A fluorescence based assay with pyranine labeled hexa-histidine tagged Organophosphorus Hydrolase (OPH) for determination of organophosphates

Abstract

Organophosphorus hydrolase has been employed extensively for catalysis based biosensor synthesis with optical, potentiometric and amperometric based detection mechanism. The present study reports the response of bioconjuagtes prepared by utilizing OPH and pH sensitive polyanionic fluorophore i.e. pyranine (8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt) in different molar ratios by the aid of electrostatic force. Two types of bioconjugates were prepared, one of them contained mature form of OPH obtained from Escherichia coli cells expressing organophosphate degrading (opd) gene from a tac promoter, whereas the variant OPH_6His, has 6× histidine tail at C-terminus. The investigation was carried out utilizing the bioconjugates of different molar ratios prepared with both the enzymes and ratio showing maximum degree of labeling with pyranine was selected for the detection of organophosphates in standard samples. The lower limit of detection for paraoxon was ∼20 ppb and for methyl parathion and coumaphos it was ∼50 ppb under optimized conditions (55-60°C) with the reaction time of 3 min. These features of the prepared conjugate make it a strong contender for the development of the field deployable biosensor for organophosphates estimation.