Stimuli responsive coatings offer a versatile method by which to manipulate interfacial interactions of proteins in a desired way. However, there exists little guidance as to how the structure of a responsive polymer coating influences adsorption of proteins. In this dissertation, the adsorption behavior of immuglobulin G (IgG) on poly (N-isopropylacryamide) (PNIPAAm) hydrogel coatings was investigated as a function of film thickness. PNIPAAm exhibits a hydrophilic to hydrophobic transition above a critical temperature of ~32°C in aqueous solutions. In this research, through the use of quartz crystal microbalance with dissipation (QCM-D) it was observed that the adsorption was thickness dependent and became non-reversible as the temperature was decreased. Interestingly, QCM-D results also suggested a similar amount of protein adsorption on both hydrated and dehydrated PNIPAAm surfaces. A rigid film analysis using Sauerbrey equation revealed a multi-layer formation on the collapsed PNIPAAm coatings. Although it is allegedly reported that PNIPAAm favors adsorption above the critical temperature due to hydrophobic interactions, there have been several studies that reported adsorption of proteins below the critical temperature. To better understand the QCM-D results, hydrodynamic shear force assays in a spinning disk configuration were performed in order to quickly measure and quantify adhesion of polystyrene (PS) probe spheres (10μm) to the PNIPAAm coatings in both the solvated (hydrophilic) and collapsed (hydrophobic) state. The influence of polymer coating thickness, polymer chain cross-link density, microsphere concentration and adsorption time on the adhesion characteristics of the coatings was investigated in relation with volume phase transition of the polymer coatings. A series of experiments on quantification of the temperature dependent adhesion of proteins adsorbed on surface attached PNIPAAm coatings of thicknesses was performed as the surface chemistry was switched from hydrophilic to hydrophobic. First, adhesion of polystyrene (PS) microspheres on PNIPAAm coatings was quantified in order to have a guideline for temperature dependent adhesion performance of these coatings. PS particles were subjected to a range of detachment shear stresses through hydrodynamic flow in a spinning disk configuration. These experiments provide an indirect method to determine the force of adhesion since it is proportional to the hydrodynamic force. Model protein, IgG, was then linked to PS microspheres and the mean adhesion strength of the IgG coated PS microspheres were determined through the detachment shear stresses. The influence of PS deposition time, PS bead concentration, PNIPAAm coating thickness and PNIPAAm cross-link density on the adhesion strength were addressed. The results indicated that in the collapsed state, the adhesion of bare hydrophobic PS microspheres depends strongly on coating thickness. For hydrophilic charged PS microspheres the adhesion was always higher on the hydrated PNIPAAm surfaces and appeared not to be strongly affected by the increase in PNIPAAm coating thickness. The adhesion of IgG was higher on the collapsed PNIPAAm surfaces and the adhesion trend did not significantly change as the PNIPAAm film thickness was increased. For PNIPAAm coatings with the cross-link density reduced by factor of 10, the adhesion was again higher on the collapsed PNIPAAm surface and scaled linearly with thickness. Moreover, the influence of thickness became prominent at the higher thickness values (165 nm-185 nm). In addition, the adhesion of carboxylated microspheres on PNIPAAm did not reach equilibrium and increased linearly with microsphere deposition time. A study on the sensing characteristics of PNIPAAm coatings in response to heavy metal ions was also conducted in this dissertation. The temperature-dependent swelling behavior of poly(N-isopropylacrylamide) and tripeptide Gly-Gly-His/poly(NIPAAm) conjugate hydrogel coatings were investigated using a quartz crystal microbalance with dissipation (QCM-D) while in contact with NaCl, ZnCl2, NiCl2, and CuCl2 solutions. To fabricate the tripeptide conjugated gels, precursor gels of poly(NIPAAm-co-3-aminopropylmethacrylamide[3.5 mole%]) were synthesized via free radical polymerization. The metal binding tripeptide, Gly-Gly-His, was subsequently synthesized in the gel via a Merrifield solid phase peptide synthesis (SPPS) technique, in which the amino group of the copolymer gel provided a functional site to support peptide synthesis. It was found that the logarithm of the transition temperature of the tripeptide Gly-Gly-His/poly(NIPAAm) conjugate hydrogel was proportional to the ionic strength, showing two distinct regions at low and high ionic strengths for the divalent ions. In the low ionic strength regime, the salting out constants were 0.08 M-1, 0.07 M-1, and 0.06 M-1 for Cu2+, Ni2+, and Zn2+, respectively, which follows the known trend for binding of the ions to Gly-Gly-His. In the high ionic strength region, when the metal-ion binding sites in the tripeptide conjugate hydrogel were saturated, the salting out constants were similar to the salting out constants associated with pure poly(NIPAAm).

Basic oxygen furnace (BOF) slag media were studied through a series of laboratory, modeling and field studies as a potential treatment material for use in on site wastewater disposal systems. Microsphere enumeration methodology was examined in a factorial experiment to evaluate the minimum density and minimum number of microspheres that should be counted to ensure accurate and precise estimations of concentration. The results suggest that to minimize variability at least 350 microspheres should be counted and a microsphere density of 25-40 microspheres field-1 is necessary. A review of existing methodologies for high-titer bacteriophage production was conducted and an amalgamation of existing methodologies was chosen that reliably achieves elevated concentration and ensures a purified suspension. A combination of batch and column studies was conducted to evaluate the removal of the bacteriophage, PRD-1, and virus-sized fluorescent microspheres by BOF media, and to delineate the relative contributions of the two principle attenuation processes, inactivation and attachment. In the batch studies, substantial removal of PRD-1 does not occur in the pH 7.6 and 9.5 suspensions, but at pH 11.4, removal of the virus was 2.1 log C/C0 day-1 for the first two days, followed by 0.124 log C/C0 day-1 over the subsequent 10 days. Two column studies were conducted after 60 and 300 days of saturation with artificial groundwater at a flow rate of 1 pore volume day-1 using two BOF mixtures. After 300 days of column saturation, microsphere concentrations approached input levels, indicating a removal of 0.1-0.2 log C/C0 and suggesting attachment processes were negligible. PRD-1 removal was more pronounced (1.0-1.5 log C/C0). The reduction of PRD-1 is likely the result of a combination of virus inactivation at elevated pH (10.6-11.4), and attachment processes. Geochemical factors controlling microsphere attachment were compared between the two sets of experiments after 60 and 300 days of column saturation. Differences in attachment efficiency may be due to higher influent DOC concentration in the second experiment, conversion of amorphous iron phases to more crystalline forms over time, reductive dissolution of preferable attachment sites on iron phases, or precipitation of calcite. Hydrus-1D, a one-dimensional numerical model, was used to quantify transport processes, inactivation and attachment/detachment, occurring in the column experiments by model inversion. Fitted microsphere breakthrough closely matched observed data, whereas PRD-1 breakthrough with realistic parameter values does not closely match the peaked nature of the observed curves. The model achieved improved fits for microsphere and PRD-1 breakthrough when both strongly- and weakly-binding sites are represented. A unique set of parameter estimates could not be determined because of overparameterization of the inverse modeling for the experimental systems. An alternative latrine incorporating BOF slag media was constructed in a periurban community located near São Paulo, Brazil. Pathogen indicator removal is approximately 4-5 orders of magnitude in less than one meter of vertical transport through the BOF slag media. In a control latrine, constructed with similar hydraulic characteristics and inert materials, comparable reductions in pathogenic indicators were observed over three meters of vertical transport.

Basic oxygen furnace (BOF) slag media were studied through a series of laboratory, modeling and field studies as a potential treatment material for use in on site wastewater disposal systems. Microsphere enumeration methodology was examined in a factorial experiment to evaluate the minimum density and minimum number of microspheres that should be counted to ensure accurate and precise estimations of concentration. The results suggest that to minimize variability at least 350 microspheres should be counted and a microsphere density of 25-40 microspheres field-1 is necessary. A review of existing methodologies for high-titer bacteriophage production was conducted and an amalgamation of existing methodologies was chosen that reliably achieves elevated concentration and ensures a purified suspension. A combination of batch and column studies was conducted to evaluate the removal of the bacteriophage, PRD-1, and virus-sized fluorescent microspheres by BOF media, and to delineate the relative contributions of the two principle attenuation processes, inactivation and attachment. In the batch studies, substantial removal of PRD-1 does not occur in the pH 7.6 and 9.5 suspensions, but at pH 11.4, removal of the virus was 2.1 log C/C0 day-1 for the first two days, followed by 0.124 log C/C0 day-1 over the subsequent 10 days. Two column studies were conducted after 60 and 300 days of saturation with artificial groundwater at a flow rate of 1 pore volume day-1 using two BOF mixtures. After 300 days of column saturation, microsphere concentrations approached input levels, indicating a removal of 0.1-0.2 log C/C0 and suggesting attachment processes were negligible. PRD-1 removal was more pronounced (1.0-1.5 log C/C0). The reduction of PRD-1 is likely the result of a combination of virus inactivation at elevated pH (10.6-11.4), and attachment processes. Geochemical factors controlling microsphere attachment were compared between the two sets of experiments after 60 and 300 days of column saturation. Differences in attachment efficiency may be due to higher influent DOC concentration in the second experiment, conversion of amorphous iron phases to more crystalline forms over time, reductive dissolution of preferable attachment sites on iron phases, or precipitation of calcite. Hydrus-1D, a one-dimensional numerical model, was used to quantify transport processes, inactivation and attachment/detachment, occurring in the column experiments by model inversion. Fitted microsphere breakthrough closely matched observed data, whereas PRD-1 breakthrough with realistic parameter values does not closely match the peaked nature of the observed curves. The model achieved improved fits for microsphere and PRD-1 breakthrough when both strongly- and weakly-binding sites are represented. A unique set of parameter estimates could not be determined because of overparameterization of the inverse modeling for the experimental systems. An alternative latrine incorporating BOF slag media was constructed in a periurban community located near São Paulo, Brazil. Pathogen indicator removal is approximately 4-5 orders of magnitude in less than one meter of vertical transport through the BOF slag media. In a control latrine, constructed with similar hydraulic characteristics and inert materials, comparable reductions in pathogenic indicators were observed over three meters of vertical transport.

Embryonic stem cells (ESCs) hold great promise for treatment of degenerative disorders such as Parkinson's and Alzheimer's disease, diabetes, and cardiovascular disease. The ability of ESCs to differentiate to all somatic cell types suggests that they may serve as a robust cell source for production of differentiated cells for regenerative medicine and other cell-based therapeutics. In order for ESCs to be used effectively in clinical settings, efficient and reproducible differentiation to targeted cell types must be demonstrated. The overall objective of this project was to engineer microenvironmental control over differentiating ESCs through the formation of embryoid bodies (EBs) uniform in size and shape, and through the incorporation of morphogen-containing polymer microspheres within the interior of EBs. The central hypothesis was that morphogen delivery through incorporated polymer microspheres within a uniform population of EBs will induce controlled and uniform differentiation of ESCs. Rotary suspension culture was developed in order to efficiently produce uniform EBs in high yield. Compared to static suspension culture, rotary suspension significantly improved the production of differentiating cells and EBs over the course of 7 days, while simultaneously improving the homogeneity of EB size and shape compared to both hanging drop and static EBs. The diffusive transport properties of EBs formed via rotary suspension were investigated using a fluorescent, cell permeable dye to model the movement of small morphogenic molecules within EBs. Confocal microscopy, cryosections and EB dissociation all demonstrated that the dye was not able to fully penetrate EB, and that the larger EBs at later time points (7 days) retarded dye movement to a greater extent than earlier EBs (days 2 and 4). Polymer microspheres capable of encapsulating morphogenic factors were incorporated into EBs in order to overcome the diffusional limitations of traditional soluble delivery. The size of microspheres, microsphere coating, microsphere to cell ratio, and rotary mixing speed were all observed to influence incorporation within EBs. The use of microsphere-mediated delivery within EBs to direct cell differentiation was examined. Microsphere-mediated delivery of retinoic acid (RA) induced formation of uniquely cystic spheroids with a visceral endoderm layer enveloping a pseudo-stratified columnar epithelium, and with spatial localization of transcriptional profiles similar to the early primitive streak stage of mouse development. Continued differentiation of RA MS EBs in defined media conditions was assessed. Gene expression demonstrated that regular serum enhanced endoderm induction, serum-free media supported ectoderm differentiation, while mesoderm was most prominent in untreated EBs in full serum. In summary, this work has realized a unique approach for stem cell differentiation through modification of the internal microenvironment of ESC spheroids. This novel inside-out method toward engineering EBs demonstrated that the mode of morphogen delivery significantly affected the course of differentiation. These studies provide the basis for ongoing work, which will utilize the choice of microsphere material, coating, and morphogen in order to uniquely study mechanisms of ESC differentiation and achieve unparalleled engineering of the EB microenvironment.

Acrylic bone cements are used in dentistry and orthopedic surgery to fix prosthetic devices into the bone. Bone cements transfer and distribute the applied load and increase the load-carrying capacity of the prosthesis/cement/bone system with the help of mechanical bonding between the device and the bone. In spite of all their advantages, bone cements have several drawbacks such as insufficient mechanical properties, high exothermic polymerization temperature, release of monomer to the environmental tissue and loosening of implant. Studies are being carried out to improve bone cement formulations with low curing temperature, good mechanical properties and good biocompatibility. In this study, bone cements with different compositions were prepared by using poly(methyl methacrylate) (PMMA) microspheres, barium sulphate (BaSO4) radiopaque agent, inorganic hydroxyapatite (HA) particles and 1-dodecyl mercaptan (DDM) chain stopping agent. Mechanical and thermal properties of the prepared bone cements were examined. When 8% hydroxyapatite was added into the formulation, both tensile and compressive strengths were increased and curing temperature was decreased. Addition of 13% BaSO4 caused 0.98% and 10.29% decrease in tensile and compressive strength values, respectively. Addition of 1%, 2% and 3% DDM, relative to the amount of methyl methacrylate monomer, decreased the maximum temperature from 101.78&deg / C to 91.80&deg / C, 78.38&deg / C and 71.35&deg / C, respectively. All compositions of the prepared bone cements fulfilled the minimum compressive strength (70 MPa) requirement and the minimum curing temperature was obtained as 71.35&deg / C. In order to have optimum desired properties, further studies to improve biocompatibility, mechanical and thermal properties of bone cements are needed.

Infections with dengue viruses (DENV) constitute both a global problem as well as locally in Florida. DENV comprise four distinct serotypes of single-stranded RNA viruses and belong to the family Flaviviridae. DENV are among the most medically important arboviruses in the world and cases may currently exceed 400 million per annum. Additionally, dengue established its first recorded endemic transmission cycle in the state of Florida in over a half century, first within the Florida Keys during 2009-10 followed by an unrelated outbreak in Martin County in 2013. The clinical profile of DENV infections ranges from a mild febrile illness to severe illness including hemorrhaging and/or shock, occasionally leading to death. Asymptomatic and mild cases also play a role in maintaining transmission cycles. The early diagnosis and management of patients at the clinical level have both proven to be major obstacles in the control of DENV and are important at both the individual and community levels. Individually, the proper management of patients that will progress to severe illness demands that they are identified in order to receive supportive treatment and mitigate associated morbidity and mortality. At the community level, early diagnosis may reduce transmission by limiting the possibility of vector contact with viremic individuals. Early diagnosis is dependent on the detection of viral markers, while a number of host factors may inform prognosis. The microsphere-based immunoassay (MIA) is capable of detecting up to 100 different targets in a single sample and therefore would be useful as a single assay for determining both. This study attempted to develop a diagnostic and prognostic MIA using the DENV NS1 glycoprotein and 5 host markers as targets. For the purposes of DENV NS1 detection in MIA, a set of monoclonal antibodies (mAbs) were subjected to immunoprecipitation and/or Western blot analysis in order to determine immunoreactivity. Two mAbs, 3A5.4 and 3D1.4, were chosen for use in MIA as a capture antibody and a detection antibody, respectively, and the results compared to a commercially available DENV NS1 ELISA. The 5 markers chosen for MIA trials included GM-CSF, IFN-γ, IP-10, IL-10, and MCP-1 and were selected from a panel of 27 markers screened initially in two in vitro models of DENV infection in addition to serum samples. The two cell lines investigated were HPMEC ST1.6R and u937 as both are thought to play important roles in models of DENV pathogenesis. The results of the DENV NS1 detection MIA were initially promising but were ultimately unsuccessful. When measuring host markers in the MIA, results pointed towards certain profiles that may be of future use. IL-10 was found to be elevated in a statistically significant manner in DENV qRT-PCR+ samples (p=0.035) when compared to negative sera. MCP-1 elevation was found to be of borderline significance (p=0.058). Other potential markers based on the results reported here include IP-10, IL-6, IL-8, VEGF, and RANTES. The ultimate goal of measuring host markers is to gain the ability to differentiate patients that will progress to severe illness from those that will recover. In conclusion, despite the failure of the MIA to detect DENV NS1 in human sera, our results in determining host markers and developments leading to successful DENV NS1 detection ELISAs elsewhere lead us to believe that this approach remains promising. Major drawbacks of this study included the lack of samples from patients that experienced severe DENV illness as a comparative group in addition to small sample sizes. Future goals should include determining the reasons for the failure of the MIA in detecting DENV NS1, selecting a panel of appropriate markers to differentiate non-severe from severe cases of DENV prior to progression, and optimizing the inclusion of these markers to an appropriate number.

Standard optical microscopes cannot resolve images below 200 nm within the visible wavelengths due to optical diffraction limit. This Thesis reports an investigation into super-resolution imaging beyond the optical diffraction limit by microsphere optical nano-scopy (MONS) and submerged microsphere optical nano-scopy (SMON). The effect of microsphere size, material and the liquid type as well as light illumination conditions and focal plane positions on imaging resolution and magnification have been studied for imaging both biological (viruses and cells) and non-biological (Blu-ray disk patterns and nano-pores of anodised aluminium oxide) samples. In particular, sub-surface imaging of nano-structures (data-recorded Blu-ray) that cannot even be seen by a scanning electron microscope (SEM) has been demonstrated using the SMON technique. Adenoviruses of 75 nm in size have been observed with white light optical microscopy for the first time. High refractive index microsphere materials such as BaTiO3 (refractive index n = 1.9) and TiO2-BaO-ZnO (refractive index n = 2.2) were investigated for the first time for the imaging. The super-resolution imaging of sub-diffraction-limited objects is strongly influenced by the relationship between the far-field propagating wave and the near-field evanescent waves. The diffraction limit free evanescent waves are the key to achieving super-resolution imaging. This work shows that the MONS and SMON techniques can generate super-resolution through converting evanescent waves into propagating wave. The optical interactions with the microspheres were simulated using special software (DSIMie) and finite different in time domain numerical analysis software (CST Microwave Studio). The optical field structures are observed in the near-field of a microsphere. The photonic nanojets waist and the distance between single dielectric microsphere and maximum intensity position were calculated. The theoretical modelling was calculated for comparisons with experimental measurements in order to develop and discover super-resolution potential.

With the aim to develop and characterize a functionalized highly magnetic polymer carrier of micrometer size and of a narrow particle size distribution that will be suitable for biological application, hypercrosslinked microspheres were prepared. Simultaneously, the relation between structure and properties of product was observed. Condition of dispersion polymerization were optimized to obtain starting monodisperse poly(styrene-co-divinylbenzene) [P(St-DVB)] microspheres. The P(St-DVB) microspheres of different degree of crosslinking were prepared and effect of some polymerization parameters such as type of solvent, initiator, concentration and mode of DVB addition on morphology, size and particle size distribution were investigated. The starting microspheres were hypercrosslinked to obtain microporous inner structure. Hyperosslinked particles had very large specific surface area (> 1000 m2/g) and a high content of micropores (ca. 0.6 ml/g). First, P(St-DVB) microspheres were chloromethylated using three different chloromethylation agents to regulate their porous properties. Hypercrosslinking was achieved by the addition of stannic chloride as a catalyst and by increasing a temperature. The hypercrosslinked microspheres were then functionalized with sulfo- or aminogroups. The functional groups captured precipitated iron oxide inside the porous structure of the microspheres and also served as a reactive site for intended immobilization of the protein. A solution of ferrous and ferric chloride was imbibed under vacuum into the porous structure and the iron oxide was precipitated by an aqueous ammonia solution. Finally, the magnetic functionalized hypercrosslinked micropsheres were integrated into a biosensor for qualitative detection of ovalbumin.

After decades of development, optical fiber technology has reached a high degree of sophistication and maturity, and currently serves as the backbone of today''s internet. Despite its technical versatility and capability, current silica fiber technology still has a significant flaw: since silica fibers only possess very weak second order nonlinearity, it has been impossible to develop a large number of important nonlinear optical devices and instruments, such as optical parametric amplifiers (OPA) and optical parametric oscillators (OPO). In this thesis,we show how to overcome this intrinsic limitation, and introduce second order nonlinearity into silica fiber devices. / Master of Science

A major limitation of silica-based high-Q microcavities is the lack of functionalities such as gain, plasmonic resonance, and second-order nonlinearity. Silica possesses third order nonlinearity but cannot produce second order nonlinearity, plasmonic resonances, or fluorescence emission. The key to overcome this deficiency is to develop versatile methods that can functionalize the surface of a silica microsphere with appropriate nanomaterials. The goal of this thesis is to present and characterize an electrostatic self-assembly based approach that can incorporate a large number of functional materials onto the surface of a silica resonator with nanoscale control. We consider several types of functional materials: polar ionic self-assembled multilayer (ISAM) films that possess second order nonlinearities, Au nanoparticles (NPs) that support plasmonic resonances, and fluorescent materials such as CdSe/ZnS core/shell QDs. A major part of this thesis is to investigate the relationship between cavity Q factors and the amount of nanomaterials deposited onto the silica microspheres. In particular, we fabricate multiple functional microspheres with different ISAM film thickness and Au NPs density. We find that the Q factors of these microspheres are mainly limited by optical absorption in the case of the ISAM film, and a combination of optical absorption and scattering in the case of the Au NPs. By controlling the number of polymer layers or the NPs density, we can adjust the Q factors of these functional microspheres in the range of 106 to 107. An agreement between theoretical prediction and experimental data was obtained. The results may also be generalized to other functional materials including macromolecules, dyes, and non-spherical plasmonic NPs. We also study the adsorption of Au NPs onto spherical silica surface from quiescent particle suspensions. The surfaces consist of microspheres fabricated from optical fibers and were coated with a polycation, enabling irreversible nanosphere adsorption. Our results fit well with theory, which predicts that particle adsorption rates depend strongly on surface geometry. This is particularly important for plasmonic sensors and other devices fabricated by depositing NPs from suspensions onto surfaces with non-trivial geometries. We use two additional examples to illustrate the potential applications of this approach. First, we explored the possibility of achieving quasi-phase-matching (QPM) in a silica fiber taper coated with nonlinear polymers. Next, we carry out a preliminary investigation of lasing in a silica fiber coated with CdSe/ZnS core/shell quantum dots (QDs). / Ph. D.