title: A high-throughput enzyme assay for organophosphate residues in milk.
abstract: A rapid, high-sensitivity, chemiluminescence (CL) enzyme assay for the determination of organophosphate (OP) residues in milk is presented. The assay for quantification of OP residues in milk is based on the inhibition of enzyme butyrylcholinesterase (BuChE). BuChE was stabilized and preloaded in 384 well plates at 30 °C. The assay permits rapid determination of OPs in milk within 12 min including an incubation step. The enzyme assay was tested for individual and mixtures of OPs such as methyl paraoxon (MPOx), methyl parathion (MP) and malathion (MT) in milk to evaluate their synergistic effect on BuChE inhibition. Good linearity was obtained in the range 0.005-50 μg·L(-1) for MPOx and 0.5-1,000 μg·L(-1) for MP as well as MT in milk. Mean recovery of 93.2%-98.6% was obtained for MPOx spiked milk samples with 0.99%-1.67% reproducibility (RSD). The proposed method facilitated rapid screening of milk samples in 384 well plate formats with further miniaturization presented in 1,536 well plates.
authors.affiliation: Biosensor Laboratory, Department of Chemistry, BITS, Pilani-K K Birla Goa Campus, Goa, 403726, India. rupeshm@bits-goa.ac.in
authors.forename: Rupesh K
authors.initials: RK
authors.lastname: Mishra
authors.forename: Kanchanmala
authors.initials: K
authors.lastname: Deshpande
authors.forename: Sunil
authors.initials: S
authors.lastname: Bhand
mesh_terms: D000818:Animals; D015374:Biosensing Techniques; D002091:Butyrylcholinesterase / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D002417:Cattle; D057075:Enzyme Assays / Q000295:instrumentation / Q000379:methods*; D004795:Enzyme Stability; D005506:Food Contamination / Q000032:analysis*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006736:Horses; D007306:Insecticides / Q000032:analysis; D008294:Malathion / Q000032:analysis; D008743:Methyl Parathion / Q000032:analysis; D008892:Milk / Q000737:chemistry*; D010755:Organophosphates / Q000032:analysis* / Q000378:metabolism; D010261:Paraoxon / Q000031:analogs & derivatives / Q000032:analysis
chemical_list: D007306:Insecticides; D010755:Organophosphates; D008743:Methyl Parathion; D002091:Butyrylcholinesterase; D010261:Paraoxon; D008294:Malathion; C008480:methylparaoxon