Primarily concerned with manipulation and study of matter at the nanoscale, the concept of nanoscience encompasses ideas such as nanomaterial synthesis, characterization, and applications to modern scientific and societal problems. These problems encompass a broad range of issues such as energy storage and conversion, medical diagnostics and treatment, environmental remediation and detection, carbon economy and as well as many others. Silica nanoparticles of porous morphology have broad application to many of these issues. In particular, the utility of silica nanoparticles is facilitated by their large intrinsic surface area, tunable surface chemistry, and synthetic variability in both their size and morphology. This facilitates applications to these problems. However, extensive characterization and deeper understanding is needed before full implementation in key applications can be realized. The work described in this thesis aims to explore fundamental and applied characterization of silica nanoparticles that might be used in biomedical and environmental applications. Fundamental studies of functionalized nanomaterials using NMR spectroscopy reveal complex, dynamic phenomena related to-and ultimately deriving from-the intrinsic and/or modified surface chemistry. Applied studies of nanomaterial-biological interfaces demonstrate free radical chemistry as dominating the toxic response of the materials when exposed to biological systems of interest. Characterization of protein adsorbed on the interface reinforces the ubiquitous nature of protein adsorption on nanomaterial surface in biological and environmental media. Overall, this work illuminates and highlights complex changes that take place in aqueous solution for silica nanoparticles of varied morphology and surface chemistry.

Engineered nanoparticles provide potential opportunities for improving current drug delivery, bioimaging and biosensing modalities. In many cases, a ligand, such as a protein, peptide or nucleic acids, is attached to the nanoparticles surface to serve as a targeting group. However, the nanoconjugation (i.e. covalently bound molecules to a nanocarrier) is not an innocuous reaction. It can change the binding affinity and interfere with the intracellular trafficking of the tethered species. The understanding of this influence to the tethered species is still lacking. Therefore, the main objective of this thesis is to investigate the effect of nanoconjugation to the biological identity of the tethered biomolecules, in terms of cellular uptake, intracellular trafficking and the ultimate biological outcomes. The Epidermal Growth Factor Receptor (EGFR) is a tyrosine kinase that regulates cell proliferation and can cause cancer if dysregulated. Continuous treatment with high doses of EGF can induce apoptosis, in EGFR overexpressing cell lines. In this thesis, Epidermal Growth Factor (EGF) was chosen as the object of investigation. Covalent attachment of EGF to gold nanoparticles (NP-EGF) was found to enhance apoptosis in EGFR overexpressing cell lines (A431, MDA-MB-468) and it is sufficient to induce apoptosis in cell lines exhibiting EGFR expression at physiological levels (HeLa). NP-EGF accumulation through the endosomal pathway was also investigated to assess the impact of nanoconjugation on the spatio-temporal distribution of NP-EGF as potential origin for the observed enhancement of apoptosis. Two orthogonal experimental approaches were applied: (1) isolation of NP-EGF containing endosomes by taking advantage of the increased density of endosomes associated with the uptake of Au NPs; (2) correlated darkfield/fluorescence imaging to map the spatial distribution of NP-EGF in endosomes as a function of time. The studies reveal that nanoconjugation prolongs the dwelling time of phosphorylated receptors in the early endosomes and that the retention of activated EGFR in the early endosomes is accompanied by an EGF mediated apoptosis at effective concentrations that do not induce apoptosis in the case of the free EGF. Investigating the nanoconjugation-enhanced EGF-induced apoptosis improves the current understanding of cell-nanomatieral interactions and provides new opportunities for overcoming apoptosis evasion by cancer cells. / 2017-01-01T00:00:00Z

Nanoparticles are currently used in a wide range of applications including industrially processes, consumer products, and as drug delivery vehicles. The potential toxicity of these nanoparticles in living organisms is concerning due to their ever-expanding applications and accumulation in the environment. The effects of properties of the human body on the potential harmful nature of these nanoparticles must be understood in order to ensure safety in workplaces and at-home products. In this thesis, the interactions between nanoparticles and the most abundant blood protein, serum albumin, were investigated. The effects of changing the aqueous environment was investigated over a range of different pH values and with different ionic salts dissolved in water. The effects of changing the nanoparticle substrate were investigated to determine if different nanoparticles affect proteins differently. Finally, the effects of changing the concentration of nanoparticles and the presence of protein were investigated in a model lung cell line in vitro. The studies over different pH values revealed that serum albumin was able to adsorb to the silica nanoparticle surface, and retained its secondary structure both as a function of pH and adsorption in a 2-hour time frame. However, adsorption was greater on the titanium dioxide nanoparticle surface and the protein lost secondary structure at acidic pH (pH 2.0). Studies with different ionic salts revealed a possible correlation between BSA adsorption and nanoparticle aggregation in that the attractive interactions between nanoparticles were least when the least amount of protein was adsorbed. To the nanoparticle surface. In vitro studies with A549 human adenocarcinoma lung cells were inconclusive in determining the potential toxicity of these nanoparticles, but preliminary results suggested that the addition of protein to the system decreased toxicity compared with nanoparticles alone. This research aims to inform the field of nanotechnology to investigate the safety and efficacy of nanoparticles before they reach the consumer.

Glycation of whey proteins results in food-grade composites with modified physicochemical properties. Here, the reaction between glucose and bovine serum albumin (BSA) is promoted under wet-heating conditions. The glycated protein is characterized in depth and compared to the native counterpart and the impact of glycation on properties like net surface charge, particle size and surface hydrophobicity are observed. Conjugation with glucose reduced the surface hydrophobicity of BSA but the interactions between albumin and curcumin became stronger, which contradicts the direct relationship between curcumin binding affinity and protein surface hydrophobicity described in the literature. Nonetheless, curcumin was still capable of quenching the intrinsic fluorescence of the protein after conjugation with glucose and leads to the conclusion that curcumin and BSA interact in a different manner upon glycation. This thesis also depicts mucin as a forthcoming model in the study of nanoparticle interactions with intestinal mucus and glycation posed no effect on such interactions.

Owing to their unique physical, chemical, and mechanical properties, nanoparticles (NPs) have been used, or are being evaluated for use, in many fields (e.g., personal care and cosmetics, pharmaceutical, energy, electronics, food and textile). However, concerns regarding the environmental and biological implications of NPs are raised alongside the booming nanotechnology industry. Numerous studies on the biological effect of NPs have been done in the last decade, and many mechanisms have been proposed. In brief, mechanisms underlying the adverse biological effect caused by NPs can be summarized as: (i) indirect adverse effect induced by reactive oxygen species (ROS) generated by NPs, (ii) indirect adverse effect induced by released toxic ions, and (iii) adverse effect induced by direct interactions of NPs with biological systems. Up to now, most efforts have been focused on the first two mechanisms. In contrast, adverse biological effects induced by direct nano-bio interactions are the least researched. This is largely because of the complexity and lack of suitable techniques for characterizing the nano-bio interface. This dissertation aims at advancing our understanding of the nano-bio interactions leading to the adverse biological effect of NPs. Specifically, it is comprised of three parts. Firstly, because the aggregation of NPs alters particle size and other physicochemical properties of NPs, the property of NPs reaching and interacting with biological cells is very likely different from that of what we feed initially. Consequently, as the first step and an essential prerequisite for understanding the biological effect of NPs, NP aggregation is investigated and models are developed for predicting the stability and the extent of aggregation of NPs. Secondly, interactions between NPs and cell membrane are studied with paramecium as the model cell. Due to the lack of cell wall, the susceptible cell membrane of paramecium is directly exposed to NPs in the medium. The extent and strength of direct nano-cell membrane interaction is evaluated and quantified by calculating the interfacial force/interaction between NPs and cell membrane. A correlation is further established between the nano-cell membrane interaction and the lethal acute toxicity of NPs. We find NPs that have strong association or interaction with the cell membrane tend to induce strong lethal effects. Lastly, we demonstrate systematic experimental approaches based on atomic force microscope (AFM), which allows us to characterize nano-bio interfaces on the single NP and single-molecular level, coupled with modeling approaches to probe the nano-DNA interaction. Using quantum dots (QDs) as a model NP, we have examined, with the novel application of AFM, the NP-to-DNA binding characteristics including binding mechanism, binding kinetics, binding isotherm, and binding specificity. We have further assessed the binding affinity of NPs for DNA by calculating their interaction energy on the basis of the DLVO models. The modeling results of binding affinity are validated by the NP-to-DNA binding images acquired by AFM. The investigation of the relationship between the binding affinity of twelve NPs for DNA with their inhibition effects on DNA replication suggests that strong nano-DNA interactions result in strong adverse genetic effects of NPs. In summary, this dissertation has furthered our understanding of direct nano-bio interactions and their role in the biological effect of NPs. Furthermore, the models developed in this dissertation lay the basis for building an “ultimate” predictive model of biological effects of NPs that takes into account multiple mechanisms and their interactions, which would save a lot of testing costs and time in evaluating the risk of NPs.

In the following work, nanoparticle quantum dot (QD) fluorophores have been exploited to measure biologically relevant analytes via a miniaturized sensor ensemble to provide key diagnostic and prognostic information in a rapid, yet sensitive manner—data essential for effective treatment of many diseases including HIV/AIDS and cancer. At the heart of this “nano-bio-chip” (NBC) sensor is a modular chemical/cellular processing unit consisting of either a polycarbonate membrane filter for cell-based assays, or an agarose bead array for detection of biomarkers in serum or saliva. Two applications of the NBC sensor system are described herein, both exhibiting excellent correlation to reference methods ((R² above 0.94), with analysis times under 30 minutes and sample volumes below 50 [mu]L. First, the NBC sensor was employed for the sequestration and enumeration of T lymphocytes, cells specifically targeted by HIV, from whole blood samples. Several different conjugation methods linking QDs to recognition biomolecules were extensively characterized by biological and optical methods, with a thiol-linked secondary antibody labeling scheme yielding intense, specific signal. Using this technique, the photostability of QDs was exploited, as was the ability to simultaneously visualize different color QDs via a single light pathway, effectively reducing optical requirements by half. Further, T-cell counts were observed well below the 200/[mu]L discriminator between HIV and AIDS and across the common testing region, demonstrating the first reported example of cell counting via QDs in an enclosed, disposable device. Next, multiplexed bead-based detection of cancer protein biomarkers CEA, Her-2/Neu, and CA125 in serum and saliva was examined using a sandwich immunoassay with detecting antibodies covalently bound to QDs. This nano-based signal was amplified 30 times versus molecular fluorophores and cross talk in multiplexed experiments was less than 5%. In addition, molecular-level tuning of recognition elements (size, concentration) and agarose porosity resulted in NBC limits of detection two orders of magnitude lower than ELISA, values competitive with the most sensitive methods yet reported (0.021 ng/mL CEA). Taken together, these efforts serve to establish the valuable role of QDs in miniaturized diagnostic devices with potential for delivering biomedical information rapidly, reliably, and robustly. / text

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In this work, a general dynamic model was proposed to simulate the dynamic motion of microtubules driven by dynein motors, which is of importance to the design of potential nano-bio machines composed of dynein motors and microtubules. The model was developed based on Newton's law of motion. By incorporating a DPD technique, the general model was applied to simulate the unidirectional motion of microtubule. The functions of dyneins and their coordination with each other, which plays an important role in the motion of microtubules, were studied. By taking into account the bending energy of microtubules, we extended the general model to study possible mechanisms responsible for the microtubule-microtubule and microtubule-wall interactions, which are essential to the design of optimal track patterns for potential nanomachine systems. This study helps to evaluate the influence of bending and rotation on microtubule joining processes, involving bumping force, bending moment and torque generation. Finally, a phenomenal modeling study based on the Monte Carlo method, was conducted to investigate the self-organization of microtubules driven by dynein motors and identify out key parameters that control the self-organized movement of microtubules, giving crucial information for nano device design. This modeling study helps to clarify several important issues regarding the interaction between dynein motors and microtubules as a power transfer medium, which provides important information for the development of potential nanobio-machines using dynein as a biological motor.

Localized surface plasmon resonance (LSPR) describes the collective oscillation of conductive electrons in noble metal nanostructures, such as gold, silver and copper; or in selected doped semiconductor nanocrystals. Nanoplasmonics is emerging as a useful and versatile platform that combines the intense and highly tunable optical responses derived from LSPR with the intriguing materials properties at the nanoscale, including high specific surface areas, surface and chemical reactivity, binding affinity, and rigidity. LSPRs in plasmonic nanoparticles (NPs) can provide large optical cross-sections, and can lead to a wide variety of subsequent photophysical responses, such as localization of electric (E-)fields, production of plasmonic hot charge carriers, photothermal heating, etc. Plasmonic NPs can also be combined with other molecular or nanoscale systems into plasmonic heterostructures to further harvest the resonant E-field enhancement or to prolong the lifetime of plasmonic charge carriers. In this dissertation, we study the photophysical properties of plasmonic Ag and Au NPs, particularly E-field localization and hot charge carrier production performances; and illustrate how they can be optimized towards plasmonic photocatalysis, development of nano-antimicrobials, and surface-enhanced Raman spectroscopy (SERS) sensing. We demonstrate that with a lipid-coated noble metal nanoparticle (L-NP) model, the E-field localization properties could be optimized through positioning molecular photosensitizers or photocatalysts within a plasmonic “sweet spot”. This factor renders the plasmonic metal NPs efficient nanoantenna for resonant enhancement of the intramolecular transitions as well as the photocatalytic properties of the molecular photocatalysts. The enhanced photoreactivity have been applied to facilitate fuel cell half reactions for the enhancement of light energy conversion efficiencies; as well as to facilitate the release of broad-band bactericidal compounds that enables plasmonic nano-antimicrobials. Localized E-fields in L-NPs also enhance the inelastic scattering from molecules through SERS. This is utilized to obtain molecular-level information on the configuration of sterol-based, alkyne-containing Raman tags in hybrid lipid membranes, which enables spectroscopic sensing and targeted imaging of biomarker-overexpressing cancer cells at the single-cell level. In contrast to the localized E-field, plasmonic charge carrier generation mechanism relies on non-radiative decay pathways of the excited plasmons that lead to production of ballistic charge carriers. The plasmonic hot charge carriers directly participate in chemical redox processes with degrees of controllability over the nature of the charge carrier produced and direction of their transfers. The implementation and optimization of these mechanisms are explored, and the significances of some relevant applications are discussed.

Surface-enhanced Raman spectroscopy (SERS) is a powerful analytical technique that combines molecular specificity of vibrational fingerprints offered by Raman spectroscopy with single-molecule detection sensitivity from plasmonic hotspots of noble metal nanostructures. Label-free SERS has attracted tremendous interest in bioanalysis over the last two decades due to minimal sample preparation, non-invasive measurement without water background interference, and multiplexing capability from rich chemical information of narrow Raman bands. Nevertheless, significant challenges should be addressed to become a widely accepted technique in bio-related communities. In this dissertation, limitations from different aspects (performance, reliability, and analysis) are articulated with state-of-the-art, followed by how introduced works resolve them. For high SERS performance, SERS substrates consisting of vertically-stacked multiple metal-insulator-metal layers, named nanolaminate, were designed to simultaneously achieve high sensitivity and excellent uniformity, two previously deemed mutually exclusive properties. Two unique factors of nanolaminate SERS substrates were exploited for the improved reliability of label-free in situ classification using living cancer cells, including background refractive index (RI) insensitivity from 1.30 to 1.60, covering extracellular components, and 3D protruding nanostructures that can generate a tight nano-bio interface (e.g., hotspot-cell coupling). Discrete nanolamination by new nanofabrication additionally provides optical transparency, offering backside-excitation, thereby label-free glucose sensing on a skin-phantom model. Towards reliable quantitative SERS analysis, an electronic Raman scattering (ERS) calibration method was developed. ERS from metal is omnipresent in plasmonic constructs and experiences identical hotspot enhancements. Rigorous experimental results support that ERS can serve as internal standards for spatial and temporal calibration of SERS signals with significant potential for complex samples by overcoming intrinsic limitations of state-of-art Raman tags. ERS calibration was successfully applied to label-free living cell SERS datasets for classifying cancer subtypes and cellular drug responses. Furthermore, dual-recognition label-SERS with digital assay revealed improved accuracy in quantitative dopamine analysis. Artificial neural network-based advanced machine learning method was exploited to improve the interpretability of bioanalytical SERS for multiple living cell responses. Finally, this dissertation provides future perspectives with different aspects to design bio-interfaced SERS devices for clinical translation, followed by guidance for SERS to become a standard analytical method that can compete with or complement existing technologies. / Doctor of Philosophy / In photonics, metals were thought to be not very useful, except mirrors. However, at a length scale smaller than wavelength, it has been realized that metallic structures can provide unique ways of light manipulation. Maxwell's equations show that an interface between dielectric and metal can support surface plasmons, resulting in collective oscillations of electrons and light confinement. Surface-enhanced Raman spectroscopy (SERS) is a sensing technique that combines enhanced local fields arising from plasmon excitation with molecular fingerprint specificity of vibrational Raman spectroscopy. The million-fold enhancement of Raman signals at hotspots has driven an explosion of research, providing tons of publications over the last two decades with a broad spectrum of physical, chemical, and biological applications. Nevertheless, significant challenges should be addressed for SERS to become a widely accepted technique, especially in bio-related communities. In this dissertation, limitations from different aspects (performance, reliability, and analysis) are articulated with state-of-the-art, followed by how innovative strategies addressed them. Each chapter's unique approach consists of a combination of five aspects, including nanoplasmonics, nanofabrication, nano-bio interface, cancer biology, statistical machine learning. First, high-performance SERS substrates were designed to simultaneously achieve high sensitivity and excellent uniformity, two previously deemed mutually exclusive properties, by vertically stacking multiple metal-insulator-metal layers (i.e., nanolaminate). Their 3D protruding nanotopography and refractive-index-insensitive SERS response enabled label-free in situ classification of living cancer cells. Tweaked nanofabrication produced discrete nanolamination with optical transparency, enabling label-free glucose sensing on a skin phantom. Towards reliable quantitative SERS analysis, an electronic Raman scattering (ERS) calibration method was developed that can overcome the intrinsic limitations of Raman tags, and it was successfully applied to label-free living cell SERS datasets for classifying cancer subtypes and cellular drug responses. Furthermore, dual-recognition label-SERS with digital assay revealed improved accuracy in quantitative dopamine analysis. Advanced machine learning (artificial neural network) was exploited to improve the interpretability of SERS bioanalysis for multiple cellular drug responses. Finally, this dissertation provides future perspectives with different aspects, including SERS, biology, and statistics, for SERS to potentially become a standard analytical method that can compete with or complement existing technologies.