Studies on the Biosensor for Detection of Methyl Parathion

Methyl Parathion (MP), an organophosphorus pesticide holds concern
due to its extensive use in agricultural practices. MP being highly toxic at trace
levels to human beings requires detection at high sensitivity, which is a
challenging task. The sensitive detection of MP by immunosensor methods
have been delineated in this thesis. Initial approach was emphasized on the
synthesis of MP hapten. Synthesized MP hapten was conjugated to a carrier
protein molecule and immunized in chicken to produce anti-MP IgY
antibodies. Antibodies were purified in bulk using affinity column
chromatographic technique. Further, antibodies were tested for cross
reactivity with structurally related OP pesticides. MP-HRP conjugate, which
have been used as a labeled marker for immunosensor applications, was
stabilized with 0.1% BSA. Furthermore, chemiluminescence (CL) parameters
were standardized for MP-HRP conjugate by different biochemical methods.
Immunosensor detection of MP requires efficient immobilization of antibodies
and dissociation of MP from the immuno-reactor column. Study revealed that
Gly-HCl (pH-2.4) in combination with 1% methanol was the best dissociating
buffer for reusability of the antibody immobilized in immuno-reactor column.
The CL and charge coupled device (CCD) based method of detections were
standardized for the sensitive detection of MP in water and different fruit
beverages. With CL techniques the detection limit was 0.005 ng/mL and with
CCD it was 0.01 ng/mL. Quantum dot based MP detection showed the
credibility of the method to detect at 0.1 ng/mL. The visual colloidal gold
based dipstick technique was also employed for the qualitative detection of
MP and detection limit was 0.1 ng/mL. Validation of all these methods over
22
existing ELISA was carried out in present investigation. The results indicated
the superiority of developed immuno-sensors against conventional techniques
for the sensitive detection of MP in water and fruit beverages.