated QD and chem ical structure of modified triblock copolymer used in QD surface coating................................................. 16 2-1 Structure of Tween20 (polyethylene gl ycol sorbitan m onolaurate) (w+x+y+z=20)......... 20 2-2 Structure of lipoic aci d and dihydrolipoic acid .................................................................. 21 2-3 1H-NMR of lipoic acid-Tween20 ester.............................................................................. 25 2-4 2D-NMR of lipoic acid-Tween20 ester.............................................................................26 2-5 1H-NMR of DHLA-Tween20 ester.................................................................................... 26 2-6 Relationship between the concentration of free ligand and the change of absorption curve. ..................................................................................................................................28 2-7 Centrifugal membrane filter unit u sed in nanocrystal purification.................................... 28 2-8 Difference in purification efficiency be tween dialysis and centrifugal filtration. ............. 29 2-9 TEM images of Au nanoparticles and CdSe/ZnS QDs......................................................29 2-10 TEM images of DHLA-Tween20 functiona lized Au nanoparticles and C dSe/ZnS QDs....................................................................................................................................30 2-11 Hydrodynamic diameters of functionalized Au nanoparticles and CdSe/ZnS QDs in water solution. ....................................................................................................................31 2-12 Thermal stability test of hydrophilic Au nanoparticles in boiling water. .......................... 31 2-13 Testing Au nanoparticle stab ility as a function of pH. ...................................................... 32 2-14 Au nanoparticle stab ility as a function of NaCl c oncentration.......................................... 33 2-15 Structure of PEGylated-DHLA..........................................................................................33 3-1 The structure of the new ligand......................................................................................... 36

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8 3-2 Gold nanoparticles before (right) an d after (left) surf ace functionalization ...................... 39 3-3 1H-NMR of lipoic acid-tyramine....................................................................................... 41 3-4 1H-NMR spectra of unidentified products in L-T-P synthesis attempt..............................42 3-5 1H-NMR and 2D-NMR of L-T-P....................................................................................... 43 3-6 P31-NMR of L-T-P.............................................................................................................44 3-7 P31-NMR of L-T-POH....................................................................................................... 44 3-8 1H-NMR of L-T-POH........................................................................................................ 45 3-9 UV-Vis. absorption of AuNPs before (in ch loroform) and after (in water) surface functionalization................................................................................................................45

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9 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science PREPARATION OF HYDROPHILIC NANOCRYSTALS US ING LIPOIC ACID BASED SYNTHETIC LIGANDS By Shuo Yang August 2008 Chair: Y. Charles Cao Major: Chemistry Over the past several decades, nanocryst als have generated a tremendous amount of interest. They are employed in biological and medical studies for sens ing, labeling, optical imaging, and drug delivery. Highquality colloidal nanocrystals are normally synthesized in organic phase with the presen ce of strongly coordinating hydro phobic organic ligands. However, using nanocrystals in biological sy stem means that these nanomateri als have to be well dispersed and stable in aqueous solution. To meet the crite ria, surface functionalization is needed to tailor the solubility and biocompatibility of nanocrystals and preserve thei r desired properties. To prepare water-soluble nanocrystals, there are generally two approaches. The first one is ligand exchange via coordinate bonding and the other one makes use of hydrophobic van der Waals interaction between amphiphilic polymers and original surface ligands. Considering both advantages and disadvantages of the above tw o approaches, a new class of dual-interaction ligand was developed by using Tween20 and lipoic acid. DHLA-Tween20 ligand modified nanocrystals are more stable than the other watersoluble nanoparticles functionalized with either ligand exchange or polymer encapsulation appr oach. By having both coordinate bonding and hydrophobic molecular interaction, this new ligand renders modified hydrophilic nanocrystals superior stability and keeps the particle size re latively small. This new surface modification

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10 approach can be used to nanocry stal with different compositions and will help develop more biomedical applications using nanomaterials. Another lipoic acid based ligand was synthesi zed for gold nanoparticle functionalization. It is expected to develop a platform for simple and rapid phosphatase detection by incorporating phosphate group into synthetic ligand structure. Although the stability is relatively low, the hydrophilic gold nanoparticles were obtained through surface modi fication using the phosphate group-containing ligand. Further work is needed to produce more stable water-soluble Au nanoparticles and test their efficien cy in phosphatase detection assay.

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11 CHAPTER 1 PREPARATION OF HYDR OP HILIC NANOCRYSTALS 1.1 Introduction Over the past several decades, nanocryst als have generated a trem endous amount of interest. This has been motivated by a desire to reach a fundamental understanding of their unique size-dependent optical, electronic, magnetic and chemical propertie s and by the potential applications involving the use of these materials. The applicati ons range from electrical devices to biological and medical research.1-15 Nanocrystals are crystalline cl usters of a few hundred to a few thousand atoms with sizes of a few nanome ters. Although more co mplex than individual atoms, their properties are different from bulk crystals.16,17 Due to their small size, much of their chemical and physical properties are dominated by their surfaces and not by their bulk volume. Nanocrystals can be synthesized from metallic ma terials and semiconductor materials. Colloidal nanocrystals are the ones dispersed in a solvent and stabi lized in a way that prevents aggregation. Nowadays, colloidal nanocrystals are employe d in biological and medical studies for sensing, labeling, optical imaging, and drug deliv ery. High-quality colloi dal nanocrystals are normally synthesized in an organic phase with the presence of strongl y coordinating hydrophobic organic ligands, such as trio ctylphosphine (TOP) or trioctylphosphine oxide (TOPO).18-20 However, using nanocrystals in bi ological system means that thes e nanomaterials have to be well dispersed and stable in aqueous solution. To meet the criteria, surface functionalization is needed to tailor the solubility of nanocry stals in different solv ents and preserve thei r desired properties. 1.2 General Methods of Nanocry stal S urface Modification High-quality metal and semiconductor nanom aterials are often prepared from organometallic precursors with surface-capping organic ligand.1, 20-24 This capping ligand binds to the metal/semiconductor clusters, prevents aggr egation of the particles into bulk material, and

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12 controls the final dimensions of the nanopartic les. The commonly used organic ligands include trioctylphosphine (TOP), trio ctylphosphine oxide (TOPO), oleic acid, and long chain alkylamines. The typical experiment se tup of injection-base d synthetic method is shown in Figure 1-1. Figure 1-1. Typical experiment setup of the injection-based method. Since biological processes are typically situat ed in an aqueous environment, using these nanocrystals (quantum dots) in biological applicati ons must somehow transform the surface coatings into hydrophilic materials. Ligand exch ange process could be used to replace the original organic ligands with hydr ophilic capping molecules. Anot her approach is to wrap the nanocrystals in an amphiphilic polymer whose hy drophobic ends interact w ith, but not replace, the organic coating on a nanocry stal. Also, silica encapsulation can be employed to prepare water-soluble and biocompatible nanocrystals as well (Figure 1-2).25 1.2.1 Ligand Exchange Method Currently, high-quality nanocrystals are synthe sized by wet chem istry procedures in organic phase in most cases.Organic capping of nanocrystals with surfactants can provide electron passivation and form a ba rrier against aggregation of crys talities. As a consequence, the

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13 synthesized nanocrystals are hydrophobic. To obt ain water-soluble partic les, ligand exchange process was developed to replace the original surface coating molecules with hydrophilic ones. These bifunctional molecules are normally hydr ophilic on one end and bind to nanocrystal surface with the other end. Functional groups such as thiols, dithiols, phosphines, and hydroxyl groups on dopamine, are involved.15, 26-29 Figure 1-2. Quantum dot surface modification strategies. In 1998, water-soluble mercaptoacetic acid modified CdSe/ZnS core-shell QD was reported by Warren Chan and Shuming Nie.15 And these highly luminescent semiconductor quantum dots were covalently coupled to biomolecules via carboxylic acid functional group for use in ultrasensitive biological detection (Figure 1-3). In compar ison with organic dye rhodamine R6G, this class of luminescent labels is 20 times as bright, 100 times as stable against photobleaching and one-third as wide in spectral line widt h. Also, these QD bioconjugates exhibited specific recognition and good sensitivity in immunoassay. Later on, using ligand exchange method with various water-soluble bifunctional molecules to make hydrophilic and biocompatible nanocryst al became a very active area of research. Examples of some bi-functional ligand s used are mercaptocarbonic acids [HS-(CH2)n-COOH, n

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14 = 1 15],15, 23 dithiothreitol (DTT),30 lipoic acid,26 oligomeric phosphines,31 peptides,32 and crosslinked dendrons.33 The general procedure of ligand exchange includes reacting the hydrophilic molecules with nanocrystals in organic phase for cer tain period of time, drying or evaporation of organic solvent, dissolution in water, and purificat ion if necessary. Overall, the process is easy to handle and not time-consuming. The obtained nanoc rystals normally have good distribution in water and can readily conjugated to biological molecules for furthe r applications. However, there are certain drawbacks should be addressed. Ligand exchange inevitably alters the chemical and physical states of the nanocrystals surface at oms; thiol-based molecules (e.g. mercaptocarbonic acids) may form disulfides over time and come off from the surface and finally the nanocrystals aggregate and precip itate out of water; 23 the cross-linking of dendrons needs low nanoparticle concentration to avoid inter-particle reactions;33 most of the achieved water-soluble nanoparticles are not stable under acidic or basic conditions or in solution containing ce rtain concentration of salt, which is a common condition antic ipated in biological applications. Figure 1-3. Schematic of a mercaptoacetic acid capped CdSe/ZnS QD coupled to a protein.

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15 1.2.2 Polymer and Silica Encapsulation Other than changing the origin al surface coa ting molecules, chemists developed methods to encapsulate the nanocrystals with polymers or in silica shell. In 2002, Benoit Dubertret and co-workers reported phospholipids micelle encapsulated CdSe/ZnS quantum dots and its application as fluorescent label for in vivo imaging (Figure 1-4).34 They found that without any surface modifications, individual ZnS-overcoate d CdSe QDs could be encapsulated in the hydrophobic core of a micelle composed of a mixture of n-poly (ethylene glycol) phosphatidylethanolamine (PEG-PE) and phospha tidylcholine (PC). This micelle structure delivers colloidal stability in water and reduces non-specific binding in complex mixture due to the presence of a dense layer of PEG polymers on the outer surface. Figure 1-4. Schematic of single-QD en capsulation in a phospholipids micelleOther examples of polymer encapsulation can be found in the publications contributed by the research groups of S. Nie, and V. Colvin.3, 35 Nie group introduced th e using of long chain length amphiphilic polymer to coat the su rface of the nanocrystals (Figure 1-5).3 This strategy of using amphiphilic polymers is generally superior to the ligand exchange on some aspects. Since there is no direct interaction w ith the particle surface atoms, th e original optical properties of nanocrystals can be well preserved. And the polym ers large number of hydrophobic side chains strengthens the hydrophobic interaction to form stable wate r-soluble structures. Also, these amphiphilic polymers are generally commercially av ailable with low prices and can be modified to achieve desired biocompatibility. However, this method normally produces water-soluble

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16 nanocrystals with larger hydrodynamic diameters on the order of 30-40 nm which could be a potential problem for biological applications. Figure 1-5. Schematic illustration of amphiphilic polymer coated QD. A) Structure of a bioconjugated multifunctional QD probe. B) Chem ical structure of modified triblock copolymer used in QD surface coating. Compared to ligand exchange and polymer coating, silica encapsula tion will incorporate functional organosilicone molecules containing pr imary amine or thiol gr oup into the shell and provide surface functionalities for biomedical applications.14, 36-39 But with the crosslinked silica layer, the size of water-soluble na nocrystals prepared th is way ranges from tens of nanometers to several micrometers. And the procedure is relatively complicated and laborious. In essence, the preparation of water-so luble nanomaterials depends on two major approaches, coordinate bonding between partic le surface atoms and appropriate functional groups and hydrophobic van der Waals interactions between original orga nic capping molecules and the hydrophobic branches of coating polymers.

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17 1.3 Biomedical Applications of Water-Soluble Nanocrystals For the last decade, nan ocrystals, especially colloidal quantum dots, have been drawing great attention as a new class of powerful tool in biological a nd medical investigations. With their superior optical properties water-soluble quantum dots are competent for the applications in cellular labeling, imagi ng, sensing and screening. Before the emergence of quantum dots ba sed assays, organic dyes are the primary fluorescent label for cellular imaging and bioa ssay detection. However, the intrinsic optical properties of organic fluorophores, which genera lly have broad absorption/emission profiles and low photobleaching thresholds, have limited their applications in long-term imaging and multiplex detection.40 Compared with organic dye molecu les, colloidal quantum dots have continuous excitation spectra and narrow emission, readily tunable luminescence, and high photobleaching thresholds.12, 41-43 The water-soluble quantum dots will be conj ugated to biomolecules (DNAs, proteins) so that the resulting conjugates ca n combine both the spectroscopic ch aracteristics of nanostructures and the biomolecular function of the surface-atta ched entities. Conjugation process for adding biocompatibility to quantum dots can be divided into three categories, (i) Us e of EDC, 1-ethyl-3(3-dimethylaminopropyl) carbodiimide, condensati on to react carboxyl gr oups on the QD surface to amines; (ii) direct binding to the QD surface using thiolated peptides or polyhistidine (HIS) residues; and (iii) adsorption or noncovalent self-assembly using engineered proteins.44 1.3.1 Cellular Labeling Cellula r labeling is where QDs use has made th e most progress and attracted the greatest interest. In a typical assay, the bioconjugated QD s will be transported into cell with different strategies.45-47 They may include non-specific uptake by endocytosis; direct microinjection of nanolitre volumes; electroporation, which uses charge to physically deliver QDs through the

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18 membrane; and mediated/targeted uptake using encapsulates QDs within lipid vesicles to facilitate entry into the cell. It has been prove d that QDs can cross into cells and bind to their intended intracellular target s via specific recognition. Also, QDs can serve as useful markers for tracking cellular movement, differentiation and fa te. Recent findings explicitly showed that QDs are not just useful alternatives to organic fluorophores in cell labeling. Their intrinsic optical properties make them unique in investigation of co mplex cellular processes, especially with their multicolor labeling capability. 1.3.2 In vivo and Deep Tissue Imaging For im aging tissue with far red/near infrar ed excitation, quantum dots can be used to achieve deeper penetration in tissue compared to the available near-infrared dye molecules demonstrated by applying near-infrared em itting QDs (840 nm) to sentinel lymph-node mapping in cancer surgery of animals.73 Due to their large two-photon cross-sectional efficiency with a two-photon fluorescence process 100,000 that of organic dyes, QDs are suitable for in vivo deep-tissue imaging us ing two-photon excitation at low intensities48, 49. In addition, QDs have been demonstrated to remain fluorescent in tissues in vivo for up to four months.74 This property makes QDs excellent choice for tr acking and visualizi ng cancer cells during metastasis.3, 48 Conjugated with tumor-targeting anti bodies, quantum dots can track cells in vivo over a long period of time without continuously sacrificing anim als. Although QDs are certainly better choice than traditional dye molecules for these applications, without thorough toxicology studies, it is debatable that whet her the QD probe is providing true in vivo physiology information.

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19 CHAPTER 2 PREPARATION OF HYDROP HILIC NANOC RYSTALS USI NG DHLA-TWEEN20 LIGAND 2.1 Introduction As discusse d in last chapter, to date, most of the high-quality nanocrystals are synthesized in organic phase with the presence of various hydrophobic surface-capping ligands. These ligands help us keep the particles from aggr egation and control the final dimensions of synthesized nanocrystals. Consequently, the synt hetic nanocrystal does not possess the intrinsic solubility in aqueous solution. To prepare water-soluble nanocrystals, su rface modification is needed to deliver hydrophilicity. There are generally two approaches to modify part icle surface for this purpose. The first one is known as ligand exchange.15, 44, 50-52 A new organic ligand with hydrophilic group on one end and an anchor functional group on the other will be added to the nanocrystals organic solution. The anchor group, such as thiol or phosphine, ha s the ability to form coordinate bonding with the nanocrystal surface atom. Theref ore, the new ligand will compete with the original ligands and partially remove them from the particle surface. Then the nanocrystal will be water-soluble because of the hydrophilic group on the other end of the substitute ligand. However, water-soluble particles prepared by this approach usually suffer from low stability and make them undesirable in in vivo imaging and tracking. The other approach makes use of hydrophobic va n der Waals interaction between, in most cases, amphiphilic polymers and original surface ligands.35, 53-55 In aqueous solution, hydrophobic branches of polymer will interact wi th, but not remove, the original hydrophobic ligands on the surface. This interaction will en capsulate nanocrystal and form a shell-like structure to provide additiona l protection for the particle inside. The hydrophilic tails of amphiphilic polymer ligand make sure the modifi ed nanocrystal is water-soluble. Although the

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20 water-soluble nanocrystals made by this method ar e normally more stable than the ones went through ligand exchange process, the size of the part icles is rather too large for some biomedical applications. Considering both advantages and disadvantages of the above two approaches, we are going to develop a new kind of ligand which could pr oduce stable water-soluble nanocrystals while keeping a relatively small size. Lipoic aci d and Tween20 (polyethylene glycol sorbitan monolaurate) were chosen to synthesize the ne w ligand. Tween20, with 20 ethylene glycol units distributed among four branches (Figure 2-1), is often used in food industry as food additive because of its relative nontoxicity.56 Also, it is widely used in bioassays, such as ELISA (enzyme-linked immunosorbent assay) and western blotting, 57, 58 to minimize nonspecific binding. These features make Tween20 a good candida te of coating nanocrsytal for biological applications. However, the interaction betw een Tween20s fatty acid chain and hydrophobic nanocrystal surface is rather too weak to stabiliz e particle in water. The bidentate structure in dihydrolipoic acid, reduced from lipoic acid, (Figure 2-2) can provide two thiol functional groups that make simultaneous capping attachme nt to two surface site s on the nanocrystal, theoretically resulting in more stable ligand/pa rticle interactions. Therefore, we will modify Tween20 structure with lipoic acid in searchi ng for a new class of ligand has both hydrophobic molecualr interaction and coordina te bonding with nanocrystals surface. Figure 2-1. Structure of Tween20 (polyethylene glycol sorbita n monolaurate) (w+x+y+z=20)

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21 Figure 2-2. Structure of A) lipoic acid and B) dihydrolipoic acid 2.2 Experimental Section 2.2.1 Material Di methylaminopyridine (DMAP, 99%), N,N -diisopropyl carbodiimide (DIPC, 99%), polyethylene glycol sorbitan monolaurate (Tween-20), sodium borohydride (NaBH4, 97%), 1dodecanethiol (97%), 1-octadecene (ODE 90%), octadecylamine (ODA, 97%), ptoluenesulfonic acid monohydrate (98%), Rhodamine 6G (99%), and trioctylphosphine oxide (TOPO, 99%), were purchased from Sigma-Aldrich. Cadmium oxide (CdO, 99.998%), selenium (Se, 99.99%), dodecyl trimethylam monium bromide (DTAB, 97%) were purchased from Alfa Aesar. Nanopure water (18.2 M cm) was prepared by a Barnstead Nanopure Diamond system. All the other reagents and solvents were purchased from Fisher Scientific International Inc. All chemicals were used without further purifica tion. CdSe/ZnS core-shell quantum dots were synthesized and provided by Ou Chen and Dr. Yongan Yang. 2.2.2 Instrumentation NMR spectra were recorded using a Varian Mercury NMR Spectrom eter (300 MHz). The samples were prepared by adding aliquots of products into a deut erated solvent (CDCl3). Absorption spectra of aliquots were co llected by a Shimadzu UV-1700 UV-Visible Spectrophotometer. The wavelength and abso rption of each aliquot were recorded.

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22 Fluorescent spectra were measured using a Jobin Yvon Horiba Fluorolog-3 Model FL3-12 Spectrofluorometer. Room-temperature fluoresce nce QY of the CdSe/ZnS core/shell QDs was determined by using R6G as standard. The nanoparticle aqueous soluti ons were filtered through a 0.22m MCE syringe filter (Fisher Scientific) first. Th e hydrodynamic sizes of nanocrystals were obtained from dynamic light scattering (DLS) (Brookhaven Instrume nts Corporation, Ho ltsville, NY) at 25 oC. TEM measurements were performed on a JEOL 200CX operated at 200 kV. The specimens were prepared as fo llows: a particle solution (10 L) was dropped onto a 200-mesh copper grid, and dried overnight at ambient conditions. 2.2.3 Experimental Procedures 2.2.3.1 Synthesis of 1-dodecanethiol-capped gold nanocrystals Gold nanoparticles were synthesized according to the literature procedure.59 In a typical synthesis, AuCl3 (0.068 g) was dissolved in a DTAB so lution (0.185 g of DTAB in 20 ml of toluene) with ultrasonication to form a dark or ange solution. Then a fr eshly-prepared aqueous solution of NaBH4 (75 mol) was added dropwise to the solution with vigorous stirring. After 20 minutes, 1-dodecanethiol (1.6 ml) was added and the stirring was continued for 10 minutes. The nanoparticles were precipitated by adding ethanol, and the solid was re-dispersed in toluene (20 ml) in the presence of 1-dodecanethiol (1.6 ml ) and refluxed for 30 minutes under nitrogen. The nanocrystals were precipitated fr om the reaction solution with ethanol (30 ml), isolated by centrifugation and re-d ispersed in CHCl3. The resulting nanoparticles have a diameter of 6.6 nm with a standard deviation of 7.0 %

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23 2.2.3.2 Synthesis of dihydrolipoic acid (DHLA) functionaliz ed Tween20 4-(N,N-Dimethylamino)pyridinium-4-toluenes ulfonate (DPTS) was prepared by mixing THF solutions of DMAP (2 M, 50 ml) and p-tolu enesulfonic acid monohydr ate (2 M, 50 ml) at room temperature with stirring. The resulting precipitate was filtered and dried under vacuum. O HOw(C2H4O) (OC2H4)XOH H C CH2(OC2H4)zCOOC11H23 (OC2H4)yOH SS OH O O Ow(C2H4O) (OC2H4)XOH H C CH2(OC2H4)zCOOC11H23 (OC2H4)yOH S S O +DIPC/DPTS [5-(1,2-Dithiolan-3-yl)-1-oxopentyl]polyet hylene glycol sorbitan monolaurate. Tween20 (4.91 g, 4.0 mmol), lipoic acid (0.83 g, 4.0 mmo l), and DPTS (1.37 g, 4.4 mmol) were mixed in CH2Cl2 (30 ml) and stirred for 10 minutes at ro om temperature. Then, DIPC (0.63 ml, 4.4 mmol) was added to the mixture. After being stirred at room temperature overnight, the precipitation was filtered and the reaction mixture was washed with water (30 ml x 4). The organic phase was dried over a nhydrous magnesium sulfate (MgSO4), filtered and concentrated. The crude product was purified by column ch romatography on silica gel (eluents: ethyl acetate/hexane 9:1 and chloroform/methanol 8:2). Yield: 80%.

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24 (6,8-Dimercapto-1-oxoocty)polyethyle ne glycol sorbitan monolaurate. Product from last step (4.96 g, 3.5 mmol) was dissolved in a mixture 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 dilute d with water (50 ml) and 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: 81% 2.2.3.3 Preparation of water-soluble gold na noparticles and CdSe/ ZnS quantum dots Hydrophobic nanoparticles (Au and CdSe/ZnS ) (25 nmol) and DHLA-Tween20 ligand (10 mol) were mixed in CHCl3 (5 ml). The solution was stirred at room temperature for 15 minutes. Then triethylamine (0.05 ml) was added into th e mixture. The resulting mixture was further stirred for 30 minutes. Equal volume of water was added into the so lution. After vigorous shaking, the organic solven t was evaporated under reduced pre ssure to give a nanocrystal water solution. The nanocrystal solution was filtered through a 0.22m MCE syringe filter (Fisher Scientific). The excess of lipoic acid-Tween20 ligand was rem oved with centrifugal filters (Millipore, 10K NMWL, 10000g, 30 min) for three times. The resulting nanocrystals were redispersed in water. 2.3 Results and Discussion 2.3.1 Ligand Synthesis The ligand synthesis includes two steps. During the first step, lipoic acid group is added to Tween20 sturcture via a sim ple esterificat ion. 4-(N,N-Dimethylamino) pyridinium-4toluenesulfonate (DPTS) and N,N-diisopropyl car bodiimide (DIPC) were used to facilitate the reaction. Thin layer chromatography (TLC) is em ployed to monitor the re action. The molar ratio between DPTS/DIPC and reactants will substantiall y affect the reaction time and yield. After the purification by column chromatography, the pure product of lipoic ac id-Tween20 ester is

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25 obtained. The structure is confirmed by 1H-NMR (Figure 2-3) and 2D -NMR (Figure 2-4). Then the pure lipoic acid-Tween20 este r will be reduced by sodium borohydride to open the disulfide bond in lipoic acid. The crude product is simply purified by extraction with chloroform. Diluted HCl is added into separatory funnel to prompt the transition of reduced form from aqueous phase into chloroform. 1H-NMR is shown to confirm the stru cture of DHLA-Tween20 (Figure 2-5). Figure 2-3. 1H-NMR of lipoic acid-Tween20 ester. 2.3.2 Preparation of Water-soluble Au Nanoparticles and CdSe/ZnS QDs The ligand exchange step is carried out in ch loro form. Triethylamine is used to facilitate the ligand exchange process by increasing the basi city of the mixture. There are two different approaches to complete the phase transfer of modified nanocrystals. One can directly dry the mixture by removing the chloroform and a thin laye r of nanocrystal will be left on the bottom of the vial. The nanoparticles can be re-dispersed in to water to give an aqueous solution followed by purification step.

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26 Figure 2-4. 2D-NMR of lipoic acid-Tween20 ester. Figure 2-5. 1H-NMR of DHLA-Tween20 ester.

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27 On the contrary, in the other approach, wate r will be added into nanocrystal chloroform solution right after 45 minutes. Then an emulsion is made by intensively shaking the mixture. Vacuum evaporation (rotavap) will be used to remove chloroform from the mixture. Eventually, a clear nanocrystal water soluti on is collected. Although these two approaches seem quite similar to each other, the different phase transfer procedures could make a difference to the quality of resulting water-soluble nanocrystals. To distingu ish these two methods, th e quantum yields (QY) of CdSe/ZnS QDs, before and after the phase tran sfer using different pr ocedure, are measured using freshly made R6G ethanol solution as fluo rescence standard. The re sult suggests that after transferred from chloroform into water, wate r-soluble QDs made from rotavap-dry procedure will have 35% of QY decrease while QDs prepared with the other approach lose about 50%. Since the conformation of Tween20 in chloroform could be different than in water, the smooth transition of its conformation under rotary eva poration should result in a stronger hydrophobic interaction between the hydrophobic tail of Tween20 ligand and the remaining original ligands which will provide better protection for the inside QDs. And it could account for the difference in QY recovery between two approaches. The obtained nanocrystals water so lution needs to be further pu rified to remove excess free DHLA-Tween20 ligands. In searching of a be tter purification method, an interesting phenomenon was discovered. Because of the enhanced molecular ac tivity, the solution containing excess DHLA-Tween20 gets turbid when it is heated to certain temperature. At which temperature (Tt) the nanoparticle solution starts to show turbidity is related to the concentration of the excess ligand in soluti on as suggested by UV-Vis measur ement of the ligand solution (Figure 2-6). Although this measurement is not enough to quantitatively measure the amount of

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28 excess ligand in the solution, it provides a tool to evaluate the effectiv eness of the purification procedure. Figure 2-6. Relationship between the concentration of free ligand and the change of absorption curve. Dialysis and centrifugal ultrafilt ration are compared to select a more effective purification method. For centrifugal u ltrafiltration, membrane filter with MW CO of 10,000 is used. Watersoluble nanocrystals solution is loaded into ultr acentrifuge tubes with membrane filters (Figure 2-7). After 3 rounds centrifugation, the purified solution is collected. Compared to the nanocrystal solution after thre e day dialysis, centrifuged solu tion contains much less free Tween20 ligands. The result is shown in Figure 2-8. Figure 2-7. Centrifugal membrane filter unit used in nanocrystal purification.

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29 Figure 2-8. Difference in purific ation efficiency between dial ysis and centrifugal filtration. 2.3.3 Stability To investigate the function of the s ynthetic DHLA-Tween20 ligand, two types of nanocrystals, 6.6nm diameter gold nanoparticles and 5.6nm diameter CdSe/ZnS quantum dots, are selected. Transmission electron mi croscopy (TEM) images are shown in Figure 2-9. Figure 2-9. TEM images of A) Au nanoparticles and B) CdSe/ZnS QDs.

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30 The water-soluble nanocrystals are prepared and purified following the above mentioned procedures. The hydrophilic nanocrystals are taken to TEM again. The images suggest that the modified water-soluble nanocrystals have nearly identical sizes and shapes compared to their hydrophobic counterparts (Figure 2-10). Figure 2-10. TEM images of DHLA-Tween20 fu nctionalized A) Au nanoparticles and B) CdSe/ZnS QDs To further understand the st ructure of DHLA-Tween20 f unctionalized nanocrystal, hydrodynamic diameters of hydrophilic gold nanoparticles and CdSe/ZnS QDs are measured by dynamic light scattering (DLS). The results s how that the hydrodynamic diameters of these nanocrystals are 17.1 nm for the gold nanoparticle s and 15.9 nm for the CdSe/ZnS QDs (Figure 2-11). After the subtraction of particle sizes from their hydrodynamic sizes, the average ligand shell thickness can be calculated as 5.2nm which is comparable to the average length of DHLATween20 ligand (4.9nm). This resu lt shows that there is only one layer of Tween20 ligand attached on the surface of nanocrystals.

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31 Figure 2-11. Hydrodynamic diameters of functiona lized A) Au nanoparticles and B) CdSe/ZnS QDs in water solution. Stability of DHLA-Tween20 ligand functiona lized nanocrystals is tested by the measurement of absorption spectroscopy under differe nt pH, temperature, and salt concentration. For hydrophilic gold nanoparticles, th ere is no change can be identi fied on the absorption spectra of the particles heated in boiling wa ter for up to 4 hours (Figure 2-12). Figure 2-12. Thermal stability test of hydr ophilic Au nanoparticles in boiling water. When testing the stability as a function of pH, functionaliz ed gold particles showed good stability at least from pH 2-13 (Figure 2-13). In addi tion, the gold nanopartic les are stable in NaCl solution of concentration up to 5M (Fi gure 2-14). Furthermore, the fluorescence quantum

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32 yield of CdSe/ZnS in water solution is measure d. It appears that after surface modification QDs still can preserve over 60% of fluorescence emission compared to the untreated hydrophobic ones. These preliminary results show the great potential of th e DHLA-Tween20 ligands functionalized nanocrystals in biol ogical applications owing to th e superior stability over wide pH, temperature and salt concentration range. Compared to the PEGylatedDHLA ligands (Figure 2-15),60, 61 this DHLA-Tween20 ligand offers better stability under various conditions The difference could be attributed to the hydrophobic van der Waals interaction between origin al surface ligands a nd the fatty-acid chain in Tween20, which is missing from PEGylated DHLA ligand. This hypothesis is further proved by the work done by Huimeng Wu.62 Taken together with the resu lts above, it shows that, in surface modification process, DHLA-Tween20 li gand presents not only coordinate bonding between its thiol groups and particle surface at oms but also a hydrophobic molecular interaction which enhances the protection to the core nanocrystal. Figure 2-13. Testing Au nanoparticle stability as a function of pH.

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33 Figure 2-14. Au nanoparticle stability as a function of Na Cl concentration. Figure 2-15. Structure of PEGylated-DHLA. 2.4 Conclusion A dual-in teraction ligand is s ynthesized by simple modification of Tween20 using lipoic acid. With optimized phase transfer and purific ation technique, high qua lity water-soluble gold nanoparticles and CdSe/ZnS QDs are prepared. Their stability under di fferent conditions is tested. The results show that the DHLA-Tween20 ligand modified nanocrystals are more stable than the other water-soluble nanoparticles functiona lized with either ligand exchange or polymer encapsulation approach.3, 29, 35, 60, 61 By having both coordinate bonding and hydrophobic van der Waals interaction, this new liga nd renders modified hydrophilic na nocrystals superior stability and keeps the particle size small enough to be em ployed into biomedical applications. This new

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34 surface modification approach can be used to nanocrystal with different compositions and will facilitate using of nanomaterials in biol ogical and medical research in the future.

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35 CHAPTER 3 PREPARATION OF HYDROPHILIC GOLD NANOPARTI CLES USING PHOSPHATE BASED SYNTHETIC LIGAND 3.1 Introduction Noble m etal nanoparticles, especially gold, ha ve been extensively investigated over the past decade due to their unique electronic, optical and catalytic properties.63, 64 These properties are different from those of bulk metal or thos e of molecular compounds as has been widely demonstrated in both experimental and theoretic al investigations. Gold nanoparticles have a strong surface plasmon resonance in aqueous so lutions, which is attributed to collective oscillations of surface electrons induced by incoming visible light. This property strongly depends on the particle size, shape and interpar ticle distance as well as the nature of the protecting organic shell. During the last ten years, we witnessed the di scovery of numerous applications using gold nanoparticles, especially in biom edical research. Colloidal gold particles adsorbed to antibodies or to other targeting agents, such as proteins or peptides, are widely used as labels for the detection or localization of molecular and macromolecular targets in immunoassay.65 They can also be applied to colorimetric detection in DNA-hybridization assay as pioneered by Mirkin group in Northwestern University.66-68 The color changes from red to bluepurple accompanying changes in the aggregation behavi or have been used to detect and monitor the programming of assemblies of two and three dimensional architectu res and to detect and quantify hybridization of gold nanoparticle-immobilized oligonucleotides. No matter for wh at purpose we are using gold nanoparticles in nanoanalytics, th eir properties greatly rely on their surface functionalization. Different types of ligands and surface modification approaches have been developed to extend the application using colloidal gold particles. However, in most cases, the tailoring of gold nanoparticles for a given functionality always in volves the binding between two molecules, for

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36 example, antigenantibody, complimentary olig onucleotides, or guesthost complexes. To present biocompatibility, pre-synthesized gold na noparticles need to be modified with new ligands or polymers having functional groups that can crosslink to certain biomolecules later. The whole process can be complicated and challe nging. As an alternative, we are trying to develop a simple and straightforward method to prepare water-soluble gold nanoparticles for enzyme sensing, phosphatase in particular. Phosphorylation and dephosphorylation play si gnificant roles in cel lular regulation and signaling processes.69 A sensitive assay to report change of the phosphorylation state will be extremely valuable for biomedical applications. As the chemical difference in the process is only a phosphate group, a new ligand is designed (Figur e 3-1). The bidentate structure possessing two thiol functional groups can make simultaneous capping attachment to two surface sites on gold nanoparticle. With the attached phosphate group, gold nanoparticle functionalized with this ligand will be well dispersed in water. Howeve r, in the presence of phosphatase which can effectively remove the phosphate group from th e ligand, the gold nanoparticle will be no longer soluble in water because of the hydrophobicity of the remaining ligand. The color change and the aggregation could potentially be used to qualitatively and quan titatively detect and measuring enzyme activity. Figure 3-1. The structure of the new ligand. In this chapter, I will focus on introducing th e effort on searching an applicable approach of ligand synthesis and water-s oluble particle preparation.

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37 3.2 Experimental Section 3.2.1 Material and Instrumentation Di methylaminopyridine (DMAP, 99%), N,N -diisopropyl carbodiimide (DIPC, 99%), lipoic acid (99%), tyramine (99%), diet hyl phosphite (98%), bromotrimethylsilane (TMSBr, 97%), sodium borohydride (NaBH4, 97%), 1-dodecanethiol (97%), and p-toluenesulfonic acid monohydrate (98%), were purch ased from Sigma-Aldrich. Dodecyl trimethylammonium bromide (DTAB, 97%) was purchased from Alfa Aesar. Nanopure water (18.2 M cm) was prepared by a Barnstead Nanopure Diamond system. All the other reagents and solvents were purchased from Fisher Scientific International Inc. All chemicals were used without further purification. Absorption spectra of aliquots were co llected by a Shimadzu UV-1700 UV-Visible Spectrophotometer. NMR spectra were recorded using a Varian Mercury NMR Spectrometer (300 MHz). The nanoparticle aqueous solutions were filtered through a 0.22m MCE syringe filter (Fisher Scientific) first and further purifie d with centrifugal membrane filters (Millipore, 10K NMWL). 3.2.2 Experiments Gold nanoparticles are synthesized by followi ng the procedure discussed in Chapter 2. 4(N,N-Dim ethylamino)pyridinium-4-toluenesulf onate (DPTS) was prepared by mixing THF solutions of DMAP (2 M, 50 mL) and p-toluen esulfonic acid monohydrate (2 M, 50 mL) at room temperature with stirring. The resulting pr ecipitate was filtered and dried under vacuum. L-T: 5-(1,2-dithiolan-3-yl)-N-( 4-hydroxyphenethyl)pentanamide: Lipoic acid (3.09g, 15mmol), tyramine (2.06g, 15mmol) and DPTS (5.60g, 18mmol) were dissolved into 25 ml

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38 pyridine. Stirred for 20 min and dropwise adde d in DIPC (2.58ml, 18 mmol). Reaction mixture was stirred overnight at room temperature. Th e solvent was removed under reduced pressure. The crude product was purified by column chroma tography on silica gel (gradually change from ethyl acetate/hexane 3:7 to ethyl acetate/hexane 7:3). Yield: 85%. L-T-P: 4-(2-(5-(1,2-dithiolan-3-yl)penta namido)ethyl)phenyl diethyl phosphate: 0.60 g L-T (1.8mmol) was dissolved in CCl4/acetonitrile (10ml/5ml) with 0.50 ml diethyl phosphite (3.6mmol). The solution was cooled in ice bath. 0.50 ml triethylamine (about 3.6mmol) was added slowly into the solution with vigorous s tirring. Then the mixture was warmed to room temperature and stirred overnight. The solven t was removed and the crude was dissolved in chloroform. The solution was washed with water, diluted HCl (three times), diluted NaOH (three times) and brine, successively. The organic phase was collected and dried over anhydrous magnesium sulfate (MgSO4). After removing chloroform, the crude product was purified by column chromatography on silica gel (ethyl aceta te/hexane 6:4, ethyl a cetate/hexane 7:3 and ethyl acetate/methanol 98:2). Yield: 77%. L-T-POH: 4-(2-(5-(1,2-dithiolan-3-y l)pentanamido)ethyl)phenyl dihydrogen phosphate: 0. 60g (1.3mmol) L-T-POH was dissolved in 10ml dry dichloromethane. 0.50ml of TMSBr (about 3.9mmol) was dropwise added in with vigorous stirring. The reaction solution was stirred at room temperature for three hours. TLC was used to monitor the reaction. Then diethyl ether/water (4.5ml/0.5ml) was added i n. After stirring for another 10 min, product

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39 precipitated out from the reaction mixture. Decan ted and washed the crude product with diethyl ether three times. After dryi ng under reduced pressure, product was collected. Yield: 65%. DHLA-T-P: sodium 4-(2-(6,8-dimercaptooctanamido)ethyl)phenyl phosphate. 0.330 g L-T-POH (0.8mmol) was dissolved in 3m l of ethanol/water (4:1), 0.038 g NaBH4 (1mmol) was added in. The mixture was stirred under room temperature for 2 hours. Then the mixture was dried under reduced pressure. The residue was dissolved into wate r. This ligand-water solution was added into gold nanoparticle chloroform solution with 0.05 ml of trie thylamine. The mixture was stirred for 45 minutes under room temperature. Then chloroform was removed with rotavap and the resulting gold nanoparticle a queous solution was filtered through a 0.22m MCE syringe filter (Fisher Scie ntific). Three rounds of centrifugal filtration w ith centrifugal membrane filters (Millipore, 10K NMWL, 10000g, 30 min) were carried out. The resulting nanoparticles were re-dispersed in water (Figure 3-2). Figure 3-2. Gold nanoparticles before (right) and after (left) surf ace functionalization. Hydrophilic AuNP in water Chlorofor m Wate r Hydrophobic AuNP in chloroform

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40 3.3 Results and Discussion Phosphate-based ligands functionalized water-soluble gold nanoparticle is obtained with the following four-step synthesis route. First, a phenol group is added to lipoic acid w ith the formation of amide. Then a phosphate or phosphor amidite group is used to modify the structure followed by hydrolysis for making correspondin g phosphoric acid. The disulfide bond can be reduced by sodium borohydride in the final step so that the resulting product will have the ability to functionalize gold nanoparticle s with two free thiol groups. For the first step, originally N -hydroxysuccinimide (NHS) and ethyl (dimethylaminopropyl) carbodiimide (EDC) are used to activate the carboxylic acid group in lipoic acid so that the activated carboxylic acid is able to readily react with amine group in tyramine. However, breaking esterification in to two steps decreases the overall yield significantly and needs extra purific ation to collect desired product. To efficiently produce lipoic acid-tyramine in one step, it is cr itical to select an appropriate so lvent that can provide resonable solubility to both lipoic acid a nd tyramine. After the failed experiments usi ng dichloromethane, tetrahydrofuran (THF) and dimethylformamide (DMF), pyridine is c hosen as the reaction solvent. Although the purification procedure involves more steps, the product is obtained with high reaction yield. The stru cture is confirmed by 1H-NMR as shown in Figure 3-3. For preparation of L-T-P, different reagen ts and procedures are attempted. In one procedure, 2-cyanoethyl N,N -diisopropylchlorophosphoramidite and N,N -diisopropylethylamine (DIPEA) are used to synthesize lipoic acid-tyramine phosphoramidite derivative.70 The reaction is carried out in THF and expected to produce desired product with simple post-reaction workup. However, even with delicate colu mn separation the pure product is still out of reach determined by proton NMR spectra (data not shown).

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41 Figure 3-3. 1H-NMR of lipoic acid-tyramine. Then another more complicated synthe sis procedure was a dopted. It involves in situ silylation with tert-butylchlorodimethylsilane (TBDMSCl) followed by treatment of the silyl ester with carbon tetrachloride, diethyl phosphite and triethylamin e leading to the formation of diethyl phosphoate crude product.71 The purification process is l ong and difficult. During column separation, as monitored by TLC, three components ar e collected separately as potential product. However, neither of them can be confirmed by 1H-NMR (Figure 3-4). At last, an ideal reaction system is developed by using diethyl phosphite in carbon tetrachloride/ acetonitrile mixing solvent. In this triethylamine-catalyzed reac tion, acetonitrile offers good solubility to both reactants and carbon tetrachloride acts as solvent/reactant. With simple extraction and column separation, pure product is obtained in high yield and confirmed by 1H-NMR, 2D-NMR and P31NMR (Figure 3-5, 6).

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42 Figure 3-4. 1H-NMR spectra of unidentified produc ts in L-T-P synthesis attempt.

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43 Figure 3-5. 1H-NMR and 2D-NMR of L-T-P.

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44 Figure 3-6. P31-NMR of L-T-P. Bromotrimethylsilane is found to be able to quantitativel y convert the alkyl phosphates into the corresponding trimethylsilyl structures which are readily transformed into the phosphoric acids by hydrolysis with neutral water.72 By using this reagent, L-T-P is hydrolyzed to its corresponding phosphoric acid as confirmed by the only signal in P31-NMR (Figure 3-7) and the absence of ethyl groups protons in 1H-NMR (Figure 3-8). Figure 3-7. P31-NMR of L-T-POH.

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45 Figure 3-8. 1H-NMR of L-T-POH. The disulfide bond in resulting phosphoric acid is then reduced with sodium borohydride in 4:1 ethanol/water. Without further purificatio n, the mixture is used to functionalize gold nanoparticle following the same procedure discussed in Chapter 2. The modified hydrophilic gold nanoparticles do not exhibit si gnificant change in their UVVis absorption (Figure 3-9). 400 500 600 700 800 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 Abs.Wavelength (nm) AuNP in water AuNP in chloroform Figure 3-9. UV-Vis. absorption of AuNPs before (in chloroform) and after (in water) surface functionalization.

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46 However, these Au nanoparticles can not surviv e the treatment with 0.5M NaCl solution or changing pH value of the solution. The particles will eventually aggregate and precipitate. The poor stability in aqueous solution makes it di fficult to utilize these Au nanoparticles in phosphatase assay at this stage. To achieve mo re stable hydrophilic Au nanoparticles, it is necessary to optimize the conditi on of disulfide reduction step a nd separate pure ligand from the crude. However, it is very difficult to apply gene ral organic separation tec hnique such as column chromatography to this step. More work is need ed in the future to improve the quality of the water-soluble particles modified by th is method (please see chapter 4). 3.4 Conclusion The experim ents are partially successful as for the purpose of making phosphate based ligand functionalized hydrophilic gold nanopartic les. The modified gold nanoparticles show basically no change in their UVVis absorption in water. However, because of the imperfection of the final reduction step, the stability of the mo dified particles is relatively low which make it difficult to carry on the phosphatase assay at this stage. To obtain high quality water-soluble Au nanoparticles, it is necessary to separate the final product from the crude mixture and optimize the surface functionalization procedure.

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47 CHAPTER 4 SUMMARY AND FUTURE WORK 4.1 Summary It has been dem onstrated that, with design and synthesis of different organic ligands, hydrophobic nanocrystals can be transferred into hydrophilic ones and functi onalized for various applications, especially in bi ological and medical studies. The interaction between synthetic ligands and nanocrystal surface can be categorized into two majo r types: coordinate binding and hydrophobic molecular interaction. Ligands can be anchored on the surface of the particle by using one or both of these two t ypes of interaction. The new ligands are also expected to deliver desired biocompatibility to the nanocrystal. Normally, one cl ass of synthetic ligands is specifically related to one cla ss of biomolecules as determin ed by the nature of specific recognition in biological system such as DNA hybridization, antigen-antibody, peptides and proteins. To be employed in biomedical app lications, one or more of the following four properties of hydrophilic nanocrystals will be need ed: good solubility and distribution; sufficient chemical stability; appropriate particle size; and high quantum yield for QDs. It was proved that a dual-int eraction ligand can be synthesi zed by simply using lipoic acid and Tween20 polymer. With the optimized surf ace functionalization technique, the modified hydrophilic nanocrystals are well di spersed in water and stable under different pH, temperature and salt concentration. At the same time, these nanocrystals have a smaller hydrodynamic size compared to those of polymer encapsulated nano particles. This surface modification approach, using both coordinate bonding and hydrophobic molecular interacti on, will facilitate the application of nanocrystals in biological labeling, targeting, imaging and sensing. In the other part of my work, another lipoic acid based ligand was synthesized. Hydrophilic gold nanoparticle was obtained by th e surface functionalizati on using this phosphate

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48 ligand. However, the modified Au nanoparticle is not very stable in its aqueous solution. This may result from the imperfection of the last step in ligand synthesis. To be used in phosphatase detection, the synthesis and surface modification procedure need to be optimized. 4.2 Future Work Quantum dots have been exte nsively used in biological a pplications because of their unique properties. But they are limite d in lots of applications due to either low chemical stability or oversize dimension after su rface functionalization. Lipoic aci d-Tween ligands have been proved for their superior stab ility and good biocompatibility.62 Using this new class of ligands to modify quantum dots may produce more opportunities for their biological applications. One can functionalize magnetic/luminescent bifunctional nanocrystals with lipoic acid-Tween ligand and target them to particular in vivo sites, such as tumors, and make novel magnetic resonance imaging agents. Also they can be used for deep-tissue imaging which needs good permeability and long-term stability in biological environment. Another in teresting area involves QDs extensive multiplexing detection potential which ha snt been fully utilized. Using lipoic acidTween ligand to protect and functionalize QDs for a multiplex labeling system can be exciting and may alter the view of in vivo labeling and imaging. For using water-soluble gold nanoparticle for phosphatase sensing, mo re stable particles are needed. To get pure ligand in the last step, other separation techniques can be used, such as HPLC or immobilized reducing ag ent gel column. Tris(2 -carboxyethyl)phosphine (TCEP) serves as excellent agent for the reduction of disulfide bonds in proteins, peptid es and other disulfide bond-containing molecules and are relatively unreactive toward other functional groups.75, 76 Immobilized TCEP disulfide reduc ing gel column may be useful in reducing disulfide in the synthetic ligand without introdu cing any impurity. If more stab le hydrophilic gold nanoparticle

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49 can be obtained, it is promising that we will be able to develop an assay for simple and rapid phosphatase detection.

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54 BIOGRAPHICAL SKETCH Shuo Yang was born in Beijing, China in 1978. He sp ent the first seven years of his life in Shanxi province and m oved to Tianjin with his parents in 1985. After spending almost ten years in Tianjin, the family moved back to Beijing in 1995. He started his coll ege life at the Peking University Health Science Center and graduated with bachelors degree in science four years later. Before he came to the Department of Ch emistry in 2005, he rece ived another masters degree from Department of Medicinal Chemistry in College of Pharmacy at the University of Florida. He would like to con tinue his study and pursue a Ph.D. degree in the near future.