|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.