Bacterial infectious diseases remain one of the major health hazards nation- and worldwide. The expedience of detection and identification of bacterial pathogens determines how early the diagnosis is, and hence, what the treatment and the outcome of the illness would be. Conventional culture-based methods that are used to diagnose bacterial colonization and infection depend on the organism's ability to grow in pre- determined conditions. Advances in biotechnology and molecular biology have provided new tools for bacterial identification that provide expedience and specificity. Such techniques as genetics based diagnostics, however, depend on initial prediction for the potential pathogens in a sample. Inherently, such high-specificity assays provide information only about the species that are targeted. Therefore, pathogens present in the sample, which are not targeted by the conducted tests, remain undetected. The first part of my doctoral research sought to address these challenges by: (1) investigating the dynamics of fluorescence enhancement caused by the bacterial uptake; (2) identifying fluorescent dyes, e.g., molecular rotors, that can selectively stain only bacteria while in blood and other biological and environmental fluids; (3) employing biomimetic functionalization of bioinert interfaces for a bacterial immobilization that brings the analysis of fluorescence dynamic staining to a single-cell level; and (4) investigating the excited-state dynamics of the cyanine dye 3, 3'-diethylthiacyanine (THIA) using transient absorption spectroscopy to provide rationale for fluorescence staining kinetics observed during bacterial uptake. In parallel, my graduate research focused on utilizing the flow dynamics in microchannels for developing lab-on-a-chip platforms for time-resolved spectroscopy. Current approaches for investigating the photophysical dynamics of chromophores require cost-prohibitive equipment thus reducing the propagation of such specialized techniques. Using steady-state excitation and imaging, we measured luminescence decay kinetics of lanthanide chelates from the spatial distribution of the emission within a microchannel. From the space-domain data for the chelates recorded at different flow rates we extracted lifetimes that we confirmed using "traditional" time-domain measurements. This validated space-domain microfluidic approach reveals a means for miniaturization of time-resolved emission spectroscopy and bringing it to lab-on-a-chip platforms.

Electrostatics, a result of one of the fundamental forces of nature, govern and underlie fundamental processes of chemistry, biology and modern technologies. Peptides and other natural macromolecules owe their structural integrity and functionality to electrostatics. (1) Anthrilamides (Aa) are favored for their large dipoles and the strong localized electric fields they generate. Furthermore, the inductive effect of substituents allows for wide tunable range of donating capability. By attaching onto various dyes, we have demonstrated multiple times these dipole-generated fields induce pronounced rectification of charge transfer. Despite its importance, the fields around Aa conjugates have not been experimentally characterized yet. By attaching electrochromic probes, such as an aminocoumarin, on either side of the electret moieties, allows us to use the induced Stark Effect for quantification of the dipole-generated electric fields. This approach toward direct quantification and mapping of the electric fields around molecular electrets can be readily adopted for facial optical probing of the electrostatic properties of biological structures, nanomaterials and materials interfaces. (2) Poly-L-lysine (PLL), along with other biological polyelectrolytes, can serve as templates for self-assembly of supramolecular structures and materials. Reported spectroscopic studies show that Eosin-Y (EY), a negatively charged dye, interacts with PLL to form structures with new electronic properties that can be potentially important for light harvesting and energy conversion. The structures of these biomimetics, however, is not well understood. Therefore, we undertook systematic studies to establish structure/ function relationships for PLL/EY self-assemblies. Circular dichroism (CD) shows feature of ordered helical structures of excitonically coupled moieties that the EY chromophores assume on the PLL templates, as the ellipticity in visible spectral region reveals. Furthermore, in the presence of EY, PLL exhibits CD features associated with protein alpha-helical conformations. In the absence of EY, PLL exists 100% as a random coil. This synergy between templated macromolecule and templated chromophores opens doors for explorations in photonics and biomimetic electronics

In order to develop and demonstrate fundamental strategies for improving the efficiency of photovoltaic devices that are commonly used for solar-energy capture and conversion, we introduced and studied anthranilamides as bioinspired electrets.

Charge transfer processes play a key role in chemical and biological systems. Photoinduced charge transfer represents the central paradigm of light-energy conversion of photosynthesis and photovoltaic devices. The Rehm-Weller equation allows for estimating the diving force of photoinduced charge transfer by employing readily measureable quantities such as the redox potentials and spectroscopic data of electron donors and acceptors. A significant part of my studies focused on the Born solvation term in the Rehm-Weller equation that introduces the electrostatic stabilization of the charge-transfer species by the surrounding media. Cyclic voltammetry, allowed me to demonstrate experimentally the effects of the supporting electrolyte on the redox potentials. These effects were especially pronounced for non-polar solvents. Most importantly, I devised an approach to address the discrepancies that the presence of electrolyte introduces to the charge-transfer analysis. Concurrently, my studies demonstrated that the Generalized Born model allows for addressing the deficiencies in the charge-transfer analysis involving redox species that are not spherical and that have heterogeneous charge distribution.

The other significant part of my studies focused on anthranilamides as bioinspired electrets that have the potential to accelerate charge separation and suppress the undesired charge recombination. My research provided the first experimental demonstration that the anthranilamides possess intrinsic dipoles. These amides with large intrinsic dipole moments (that is, electrets) can generate electric field, which enhances electron transfer from their N- to their C-termini and impedes it in the backward direction. To test the ability of the electrets to modify the direction of electron transfer, I incorporated an anthranilamide monomer in electron donor-acceptor (DA) dyads. Comparison between the charge-transfer kinetics of electret-acceptor dyads, revealed a faster initial photoinduced charge separation and a slower charge recombination when electron was transferred toward C-terminus. These findings were consistent with the orientation of the intrinsic dipole moment of the anthranilamide monomer. Aside from previous work employing polypeptides, this is the first demonstration of rectification of charge transfer by dipole moments of synthetic bioinspired derivatives.

In summary, the most important contributions from my doctoral work were: (1) developing methods for reliable interpretation of experimental results pertinent to charge-transfer kinetics and thermodynamics; (2) demonstrating rectification of photoinduced charge transfer induced by the anthranilamide intrinsic dipole; and (3) demonstrating that the photoinduced processes result in charges residing on the anthranilamides (i.e., radical ions) which is essential for attaining hopping mechanism for long-range charge transfer.

Fluorescence is a radiative-deactivation phenomenon of broad importance for opticalimaging and sensing. Fluorescence also can serve as a signal for characterizing excited-state dynamics and the kinetics of other parallel processes such as energy transfer and charge transfer (CT). The importance of CT cannot be overstated. In addition to sustaining life on earth in photosynthesis, cellular respiration and redox enzymatic conversions, CT ensures the modern ways of living possible as a part of electronics photonics and energy conversion. My work focuses on developing methods for characterizing fluorescent chromophores and redox species that can undergo CT. The viscosity of the microenvironment can have a huge impact on the fluorescence properties of photoprobes of biological importance. After a brief introduction, the second chapter of my dissertation describes the development of methodology for utilizing a thermoset transparent polymer as a room-temperature solid solvent for spectroscopy applications. With ultimately large viscosity, this solid-state media shows significant effects on suppressing non-radiative pathways of deactivation and enhancing the fluorescence quantum yields of certain dyes by orders of magnitude. The third chapter describes a method for expanding the applicability of cyclic voltammetry (CV), which is the most widely used technique in electrochemical analysis. My work demonstrates how to obtain useful information, in terms of standard electrochemical potentials, from voltammograms showing irreversible behavior. Such information is essential for characterizing electron donors and acceptors for CT systems. The fourth chapter demonstrates the utility of spectroscopic and electrochemical studies for CT. Fundamental scientific studies show the importance of hydrogen bonding for transferring holes and electrons in peptide conjugates. Deciphering structure-function relationships also demonstrates how hydrogen-bonding propensity of fluorescent probes, which can act as electron donors and acceptors, can induce well-defined folds in oligopeptides that are as short as four residues. These fundamental-science studies set an important foundation for electronics, photonics and bioinspired engineering.

Due to the considerable challenges for reliable and expedient detection of virulent vegetative bacterial, bacterial endospores and other hazardous biological agents, accidental exposure is a principal cause of fatality due to wound infection or slow diagnosis. Staining techniques, due to their simplicity, form a set of important tools for pathogen monitoring and control. The staining assays, such as immunoassays, however, provide only Boolean information: i.e., stain vs. does not stain. As a result, only the species being sought may have a chance to be identified, leaving key determining factors for the diagnosis quite susceptible to human error.

Our principle motivation for this project is to expand the current analytical techniques beyond their Boolean nature, and to address the apparent need for simple, expedient and inexpensive assays. We describe the development of an assay system based on the dynamics of fluorophore uptake that will serve as a robust platform for detection of both vegetative and endospore bacterial contaminants. The kinetics of fluorescence staining depends on the coatings and protein content, which reflect the species phenotype. Hence, such kinetic measurements gain access to genetic information, in less than a minute, without conducting amplification procedures such as PCR.

We have demonstrated for the first time that the kinetics of emission enhancement, caused by cell uptake of fluorophores, provides statistically significant discernibility between different and closely related vegetative bacteria and bacterial endospore species respectively. We observed that the time course of the fluorescence signal provides a unique species-specific signature that can prove indispensible for the identification of dangerous and life-threatening spores.

Charge transfer (CT) is an essential part of life. Ubiquitous in nature, CT is the principal foundation for biological processes, solar-energy conversion and electronics. Understanding CT processes at the molecular and nanoscale levels and interface between them is essential for energy science, biomedical advances, and organic electronics. As important as interfacing, or “wiring,” molecular moieties with conducting solid substrates is for materials and device development, it still remains a formidable challenge. Over the last decade lead halide perovskite compositions have attracted tremendous interests due to their uniquely promising electronic and optical properties. The dynamic nature of ligand binding to such materials makes the wiring of CT molecular moieties to them especially challenging.

Here we show how effective interfacing between semiconducting all inorganic CsPbBr3 perovskite nanocrystals and electron donating molecules facilitates charge transfer. We illustrate the important dual benefits that a binding motif offers: (1) electronic coupling essential for efficient interfacial CT and (2) surface-trap passivation eliminating non-radiative pathways of exciton deactivation. Aliphatic amines show the strongest known propensity for perovskite surfaces. We show that an increase in the amounts of such Lewis bases etches the perovskite material, changing the shape and size of the nanocrystals and degrading them. Our results demonstrate that concentration control allows for optimal loading of amine moieties on perovskite nanomaterials without compromising their morphology, permitting efficient interfacial CT even in non-polar media. These findings reinforce and open doors for a wide range of photonics and electronics applications including solar-energy conversion, photocatalysis and molecular electronics.

Molecular-level control of charge transfer is essential for organic electronics and solar energy conversion, as well as for comprehending a wide range of biological processes. Photoconversion efficiency is mainly hindered by interfacial charge recombination. In nature, charge recombination is suppressed by incorporating molecular electrets, systems with ordered electric dipoles. Local electric fields originating from molecular dipoles can thus aid in overcoming charge recombination while promoting the desired forward charge transfer. Protein alpha helices present the best example for natural molecular electrets, that is, they have intrinsic dipoles originating from the ordered amide and hydrogen bonds.

A principal challenge of polypeptide charge transfer systems composed of native amino acids is the inherent distance limitation for efficient transduction of electrons and holes. Along their backbones and hydrogen bonds, protein -helices mediate electron transfer via tunneling, the rate constants of which, ket, fall off exponentially with an increase in the length of the electron-transfer pathways, ret, i.e. ket∝exp(−β ret). Specifically, for protein -helices, β is about 1.3 Å-1. The inherent presence of competing nanosecond processes, such as fluorescence, internal conversion and intersystem crossing, therefore, places a practical limit of about 2 nm for attaining photoinduced charge transfer with acceptable efficiency in such bimolecular structures.

Because of the importance of long-ranger charge transfer via hole hopping, we focus on the development of suitable electron deficient sensitizer for initiation hole transfer. Hence, the photosensitizer must absorb away from the electron donor in the visible region of the spectrum, and have a carboxylic acid (or amine) functionality for covalent attachment to molecular electrets that are polypeptides themselves.

Therefore, my initial studies focused on characterization of non-native aromatic beta-amino acid residues, derived from anthranilic acid, as building blocks of hole-transfer molecular electrets. The aromatic moieties provide the means for attaining long-range hole transfer. Chemical modification of the distal position of the anthranilamides allows for adjusting the reduction potentials of oxidation over a range of one volt. My major contribution to this work was discovering what makes oxidized N-Acylanthranilamides stable. By correlating the electrochemical data with the spin-density distribution of the radical cation, I determined that for radical cations of the anthranilamide to be stable 1) their reduction potential must be at about 1.5 V or below vs. S.C.E., and 2) the electron spin density should not extend over the C-terminal amide. Indeed, this proved valuable for increasing our library of building blocks for molecular electrets that have practical feasibility.

A significant part of my studies encompass various aspects of synthesis of electron deficient photosensitizers for initiating the hole transfer in the anthranilamides, focusing on pyrene, nitropyrene and diketopyrollopyrolle chromophores. From my pyrene work I was able to shed light on the effect of the orientation of amide bonds on the electronic properties of polycyclic aromatic molecules. Incorporating an electron deficient diketopyrollopyrollo chromophore as an electron acceptor in donor-acceptor conjugates, where we use anthranilamides as donors, allowed us to determine the two important requirements for enhancing dipole effects on charge transfer and how to harness them. 1) The electric dipole has to be as close as possible to the charge-transfer systems. Indeed, incorporating the dipoles in the components of the charge-transfer systems presents the ideal case. 2) The media polarity should be lowered. While non-polar media impedes charge-separation processes, it also enhances the permiation of the dipole-generated electric fields. We demonstrated that the later of those two opposing effects can prevail. Changing the solvent from acetonitrile to toluene leads to a six-fold increase in charge separation rates when the electron transfers along the dipole. The same change in the solvent polarity completely shuts down the charge transfer when the electron is pushed to move against the dipole. These findings show rectification of photoinduced charge separation that is practically infinity and have important implications about the utility of dipole effects for electronic and energy materials and devices.

The most significant contributions from my doctoral research include: (1) the determination of the effects of location and strength of electron donating substituents on the stability of the radical cations of the non-native amino acids for building molecular electrets; (2) demonstrating that amide bonds are not just linker but they can significantly affect the properties of the moieties they are attached to; and (3) developing of electron-deficient and conjugating them with anthtanilamides, which showed us to demonstrate dramatic dipole effects on charge transfer.

Bacterial infections continue to be one of the major health risks in the United States. The common occurrence of such infection is one of the major contributors to the high cost of health care and significant patient mortality. The work presented in this thesis describes spectroscopic studies that will contribute to the development of a fluorescent assay that may allow the rapid identification of bacterial species. Herein, the optical interactions between six bacterial species and a series of thiacyanine dyes are investigated. The interactions between the dyes and the bacterial species are hypothesized to be species-specific. For this thesis, two Gram-negative strains, Escherichia coli (E. coli) TOP10 and Enterobacter aerogenes; two Gram-positive bacterial strains, Bacillus sphaericus and Bacillus subtilis; and two Bacillus endospores, B. globigii and B. thuringiensis, were used to test the proposed hypothesis. A series of three thiacyanine dyes--3,3'-diethylthiacyanine iodide (THIA), 3,3'-diethylthiacarbocyanine iodide (THC) and thiazole orange (THO)--were used as fluorescent probes. The basis of our spectroscopic study was to explore the bacterium-induced interactions of the bacterial cells with the individual thiacyanine dyes or with a mixture of the three dyes. Steady-state absorption spectroscopy revealed that the different bacterial species altered the absorption properties of the dyes. Mixed-dye solutions gave unique absorption patterns for each bacteria tested, with competitive binding observed between the bacteria and spectrophotometric probes (thiacyanine dyes). Emission spectroscopy recorded changes in the emission spectra of THIA following the introduction of bacterial cells. Experimental results revealed that the emission enhancement of the dyes resulted from increases in the emission quantum yield of the thiacyanine dyes upon binding to the bacteria cellular components. The recorded emission enhancement data were fitted to an exponential (mono-exponential or bi-exponential) function, and time constants were extracted by regressing on the experimental data. The addition of the TWEEN surfactants decreased the rate at which the dyes interacted with the bacterial cells, which typically resulted in larger time constants derived from an exponential fit. ANOVA analysis of the time constants confirmed that the values of the time constants clustered in a narrow range and were independent of dye concentration and weakly dependent on cell density.

Photon conversion efficiency is primarily impaired by interfacial charge recombination. Natural light harvesting systems address this challenge by incorporating molecular electrets. Protein alpha helices are the best examples of molecular electrets since the ordered amide and hydrogen bonds naturally generate a co-directionally ordered permanent electric dipole.

Although the local electric fields generated by the protein's dipole provides a means for “steering” electron transduction, they cannot be directly incorporated as electronic material because 1) electron transfer is mediated via tunneling, 2) they contain large band gaps and 3) are conformationally sensitive.

Therefore, a significant part of my studies focused on developing a library of non-native, aromatic beta-amino acid residues composed of anthranilic acid derivatives. The aromatic moiety is designed to provide an electronic framework that supports a hole hopping mechanism resulting in a system that captures the benefits of natural systems. Furthermore, altering the attached substituents results in a range of reduction potentials over 1 volt.

Electrochemical examination revealed the structure-function relationship between the position and strength of the electron donating substituent. These findings provided the criteria for generating a long-lived radical cation to be 1) the reduction potential must be under 1.5 V vs. S.C.E and 2) the electron spin density must not extend over the C-terminal amide.

From these findings, I development a set of ad hoc hole-transfer molecular electrets. Based on a fluorinated aminoanthranilamide templet, each residue contained a both an electron withdrawing and donating group. The spin-density-distribution and electrochemical analysis revealed that the fluorine atom induced a positive shift in the reduction potentials without destabilizing the oxidized residue. Additionally, the regio-selective nucleophilic aromatic substitution of the starting material provides a straightforward synthesis.

Overall, the most significant contributions from my doctoral research are: (1) the design and development of a synthetic amino acid library with a wide range of reduction potentials; (2) the determination of the effects of location and electron donating strength on the stability of anthranilamide radical cations; and (3) the implantation of nucleophilic aromatic substitution to synthesize residues for hole hopping.