Effectiveness of plant esterase immobilized on multi-walled carbon nanotube screen-printed electrode for detection of pesticides

Abstract

Organophosphorus compounds (OP) account for the most toxic substances used to destroy pests by inhibiting acetylcholinesterase (AChE) of the central nerve. Since OP resulted in the accumulation of its residue and finally increased the exposure to humans, therefore, various acetylcholinesterase (AChE) based biosensors from animal sources had been developed for this purpose. However, Alpha naphthyl acetate esterase (ANAE) enzyme from wheat flour was more economical alternative due to its simpler procedure of extraction and purification. OP has an inhibitory effect on ANAE as also with AChE, thus ANAE based biosensors’ sensitivity is comparable to the AChE biosensors. The crude enzyme extract was first filtered and purified using an aqueous two-phase separation system (ATPS). A purification fold of enzyme 4.36 was obtained with an enzyme yield of 56.89% of atta flour. The molecular weight of the target esterase was found to be around 63 (kDa). The incubation time within 15 minutes at 30°C with pH 6.5 of phosphate buffer are the optimum condition at which ANAE resulted in the highest activity. The Michaelis-Menten parameters of the purified enzyme were 9.765 mM and 0.084 mMmin-1, respectively for Km and Vmax. To analyze and characterize the immobilization of ANAE on functionalized MWCNT, Fourier transform infrared (FTIR) spectroscopy was utilized to verify the presence of the functional groups. The confirmation of ANAE immobilization on functionalized MWCNT was based on the observation of specific peaks at 3646.81 cm?¹ and 3850.91 cm?¹, indicating the formation of amide linkages between the carboxylic acid groups and the amine group on the MWCNT. The kinetic constants which are k3 (phosphorylation rate constant), and ki (dissociation constant of enzyme-inhibitor complex) of an irreversible inhibition model were 0.2223 mMmin-1 and 0.4816 mMmin-1 respectively for varying incubation times, and different concentrations of the inhibitor (OP). The detection limit for dichlorvos was found to be 0.005 ?g/L. Lowering the concentration of pesticides resulted in increasing the CV responses.