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Nanoparticle toxicity has become a major topic of interest due to the inevitable exposure of these nanomaterials to both humans and the environment. Nanotechnology is a rapidly growing industry with diverse material resources and an extensive market for commercialization and introduction of nanomaterials into consumer products. The problem with this flourishing technology is that it has far outgrown research based on the safety and toxicity of the nanomaterials, which in bulk are generally nontoxic. The need for research in determining the toxic effects on cells and the implications it may have on the environment have grown but the different techniques, cell systems and nanoparticles employed are generally to diverse and conflicting in overall results that determination toxicity is nearly impossible. The need for a universal technique to study the interaction of nanoparticles with cells and decouple the molecular effects (chemical properties) from the“nanospecific" effects (including size, concentration, surface charge, functionality and polarity) is apparent. It is additionally necessary to determine the mechanisms associated with nanoparticle-induced cytotoxicity in order to better understand the problems posed to both human and environmental health and then develop new safer nanoparticles. Therefore, the focus of this study is to determine the nano-specific (physical) properties, including size and functionalization that cause toxicity, specifically through interaction with a cell membrane. A supported lipid bilayer (SLB) composed of L-α-phosphatidylcholine (egg PC) was used as a model cell membrane to test the effects of 2, 5, 10 and 40 nm gold nanoparticles (AuNPs). Given the imminent exposure of nanoparticles to the environment it is important to determine how nanoparticles would behave in the presence of natural organic matter or polymers which are naturally present in environmental systems. Poly(methacrylic acid) (PMA) can be used to represent the polymers normally found in the environment. AuNPs were diluted in PMA in order to simulate fundamental environmental conditions. Analysis was done using a quartz crystal microbalance with dissipation (QCM-D), which measures the frequency (f) and dissipation (D) changes directly associated with mass and conformation changes of the SLB. Different overtones for f and D allow for theoretical interpretation of changes correlated to different layers of the membrane. The 2 and 5 nm particles were found to interact strongly with the lipid bilayer by adsorbing to and/or partially/completely penetrating into the lipid bilayer presumably due to a hydrophobic coating caused by PMA adsorption to the NP surface. The penetration caused a much more rigid membrane due to higher lipid packing caused by nanoparticle addition. The 10 and 40 nm particles interaction with the bilayer were not affected by the presence of PMA. Both AuNP sizes removed mass from the membrane with losses similar in de-ionized water and PMA solution. Removal of membrane mass (lipids/hydration) caused a more flexible membrane. It was determine that sized is the limiting factor for nanoparticle solubilization into the membrane. It can be concluded from the results that size coupled with natural organic matter affects the cytotoxicity of the nanoparticles to the membrane. A study was done with 12 nm functionalized AuNPs in the presence of humic acid, a well-known and more complex and realistic model for natural organic matter. A PC lipid bilayer was used to simulate a model cell membrane and QCM-D techniques were utilized in the determination of toxicity and mechanistic interaction of nanoparticles with a lipid bilayer. Functionalized AuNPs were shown to decrease the rigidity of the lipid bilayer by increasing the dissipation and decreasing the mass associated with the adsorbed film (SLB). The presence of humic acid stabilized the nanoparticles and provided increased electrostatic repulsion which resulted in decreased mass losses from the membrane and much smaller decreases in membrane rigidity. It was concluded that presence of humic acid reduces the effects of functionalized nanoparticle interaction with a lipid bilayer. These results may mean that natural organic matter has the ability to reduce the cytotoxic effects of nanoparticles released into the environment. Overall, the QCM-D was found to provide valuable information regarding the possible toxic properties and mechanisms in which different gold nanoparticle interact with a supported lipid bilayer under environmental conditions. The information provided by the studies performed has shed much light on the interaction of gold nanoparticles with a supported lipid bilayer in the presence of model natural organic matter. The experiments done in this study are the first steps towards developing an assay with the ability to determine the toxic physical properties and mechanisms by which nanoparticles interact with lipid bilayers will greatly aid in development of non-toxic nano-materials. The technology and techniques used in this study will greatly improve the field by solidifying one technique to use in the quantitative approach studying nanoparticle/cell interactions. The use of AFM techniques in conjunction with the QCM-D would be highly beneficial by facilitating better understanding of the exact mechanisms by which nanoparticles induce cytotoxicity.

Studies of liquid crystal (LC) /Au nanoparticle (NP) composites have been pursued in columnar and in nematic phases of thermotropic LCs. Using LCs forming a columnar phase, we found that different functionalities on the corona of the Au NPs (hydrophobic vs. hydrophilic) display unique effects on the stability and ordering of the columnar LC phase. Doping nematic LCs with non-chiral or chiral Au NPs causes the formation of textures commonly observed for chiral nematic LCs, i.e., the formation of somewhat uniform stripe textures or patterns separated by areas of homeotropic alignment of LC molecules. Two scenarios are proposed. In the first scenario, the Au NPs form topological chain-like defects and the remaining Au NPs reside at the interface inducing vertical alignment of the LC molecules. In the second scenario, chiral Au NPs transfer chirality to the nematic LC host. Further, induced circular dichroism studies proved the second scenario. Using the same chiral Au NP systems, the origin of chirality of Au NPs has also been studied, and a powerful methodology has been proposed to unravel the puzzle of chirality of chiral ligand-protected Au NPs. Further investigations of these texture phenomena led to the discovery of using metal NPs to control the orientation and alignment of LCs. In due course, a dual alignment and electro-optical switching behaviour was found using alkylthiol-capped Au NPs doped into a nematic LC with positive dielectric anisotropy in planar namatic LC cells. This study was also expanded to Ag and CdTe NPs, which showed the same phenomenon, and all investigated NPs significantly reduced the voltage needed to re-orient the LCs in an electric field (threshold voltage). Starting from basic and moving on to more application-oriented research, we finally also initiated structure-property relationship studies of LC/NP composites.

Studies of liquid crystal (LC) /Au nanoparticle (NP) composites have been pursued in columnar and in nematic phases of thermotropic LCs. Using LCs forming a columnar phase, we found that different functionalities on the corona of the Au NPs (hydrophobic vs. hydrophilic) display unique effects on the stability and ordering of the columnar LC phase. Doping nematic LCs with non-chiral or chiral Au NPs causes the formation of textures commonly observed for chiral nematic LCs, i.e., the formation of somewhat uniform stripe textures or patterns separated by areas of homeotropic alignment of LC molecules. Two scenarios are proposed. In the first scenario, the Au NPs form topological chain-like defects and the remaining Au NPs reside at the interface inducing vertical alignment of the LC molecules. In the second scenario, chiral Au NPs transfer chirality to the nematic LC host. Further, induced circular dichroism studies proved the second scenario. Using the same chiral Au NP systems, the origin of chirality of Au NPs has also been studied, and a powerful methodology has been proposed to unravel the puzzle of chirality of chiral ligand-protected Au NPs. Further investigations of these texture phenomena led to the discovery of using metal NPs to control the orientation and alignment of LCs. In due course, a dual alignment and electro-optical switching behaviour was found using alkylthiol-capped Au NPs doped into a nematic LC with positive dielectric anisotropy in planar namatic LC cells. This study was also expanded to Ag and CdTe NPs, which showed the same phenomenon, and all investigated NPs significantly reduced the voltage needed to re-orient the LCs in an electric field (threshold voltage). Starting from basic and moving on to more application-oriented research, we finally also initiated structure-property relationship studies of LC/NP composites.

Proteins and organothiols are known for their high binding affinity to noble metal surface including gold nanoparticles (AuNPs). Numerous reports have been dedicated to AuNP interaction with protein or organothiol alone. Competitive protein and organothiol (OT) interaction is, however, mostly an unexplored area. The research reported here focused on developing a fundamental understanding of sequential and simultaneous protein and organothiol interaction with AuNPs in which protein and OT are added either simultaneously or sequentially into the colloidal AuNP solutions. In studies of OT interactions with bovine serum albumin (BSA) stabilized AuNPs, we found that the protein coating layer is highly porous and permeable for small molecules such as mercaptobenzimidazole (MBI), cysteine (Cys), homocysteine (Hcy), and glutathione (GSH). Based on the amounts of MBI adsorbed and the kinetics of MBI adsorption onto BSA stabilized AuNPs, we were able to get an insight into protein conformational changes on the AuNPs. The competitive and sequential studies of protein and OT interactions with AuNPs involving eight model organothiols showed that the protein and OT cosorption onto AuNPs is a kinetically controlled process. The AuNP stability against ligandsorption-induced AuNP aggregation differed significantly among the AuNP/OT and AuNP/BSA/OT mixtures where the AuNP stability order increased from (AuNP/OT)/BSA to AuNP/(BSA/OT), and finally (AuNP/BSA)/OT samples (the two components inside the parenthesis are mixed first followed by the addition of the third component). The studies on the role of cysteine in protein-AuNP interactions found that the cysteine has no significant effect on the kinetics of protein adsorption onto AuNPs. However the stability of the protein-AuNP complex against the organothiolsorption induced AuNP aggregation increased as the number of cysteine residues increased from zero to two. Besides providing new insights on protein interaction with AuNPs, this research is important for AuNP biological/biomedical applications because AuNPs in biofuids encounter a mixture of proteins and OTs in addition to other molecular species.

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La synthèse de nanoparticules (NPs) d’or fonctionnalisées en phase aqueuse est encore aujourd’hui un enjeu majeur de la recherche dans le domaine des nanomatériaux. Depuis les travaux de J. Turkevich de 1951, la synthèse utilisant le citrate comme ligand et agent réducteur est la méthode de choix pour obtenir des NPs d'or. Cependant cette synthèse nécessite une étape supplémentaire de modification de surface par échange de ligand, pour pouvoir accrocher des molécules d’intérêt. Afin de simplifier la procédure, notre projet propose de synthétiser en une seule étape des NPs qui possèdent un groupement permettant une post-fonctionnalisation. La nouvelle voie de synthèse fait intervenir des composés bifonctionnels de la famille des 1-hydroxy-1,1-méthylène bisphosphonates (HMBP). Ainsi la base conjuguée de l'acide (1-hydroxy-1-phosphonopent-4-ènyl) phosphonique (HMBPène), qui possède une fonction éthylénique terminale nous a permis d'obtenir des dispersions de nanosphères de tailles contrôlées et nous avons pu rationaliser le mécanisme de synthèse utilisant ce type de molécules. Nous avons ensuite évalué plusieurs modalités de post-fonctionnalisation de notre nanoplateforme et validé une approche par chimie « Click » la via cycloaddition de composés tétrazine. En utilisant une nouvelle classe de HMBP couplés à une chaine polyéthylène glycol, des NPs stables en milieu physiologique ont pu être synthétisées selon le même modèle. Elles offrent également des possibilités de post-fonctionnalisation par couplage carbodiimide, que nous avons illustré par le couplage d'un fluorophore. Nous développons en dernière partie les résultats préliminaires sur deux types NPs d'or synthétisées à l'aide des HMBP pour des applications thérapeutiques. / In the ever growing fields of nanoscience the control of the synthesis of gold nanoparticles (GNPs) owing to their large variety of applications has emerged as an important domain. Among all methodologies Turkevich-Frens synthesis using citrates that act as ligand and reducing agent remains a method of choice for the obtaining of water soluble GNPs. Nevertheless, in post-synthesis, citrates are often exchanged with other ligands to enhanced stabilization and allow further functionalisation. In our work we present a new class of bi-functional molecules (1-hydroxy-1,1-methylene bisphosphonates HMBP) that can both reduce Au(III) and act as an efficient stabilizer of the formed GNPs in water. The first size controlled GNPs “one pot” synthesis was achieved by using an alkene conjugated HMBP, the (1-hydroxy-1-phosphonopent-4-enyl)phosphonic acid (HMBPene). We moreover, rationalized the mechanism of the GNPs synthesis using this type of molecule. We then, evaluated several methodologies for the post-functionalization of our nanoplateform and developed a « Click » chemistry approach to nanoparticle coating by tetrazine cycloaddition. Other nanoplatforms were synthesized using pegylated hydroxyl methylene bisphosphonates. This new class of bisphosphonate coated GNPs showed an improved stability in biological media and brought reactive groups available for post-functionalization as well, illustrated by the coupling of a fluorescent dye. The last part of this was dedicated to our latest results on GNPs synthesis for biomedical applications with HMBP compounds.

Electrolyte interactions with colloidal nanoparticles (NPs) in aqueous solutions have been implicated in a wide range of research and applications. Existing studies on electrolyte interactions with NPs are primarily based on the electrical double layer (EDL) theory. However, the EDL model provides very limited information on how electrolytes directly bind to NPs, electrolyte impact on charge distribution on NPs, and NP morphological modification upon electrolyte binding. Furthermore, the previous reports have mainly focused on either cations or anions binding onto NPs, while the potential cation and anion coadsorption onto NPs and NPacilitated cation-anion interactions remain largely uncharted. Filling these knowledge gaps are critical to enhance the fundamental understanding of interfacial interactions of electrolytes with NPs. Experimental characterization of cations and anions at the solid/liquid interface is a challenging analytical task. In the first study, we demonstrated the first direct experimental evidence of ion pairing on gold nanoparticles (AuNPs) in water by using surface enhanced Raman spectroscopy (SERS) in combination with electrolyte washing. Unlike ion pairing in aqueous solutions where the oppositely charged ions are either in direct contact or separated by a solvation shell, the ion pairing on AuNPs refers to cation and anion coadsorption onto the same NP surface regardless of separation distance. Ion pairing reduces the electrolyte threshold concentration in inducing AuNP aggregation and enhances the competitiveness of electrolyte over neutral molecules in binding to AuNPs. In the second study, we demonstrated that binding, structure, and properties of an ionic species on AuNPs are significantly dependent on the counterion adsorbed on AuNPs. These counterion effects include electrolyte-induced AuNP aggregation and fusion, quantitative cation and anion coadsorption on AuNPs, and SERS spectral distortion induced by the ionic species on AuNP surfaces. In the final study, we proposed that ion pairing as the main mechanism for reducing electrostatic repulsion among organothiolates self-assembled on AuNPs in water by using a series of experimental and computational studies. The work described in this dissertation provides a series of new insights into electrolyte interfacial interactions with AuNPs.

Gold nanoparticles (AuNPs) have been of interest due to their biocompatibility and surface plasmon resonance. Biomolecules can spontaneously adsorb to their surface, a trait that could be exploited for drug targeting. It is unclear, however, whether protein-AuNP interactions at the nanoparticle surface are dependent on nanoparticle size. In this project, we investigate whether surface curvature can induce protein unfolding and multilayer binding in citrate-coated AuNPs of various sizes. An NMR-based approach was utilized to determine the adsorption capacity, and protein NMR spectra were compared to determine whether nanoparticle size influences protein interactions. Transmission electron microscopy (TEM) was used to support the results. Over a range of AuNP sizes (15-100 nm) proteins appear globular on the nanoparticle surface. Additionally, a single layer of proteins is adsorbed regardless of AuNP size. Our results are consistent for two differently sized proteins, GB3 (6 kDa) and bovine carbonic anhydrase (BCA, 29 kDa).

This dissertation presents the development of an alternative aptamer screening process, Competition-Induced Selection of Ligands (CISL), and its use in screening for ssDNA aptamers for gold substrates. Gold substrates are presented as the nonnucleotide target for implementing CISL as a novel aptamer screening approach. Chapter 1 provides an overview of the in vitro selection of oligonucleotide aptamers, the polymerase chain reaction that is a key step in the aptamer screening process, the synthesis and properties of gold nanoparticles and the biomolecule-mediated formation of inorganic nanoparticles. Chapter 2 presents the goals and objectives of this thesis along with an organizational overview of the dissertation. Chapter 3 describes the experimental techniques and optimizations pertinent to the development of the CISL aptamer screening process. Chapter 4 investigates the effects of various nucleic acid additions during the seed-mediated growth of gold nanoparticles. Chapter 5 discusses the use of CISL in screening for ssDNA aptamer candidates for spherical gold nanoparticles (AuNPs) and the primary and secondary structure analysis of identified sequences. Chapter 6 presents the use of CISL in screening for ssDNA aptamer candidates for planar gold substrates (PlanarAu) and also includes primary and secondary structure analysis of identified sequences accompanied with an incubation study to provide a “frequency” ranking of aptamers as adsorbate species on PlanarAu. Chapter 7 offers concluding remarks and ideas for future expansion and applications of this work.