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In this work the development of affinity sensors for the detection of microcystin-LR based on a computationally designed artificial receptor is presented. Microcystin-LR is a cyclic heptapeptide hepatotoxin produced by Cyanobacteria (aquatic organisms also known as blue-green algae), which during blooms period can release toxins in water. Clinical signs of hepatotoxicosis have been observed in domestic animals and livestock and recently also in humans. At present, analysis of these toxins is achieved largely using conventional, time consuming and expensive techniques such as chromatographic methods (HPLC, TLC) and immunoassay. Therefore, the necessity of an easy and inexpensive method of analysis such a biosensor is becoming urgent. In this work an artificial receptor for microcystin-LR was synthesised using a combined approach of molecular imprinting and computer modelling. A computer-aided rational design was applied to study microcystin-LRlmonomers interactions in order to find an optimal composition for the synthesis of the receptor. The optimised composition, suggested by computer modelling, consisted in 1 mol of2-acrylamido-2-methyl-propanesulfonic acid and 6 mol ofurocanic acid ethyl ester for 1 mol of template. This monomer composition was then used to synthesise a molecularly imprinted polymer (MIP) and an enzyme- linked competitive assay was developed to characterise the computational receptor. In the assay, computational MIP was able both to detect 0.1 ~g rl of microcystin-LR and to distinguish the analyte among analogues such as microcystin-YR, microcystin-RR and nodularin. The computationally designed receptor was then used as a sensing element for the construction of sensor devices. A MIP-based piezoelectric sensor, capable of detecting 35 ~g rl of toxin in water, was developed. In order to improve the system sensitivity, the computational polymer was also used as a material in solid-phase extraction (SPE) for samples pre-concentration. The receptor was able to pre-concentrate up to 1,000 fold tap water samples spiked with only 1 J.1g rl of toxin. By combining MIP-based SPE and piezoelectric sensor an improved system with a minimum detectable concentration of toxin of 0.35 ~g rl was achieved. Encouraging preliminary results were also obtained in developing a MIP-based electrochemical sensor.

For the successful detection of selected DNA sequences or mutated genes associated with human disease, there are several challenges that the current research aims to overcome - higher sensitivity, greater selectivity and rapid assaying time. An electrochemical device using redox-active intercalators to transduce DNA hybridization via long-range electron transfer is presented in this thesis which aims to address the above challenges. The DNA recognition interface is composed of thiolated single-stranded DNA (ss-DNA) and a diluent component both of which are self-assembled onto a gold electrode. This project seeks to advance fundamental insight into issues that impact the structure and behavior of surface-immobilized DNA towards hybridization with target complementary ss-DNA. After the optimal conditions have been identified for the construction of a reproducible DNA recognition layer, a stepwise detection scheme using an anionic intercalator, as the redox molecule is introduced for the DNA transduction. The stepwise detection relies on the absence of any electrochemistry prior to DNA hybridization. Upon hybridization, the perfectly stacked DNA is capable of mediating the electrochemical oxidation and reduction of intercalated species and hence voltammetric peaks become evident. Although excellent selectivity towards single-base mismatch detection is achieved, this detection scheme has a high detection limit and slow assaying time. However, this is overcome by a novel in situ approach where the electrochemistry is performed in the presence of both complementary target DNA and intercalator. The effect of different DNA recognition interfaces on hybridization is also investigated using electrochemical and gravimetric techniques where the hybridization efficiency, kinetics and affinity constant of hybridization were assessed. These measurements showed that the length of the diluent layer has a large impact on the time taken to form a perfect duplex but no impact on the initial recognition of the target DNA by the immobilized probe DNA. Fundamental aspects of the DNA technology towards assaying small molecules which have binding affinity to DNA are also investigated. The probe ss-DNA sensing interface was found to be highly sensitive towards detection of Cd2+. The long-range electron transfer approach was also utilized in gaining more insight knowledge of the interaction of cisplatin, an anti-cancer drug with the DNA.

Methodologies that detect biologically active substances have important potential applications for medical diagnostics, drug discovery, and chemical and biological anti-terrorism efforts. The wide spectrum of potential analytes that induce a physiological response dictates that novel techniques be developed to more rapidly screen and characterize agents that are more economical and have greater sensitivity than current practices. The research presented in this dissertation describes the development of a biosensor methodology that utilizes optical changes in naturally pigmented chromatophore cells from fish to detect and measure an array of biochemicals and protein toxins. The chromatophores used in this biosensor were harvested from teleost fish sources and the observed patterns of pigment aggregation and dispersion in response to added chemical modulators were used as a reporter mechanism. Differential responses between chromatophore subtypes were utilized as simple cellular sensors for the detection of cholera toxin and in the study of the calcium signaling requirements in these cells. A multi-cellular biosensor was developed that couples pigmentation changes in erythrophores from the teleost Betta splendens with mammalian nerve cell secretory activity. An apparatus was developed that placed PC12 cells, a neuroendocrine cell line, and erythrophores in adjacent chambers connected by a fluid network that allowed erythrophores to be exposed to effluent from PC12 cells; neurotransmitters secreted from PC12 cells induced pigment aggregation in erythrophores. By analyzing the extent of this erythrophore response, this method was capable of detecting the occurrence of substances that altered neurotransmitter secretion levels. A demonstration of this biosensor is presented that detected the inhibition of neurosecretory activity caused by the pathogenic bacterial toxin botulinum, the causative agent of human botulism. / Graduation date: 2002

This thesis presents broadly the applications of molecularly imprinted polymers in sensors and solid phase extraction. Sensors for creatine and creatinine have been reported using a novel method of rational design of molecularly imprinted polymers (MIPs), and solid phase extraction of aflatoxin-B 1 has also been described in the thesis. A method for the selective detection of creataine and creatinine is reported in this thesis, which is based on the reaction between polymerised hemithioacetal, formed by allyl mercaptan, o-phthalic aldehyde, and primary amine leading to the formation of fluorescent isoindole complex. This method was demonstrated for the detection of creatine using creatine-imprinted MIPs. Since MIPs created using traditional methods were unable to differentiate between creatine and creatinine, a new approach to the rational design of a MIP selective for creatinine was developed using computer simulation. A virtual library of functional monomers was assigned and screened against the target molecule, creatinine, using molecular modeling software. The monomers giving the highest binding score were further tested using simulated annealing in order to mimic the complexation of the functional monomers with template in the monomer mixture. The result of this simulation gave an optimised MIP composition. The computationally designed polymer demonstrated superior selectivity in comparison to the polymer prepared using traditional approach, a detection limit of 25 μM and good stability. The 'Bite-and- Switch' approach combined with molecular imprinting can be used for the design of assays and sensors, selective for amino containing substances. MEP for the selective binding properties for aflatoxin-B 1 was prepared using the computational approach. The results obtained demonstrate that the MISPE offers a simple, convenient and a rapid methodology for solid phase extraction of aflatoxin-B 1 even at very low concentrations of 2 ppb. The commercially available C-18 cartridges were able to recover only about 52% of aflatoxin-B 1 at concentrations of 2 ppb when compared with almost complete recovery by the MIP. We have proved here that, MIPs as a solid phase extraction materials offer important and practical advantages with respect to other solid phase extraction methodologies.

Dissertation (Ph.D.)--Auburn University, / Abstract. Vita. Includes bibliographic references (p.119-123).

Thesis (Ph.D.) in Chemical Engineering--University of Maine, 2007. / Includes vita. Includes bibliographical references (leaves 141-151).

Thesis (Ph. D.)--University of Texas at Austin, 2005. / Supervisor: Raul G. Longoria. Vita. Includes bibliographical references.

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2007. / Includes bibliographical references. Also available in electronic version.