Development of Enzyme-based Biosensors for the Detection of Organophosphate Neurotoxins
Type of DegreeDissertation
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Elevated concerns of ecological safety and environmental pollution have facilitated massive research in the area of chemical and biological agent detection. Large numbers of anthropogenic chemicals were introduced in the environment within last 50 years, which posed a serious threat to the natural ecological balance. The series of unfortunate events in the past could have been avoided or minimized by the availability of adequately selective and sensitive detectors, capable of discriminating between common use pesticides and chemical warfare agents. Organophosphates (OPs) in particular are more of a concern because of their toxicity and inhibition of esterase enzymes which are involved in nerve transmission. They have been an integral part of agricultural industry for past decades owing to their specificity. In addition, to their wide usage, they have also been exploited for the development of chemical warfare agents. Consequently, there is an emergent need for rapid and sensitive detection methods. Enzyme-based studies have been investigated in an effort to develop simple, rapid, user-friendly and sensitive detection of OPs. The methods described have been developed using OPH, an enzyme capable of hydrolyzing OPs. A rapid detection method using a fiber-optic waveguide and a portable fluorimeter Analyte 2000 was developed. Immobilization of OPH was accomplished using avidin-biotin chemistry. Change in fluorescence intensity was observed upon hydrolysis of substrate by OPH, concentration as low as 0.05µM paraoxon (PX) was qualitatively measured. A novel detection method for p-nitrophenol and p-nitrophenyl substituent OPs based on fluorescence quenching of coumarin1, a dye similar in structure to some OPs have also been developed. Decrease in fluorescence intensity of coumarin1 was proportional to paraoxon concentration in the range of 0.7–170 µM. To preserve OPH activity and stability, encapsulation using lysozyme-mediated silica nanoparticles was accomplished and detection limit for PX was found to be 20µM. To improve site accessibility and sensor sensitivity, orientation-specific immobilization of OPH was achieved using site-specific antibodies and different variants of OPH. Finally, the potential use of carbon nanotubes for the electrochemical biosensing of OPs has been exploited OPH was covalently immobilized on the surface of these nanotubes and a detection limit of 2.3 µM was obtained for PX. Results demonstrated that the immobilized enzyme retained a significant degree of enzymatic activity, and displayed remarkable stability with only 8% decrease in signal over a period of 10 days.