title: Screen-printed bienzymatic sensor based on sol-gel immobilized Nippostrongylusbrasiliensis acetylcholinesterase and a cytochrome P450 BM-3 (CYP102-A1) mutant.
abstract: Here, we describe the development of a bi-enzymatic biosensor that simplifies the sample pretreatment steps for insecticide detection, and opens the way for a highly sensitive detection of phosphorothionates in food. These compounds evolve their inhibitory activity towards acetylcholinesterases (AChEs) only after oxidation, which is performed in vivo by P450 monooxygenases. Consequently, phosphorothionates require a suitable sample pretreatment by selective oxidation to be detectable in AChE based systems. In this study, enzymatic phosphorothionate activation and AChE inhibition were integrated in a single biosensor unit. A triple mutant of cytochrome P450 BM-3 (CYP 102-A1) and Nippostrongylus brasiliensis AChE (NbAChE) was immobilized using a fluoride catalyzed sol-gel process. Different sol-gel types were fabricated and characterized regarding enzyme loading capacity and enzyme activity containment. The enzyme sol-gel itself already proved to be suitable for the highly sensitive detection of paraoxon and parathion in a spectrometric assay. A method for screen-printing of this enzyme sol-gel on thick film electrodes was developed. Finally, amperometric biosensors containing coimmobilized NbAChE and the cytochrome P450 BM-3 mutant were produced and characterized with respect to signal stability, organophosphate detection, and storage stability. The detection limits achieved were 1 microg/L for paraoxon and 10 microg/L for parathion, which is according to EC regulations the highest tolerable pesticide concentration in infant food.
authors.affiliation: Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Waibel
authors.forename: Holger
authors.initials: H
authors.lastname: Schulze
authors.forename: Norbert
authors.initials: N
authors.lastname: Huber
authors.forename: Till T
authors.initials: TT
authors.lastname: Bachmann
mesh_terms: D000110:Acetylcholinesterase / Q000737:chemistry*; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics; D004563:Electrochemistry / Q000295:instrumentation* / Q000379:methods; D004800:Enzymes, Immobilized / Q000737:chemistry; D004867:Equipment Design; D019544:Equipment Failure Analysis; D007306:Insecticides / Q000032:analysis*; D009559:Nippostrongylus / Q000201:enzymology*; D009938:Organizations; D010755:Organophosphates / Q000032:analysis*; D010105:Oxygenases / Q000737:chemistry* / Q000235:genetics; D044367:Phase Transition; D011327:Printing / Q000379:methods; D011994:Recombinant Proteins / Q000378:metabolism; D014159:Transducers
chemical_list: D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D007306:Insecticides; D010755:Organophosphates; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; C086254:P450SU1 protein, Streptomyces griseolus; D000110:Acetylcholinesterase