The objectives of this research were to identify the individual ionization constants (pKa values) of lipopeptide (daptomycin), evaluate the factors of pH, concentration, temperature, and calcium ions on daptomycin aggregation in aqueous solutions, and elucidate the effects of conformation and aggregation on ionization and the interaction mechanism between polyamidoamine (PAMAM) dendrimers and daptomycin. Daptomycin is a cyclic anionic lipopeptide antibiotic. It is composed of 13 amino acids with six ionizable groups, four side-chain carboxylic acids and two side-chain amine residues. The pKa values for individual daptomycin residues have not been elucidated. The sequence-specific pKa values for the four acidic residues and one aromatic amine (Kyn-13) in daptomycin were determined in the monomeric state by TOCSY 2D 1H NMR. From the NMR pH titration, the estimated pKa values for Asp-3, Asp-9, and mGlu-12 were determined to be 4.15, 3.85, and 4.55 in the absence of salt, and 4.07, 3.83, and 4.39 in the presence of 150 mM NaCl, respectively. The pKa value for Asp-7 is estimated to be ~1.01 in the absence of salt and 1.31 in the presence of salt. The estimated Hill coefficients for Asp-7 were 0.72 and 1.31 in the absence and presence of salt, respectively. The increase in Hill coefficients from 0.72 to 1.31 with increasing salt concentration is consistent with the estimated lower pKa in the absence of salt and suggests that a salt bridge is formed in solution possibly between Asp-7 acidic group and the neighboring Orn-6 basic group. The pKa value of the aromatic amine (Kyn-13) was confirmed using UV and fluorescence spectroscopic titrations. Aggregation behavior and critical aggregation concentration (CAC) values of daptomycin were evaluated in the different pH aqueous solutions by using the complementary analytical techniques, fluorescence, dynamic and static light scattering, and NMR spectroscopy. Based on fluorescence resonance energy transfer (FRET) from donor Trp-1 to acceptor Kyn-13, the CAC values were determined by an upward inflection of the intrinsic fluorescence emission from Kyn-13 at 460 nm as a function of increasing daptomycin concentration. The pH-dependent CAC values were determined to be 0.14 mM at pH 3.0, 0.12 mM 4.0, and 0.20 mM at pH 2.5 and 5.0. The CAC values obtained by fluorescence spectroscopy were confirmed by dynamic light scattering and NMR spectroscopy. The effects of temperature and calcium ion on daptomycin aggregation were also discussed. The interaction mechanism between daptomycin and PAMAM dendrimers generation 5 and 6 was studied using fluorescence spectroscopy. The shapes of binding isotherms daptomycin were quantitatively described by one- and two-site binding models to estimate binding capacity and dissociation constants. Both solvent pH values and PAMAM generation size were shown to affect the binding model and parameters. The interaction between daptomycin and PAMAM dendrimer was proposed wherein the ionized Asp-3 and Asp-9 residues of daptomycin interact with PAMAM cationic surface amine.

The study of the binding between estrogen receptors (ER) and their ligands in vitro has long been of interest mainly because of its application in anti-estrogen drug discovery for breast cancer treatment as well as in the screening of environmental contaminants for endocrine disruptors. Binding strength was conventionally quantified in terms of equilibrium dissociation constant (KD). Recently, emphasis is shifting towards kinetics rate constants, and off-rate (koff) in particular. This thesis reported a novel method to measure such binding kinetics based on fluorescence polarization complemented with total internal reflection fluorescence (FP-TIRF). It used tethered receptors in a flow cell format. For the first time, the kinetics rate constants of the binding of full-length human recombinant ERα with its standard ligands were measured. koff was found to range from 1.3 10-3 to 2.3 10-3 s-1. kon ranged from 0.3 105 to 11 105 M-1 s-1. The method could also be used to screen potential ligands. Motivated by recent findings that ginsenosides might be functional ligands of nuclear receptors, eleven ginsenosides were scanned for binding with ER and peroxisome proliferator-activated receptor gamma (PPAR). None of the ginsenosides showed significant binding to ER, but Rb1 and 20(S)-Rg3 exhibited significant specific binding with PPAR.

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Polycyclic aromatic hydrocarbons (PAH) are important environmental pollutants generally formed during incomplete combustion of organic matter containing carbon and hydrogen. Introduced into the human body by adsorption through the skin, ingestion or inhalation, the biotransformation processes of PAH lead to the formation of multiple metabolites. Due to the short elimination lifetime from the body, the quantitative determination of monohydroxy-PAH (OH-PAH) in urine samples provides accurate information on recent exposure to environmental PAH. Urine analysis of OH-PAH with established methodology relies on sample clean-up and pre-concentration followed by chromatographic separation and quantification. Although chromatographic techniques provide reliable results in the analysis of OH-PAH, their experimental procedures are time consuming and expensive. Additional problems arise when laboratory procedures are scaled up to handle thousands of samples under mass screening conditions. Under the prospective of a sustainable environment, the large usage of organic solvents is one of the main limitations of current chromatographic methodology. It is within this context that new analytical approaches based on easy-to-use and cost-effective methodology become extremely relevant. This dissertation focuses on the development of screening methodology for the routine analysis of PAH metabolites in numerous samples. It explores the room-temperature fluorescence properties of six metabolites originating from parent PAH included in the Environmental Protection Agency priority pollutants list. 1- hydroxyfluorene, 1-hydroxypyrene, 6-hydroxychrysene, 9-hydroxyphenanthrene, 3- hydroxybenzo[a]pyrene and 4-hydroxybenzo[a]pyrene are used as model biomarkers to investigate the analytical potential of new methods based on solid-phase extraction (SPE) and iii room-temperature fluorescence (RTF) spectroscopy. Quantitative determination of metabolites is carried out either in the eluent extract[1, 2] or on the surface of extraction membranes[3, 4] . The direct determination – i.e., no chromatographic separation - of the six metabolites is based on the collection of excitation-emission matrices and synchronous fluorescence spectra.

Yes / Wastewater treatment is an energy-intensive operation. Energy consumption is forecast to increase by 60% in the forthcoming decade due to tightened legislation surrounding the discharge of final effluent to watercourses. Treatment plants rely on the time-consuming and unreliable biochemical oxygen demand to assess the quality of final effluent, leading to process inefficiencies. Here, the authors show that fluorescence spectroscopy is a robust technique for real-time monitoring of changes in effluent quality. Three portable fluorimeters were installed for one month at the final effluent discharge point of a large municipal wastewater treatment plant. The authors show that organic matter composition of the wastewater varies diurnally depending on the flow rate and antecedent rainfall. High fluorescence intensity and ammonia are attributed to sewage sludge liquor, which is regularly discharged to the treatment plant. Moreover, elevated fluorescence intensities were recorded as a result of process failure following a power outage. The study shows that online fluorescence analysis is capable of detecting both minor changes in effluent quality and issues with treatment process performance. / European Commission Framework Programme 7, Marie Curie IEF (PIEF-GA-2012-329962) and the Core Program, ANCS (PN 16.40.01.01).

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Rapid advances in computer technology over the last few years and their integration into analytical instruments have led to tremendous increases in data collection rates. The need for tools to assist analytical chemists, and especially spectroscopists, in their task of interpreting such vast quantities of data is immediate. This work focuses on the development of an algorithm based on an alternative to the Fourier transform, the Hartley transform, for the qualitative and quantitative analysis of multi-component mixtures using Excitation Emission Matrices. The algorithm involves the reverse search of a compressed reference spectral library for the identification of possible components of the mixture and the method of Non-Negative Least Squares for the quantification of the components. A number of techniques for pre-processing of three dimensional fluorescence spectra along with several spectral encoding methods for the compression of the spectra were investigated. Both simulated and real data collected with a fluorescence spectrophotometer were used in this study. The algorithm proved capable of analyzing mixtures of five components with relative concentrations ratio of about 100:1 and significant spectral overlap. At the same time a compression ratio of about 10:1 for the spectra in the reference library was achieved. Finally, a library of three dimensional fluorescence spectra of some aromatic and poly-aromatic hydrocarbons was developed to be used with the algorithm. Such a library, along with the algorithm, provides a tool for the quick and simple qualitative and quantitative determination of mixtures of aromatic and poly-aromatic hydrocarbons. / Ph. D.

Due to the frequent use of plastics in daily life, encountering microplastics as pollution and trace evidence in forensic investigations is a common occurrence. Nylons are a plastic class known to be strong and durable, and they are used in a variety of products. Nylon is also a popular choice among 3D printing materials, and its strength and durability lend themselves to printing tools like keys and firearms. The analysis of microplastics is typically performed using any combination of microscopy, infrared spectroscopy, Raman spectroscopy, and mass spectrometry. Nylons are known to have fluorescent impurities created during the polymer’s synthesis and processing, so this work set out to develop a method to analyze nylon microplastics non-destructively using room-temperature fluorescence spectroscopy. By analyzing impurities in the sample, fluorescence spectroscopy can provide additional information about the microplastic’s source that other non-destructive methods do not. Excitation-emission matrices, excitation and emission spectra, and synchronous fluorescence spectra were collected from pellets of four different nylons. Using these spectra, all nylons could be distinguished from one another. A trace evidence simulation was then performed with fragmented pellets and a 3D printed key, and the trace microplastics were shown to have spectra that were consistent with those of the bulk plastic. This method was then improved by applying chemometric algorithms to the fluorescence data. Fluorescence spectra, excitation spectra, and excitation-emission matrices collected from Nylon 6 and Nylon 6/12 were found to be indistinguishable by visual comparison. Using synchronous fluorescence spectra from the two nylons in conjunction with Principal Component Analysis and Common Dimension – Partial Least Squares – Discriminant Analysis, the two nylons could be fully discriminated. Overall, this work presents a powerful new method to non-destructively analyze nylon microplastics and generate more reliable conclusions about the source of the microplastic than current methods alone.

We have investigated the use of deep UV laser induced fluorescence for the sensitive detection and spectroscopic lifetime studies of terbium doped dipicolinic acid (DPA-Tb) and used this to study the optical characteristics of DPA which is a chemical surrounding most bacterial spores. Background absorption spectra, fluorescence spectra, and Excitation Emission Matrix (EEM) spectra were made of the DPA-Tb complex, using both fixed 266 nm wavelength and tunable (220 nm -- 280 nm) UV laser excitations. Of importance, the fluorescence lifetimes of the four main fluorescence peaks (488 nm, 543 nm, 581 nm, and 618 nm) of the DPA-Tb complex have been measured for the first time to our knowledge. The lifetimes of all the fluorescing lines have been measured as a function of DPA-Tb concentration, solvent pH, and solvent composition, including that for the weakest fluorescing line of DPA-Tb at 618 nm. In addition, a new spectroscopic lifetime measurement technique, which we call "Transient Fluorescence Spectroscopy" was developed. In this technique, a weak, quasi-CW, amplitude modulated UV laser (8.5 kHz) was used to measure the lifetimes of the fluorescence lines, and yields insight into energy transfer and excitation lifetimes within the system. This technique is especially useful when a high power laser is not either available or not suitable. In the latter case, this would be when a high power pulsed deep-UV laser could produce bleaching or destruction of the biological specimen. In addition, this technique simulated the excitation and fluorescence emission of the DPA-Tb using a 4-level energy model, and solved the dynamic transient rate equations to predict the temporal behavior of the DPA-Tb emitted fluorescence. Excellent agreement between the experiments and the simulation were found. This technique has the potential to provide a more accurate value for the fluorescence lifetime values. In addition, with the use of asymmetric excitation waveforms, the dynamic transient rate equation analysis may allow for detailed studies of selected transfer mechanisms in a wide range of other spectroscopic applications including rare-earth solid-state lasing materials and biological samples.