The green fluorescent protein chromophore exhibits remarkably different properties upon removal from the protective beta-barrel. This work focuses on the synthesis of these chromophores as wells studying the photophysics as to why they readily deactivate. Following these initial discoveries, these chromophores can be applied to many different environments providing a fluorescence "turn-on" and thus proving to be applicable in a number of different environments and fields.

In 2008 the Nobel Prize in Chemistry was awarded to the scientists who revolutionized biomedical technology by isolating, characterizing, and pioneering the use of a green fluorescent protein (GFP) from a humble hydrozoan jellyfish. Now numbering in the hundreds of colors and applications, fluorescent protein (FP) tools have facilitated the explosion of biological knowledge elucidated by a technology that can label DNA or RNA, track protein expression, and identify protein interactions. The development of the large variety of FP biotechnology available today has been due to the need for expanded color palettes and applications, and more efficient functionality. Yet, as our understanding of the biochemical and spectral characteristics of these genetically-encoded, self-assembling proteins has expanded, our comprehension of the biological function of FPs in the host organisms has remained inadequate. While the need for novel FP laboratory applications still continues, the new focus in the field of fluorescent proteins is moving to also characterize their biological functions. In this research compilation, the identification of three groups of new fluorescent proteins from marine copepods and hydrozoans has provided a collection of eleven FPs exhibiting previously uncharacterized colors, and biochemical and structural features. The green FPs from copepods are the brightest wild-type FPs identified and support the hypothesized biological function of fluorescence as counter-shading in the marine environment where these animals live. The FPs from the siphonophore and anthoathecate jelly, both hydrozoan animals, are comprised of tandemly expressed fluorescent protein units, a solution to the oligomeric structure common to most FPs that suggests a novel structure-function relationship. The fluorescent proteins from Obelia reveal a novel hydrozoan cyan FP, previously uncharacterized higher-order structural complexes, and have initiated the work to describe the biological function of these proteins as potential regenerators of their internal bioluminescent light sources. All eleven fluorescent proteins may also be adapted for FP technology. / text

Chemical sensors capable of detecting a specific atom or molecule under various conditions have been utilized in biological and environmental analyses. Fluorescence based sensors are particularly advantageous in these studies because of their high sensitivity, relative ease in handling, and low technical costs. This dissertation focuses on the detection of two analytes, H+ and hypochlorous acid, which are of interest in biology because the presence of abnormal quantities of these analytes may be indicative of disease. We have established a new platform for which sensitive changes in various regions of pH can be detected using fluorescence. The aminomethylrhodamine (AMR) scaffold is highly versatile, i.e. the pH range in which the sensor is active can be tuned by introducing different substituents on the amine moiety. Overall this systematic approach to the design of pH sensitive fluorophores has allowed for a library of compounds that are responsive over a broad range of pH (pH 3 - 10) by simply changing the substituent on the amino group. We report the synthesis and characterization of a silicon analog of rhodamine for the fluorescence based detection of hypochlorous acid. This fluorophore exhibits a 90 nm bathochromic shift in its absorption and emission, relative to its oxygen counterpart. Hypochlorous acid is a biological agent linked to certain diseases. Therefore, the longer wavelength properties of the this far-red fluorescent sensor will be of significant benefit to imaging experiments of this analyte in biological media and tissue due to its spectral proximity of the so called NIR optical window. Furthermore, the novel synthetic methodology of this sensor possesses a key intermediate, which could potentially lead to future fluorescence based sensors. The characterization of a fluorescent probe designed for the detection of hypochlorous acid (HOCl) using a silicon analog of fluorescein (SiF) was also reported. Over a range of pHs, the probe reacts with a stoichiometric amount of HOCl resulting in a mixture of two pH dependent fluorescent species, a SiF disulfide product and a SiF sulfonate product. The unique colorometric properties of the individual SiF fluorophores were utilized to perform simultaneous detection of HOCl and pH. When an excess of HOCl is present, the SiF fluorophores become chlorinated, via an intermediate halohydrin, resulting in a more pH independent and red-shifted fluorophore. Finally, an attempt was made at developing a pH responsive photodynamic therapy agent. This system was designed to target the relatively low extracellular pH found around tumors. A di-bromohydroxymethylrhodamine system was synthesized and the photophysical properties were characterized. This system absorbs weakly under acidic conditions (ca. pH 3), however was shown to be a moderate photosensitizer under acidic conditions.

In the first study, a new µ-HPLC column was developed using a monolithic silica gel as a column substrate for chiral separation by covalently modifying with (S, S)-Whelk-O1 chiral selector. The monolithic stationary phase was generated through a sol-gel process and prepared in situ in a 100 µm i.d. fused silica capillary tubing. The chromatographic performance was characterized in terms of retention factor, column efficiency, enantioselectivity and resolution, as well as the kinetics parameters affecting the separation. Comparison with a commercial particle packed HPLC column demonstrates a promising enantioselective resolving ability of the monolithic Whelk-O1 capillary column. The second project focuses on characterization of fluorescent sensor for zinc detection. In this work, we have examined the photophysical properties of the fluorescent probe sensor that has been developed in our laboratory for Zn2+ recognition via a photo-induced electron transfer (PET) sensing mechanism. To characterize the fundamental function of sensor, response curves have been conducted, using acetone/methanol (199:1), 1,4-dioxane, acetone, methanol and aqueous buffer as the solvent system. Similar to prior work from our group, the sensor was found to respond selectively to Zn2+ ions with fluorescence enhancement. The fluorescence properties and binding response were evaluated in the presence of water and a Lewis base, which we found to have a marked effect on the fluorescence signal. The selectivity of the sensor for Zn2+ was also observed and compared to other divalent metal such as Ca2+, Mg2+, Cu2+ and Hg2+ with the goal of learning fundamental information on the system that can aid in the development of future PET based sensors.

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This thesis presents an overview of the research activities undertaken during my PhD under the supervision of Dr. F. Iza from the School of Electronic, Electrical and Systems Engineering and Dr. B. Buckley from the Chemistry Department at Loughborough University. The thesis is divided as follows. The first chapter of the thesis presents an introduction to plasma and chemical probes as well as the motivation for developing fluorescent probes for plasma characterisation. Analytical techniques used during this work to analyse chemical substances are described in the second chapter. Results and discussions from the experiments are discussed in chapters 3 to 7. Conclusions and future work are presented in chapter 8. In chapter 9, experimental data is presented. In the last century, plasma has attracted the attention of numerous researchers. Due to the wide-range of applications of this ionised gas, people from different fields have focused their effort on studying plasma. Low-temperature plasmas have received growing attention in the last 50 years when the development in cold plasma devices made them more controllable. Plasma played (and continues to play) a critical role in the fabrication process of integrated circuits and recent advances in the generation of low-temperature atmospheric-pressure plasmas have resulted in the emergence of new applications including treatment of temperature sensitive surfaces and biological targets. During the first months at Loughborough I worked on the ozonolysis of various alkenes with air plasmas. This allowed me to familiarised myself with plasma as this was new to me and get a feeling of some of the challenges lying ahead. Nonetheless, the data I obtained was encouraging and I presented the results of batch and flow plasma-based ozonolysis of alkenes at the Technological Plasma Workshop held in Manchester in January 2012. Once I had familiarised myself with the plasma system, I worked on synthesising fluorescent probes to detect ozone, one of the many reactive species that are typically generated in oxygen containing plasmas. Details of the experiments conducted to date and most significant findings are discussed in this thesis.

Towards the construction of protein-based biological sensors, chimeric proteins comprised of an antibody single chain antigen-binding protein (scFv) and the green fluorescent protein (GFP) were constructed. Although correct folding of the scFv domain typically requires the oxidizing conditions of extracellular compartments, such as the periplasmic space of E. coli, GFP is unable to mature under these conditions. Using DNA recombinant technology, fusion constructs were made in the cytoplasm under control of the araBAD promoter. Weak fluorescence of the GFP domain and antigen binding activity of the sFv domain were obtained in the cytoplasm of E. coli BL21, but improved expression and activities of both domains were obtained by using a trxB- mutant of E. coli, as well as by modifying physical and genetic conditions for expression of the fusion proteins. Assessment of the fluorescence and antigen binding activity of the fusion proteins indicates that GFP fluorescence can serve as an indicator of correct folding of fusion proteins.

The brightness and photostability of semiconductor quantum dots (QDs) has prompted the exploration of their use in a wide variety of fields. Several examples of QD-based biosensors have been reported but none have actually replaced their preexisting technologies. This work reveals the barriers hindering widespread use of QD based biosensors and examines how QDs can be engineered for improved utility in bioassay designs. The first portion of this project aims to improve Förster Resonance Energy Transfer (FRET) that use QDs as both the donor and acceptor. FRET-based sensors often use fluorescent dyes (FD) or proteins (FPs), but their photo- and chemical instability can be problematic. Contemporary QD-QD FRET systems suffer from unacceptably high background signal due to direct acceptor excitation. Materials engineering is used to create QD donors that are brighter than their QD acceptors to mitigate this effect. First, CdSe/xCdS/xZnS QDs of increasing shell thickness were synthesized and tested in a QD-fluorescent dye system to elucidate the effect of increased donor size on the performance of a FRET sensor. The optimal donors were medium-sized and 8 times brighter than commercially available QDs while retaining ~60% FRET efficiency. When used in a sensor, changes in sensor brightness were visible by eye. Moving towards QD-QD systems, a pH-based aggregation assay was used to test how QD heterostructures comprised of different semiconductor materials perform as FRET donors or acceptors. The fundamental principles uncovered are used to improve contemporary QD-QD FRET sensing and show that sensors can be designed to use color change as a visible, easy-to-decipher readout. Color change-based sensor output is further explored in an allosteric transcription factor-based small-molecule sensor that employs QDs as the sole fluorescent label. A highly modular design is presented that achieves a nanomolar concentration visual limit of detection. The ease of use, and fast, instrument-free readout of the sensor shows promise for its development into a fully integrated point-of-care device, endorsing the value of further developing QD-based in vitro biosensors for clinical or commercial translation. / 2020-06-04T00:00:00Z