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OP high doses are acutely neurotoxic

This study explores the potential of using Esterase 2 (EST2) from A. acidocaldarius as a bioactive component in biosensors for the detection of organophosphates. The current methods of organophosphates detection are expensive, time-consuming, and require trained personnel. The aim is to develop a small, cheap, and fast biosensor that offers real-time detection and high selectivity.

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OP high doses are acutely neurotoxic

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  1. Use of EST2 from A. acidocaldarius as bioactive part in biosensors for organophosphates detection Ferdinando Febbraio Institute of Protein Biochemistry of CNR, Italy

  2. OP high doses are acutely neurotoxic OP low doses have several suspected mechanisms of action, including disruption of nuclear transcription factors, interference with neural cell development and neurotransmitter systems, and altered synaptic formation.

  3. Organophosphates detection AChE biosensors GC-MS HPLC-MS UPLC-ESMS Expensive Insufficient for in situ and real-time detection Unable to provide any information concerning the toxicity of the sample Require highly trained personnel Not sufficiently robust to deal with raw samples Do not offer adequate selectivity High resolution Automation Identification of a large number of metabolite in a single measure Comparable or even better analytical performances than traditional systems Small, cheap and fast. Easy to handle (do not require trained technicians to be used) Real-time detection

  4. Esterase 2 from Alicyclobacillus acidocaldarius EST2 is a monomeric carboxylesterase of about 34 Kda belonging to the hormone sensitive lipase (HSL) family. EST2 hydrolyses monoacyl esters of different acyl chain lengths, displaying maximal activity on p-nitrophenyl esters characterized by an acyl chain length of 6-8 carbon atoms, at an optimal temperature of 70 °C. Manco et al., Biochem. J. 1998

  5. E + I-OH k cat H2O (k3) ki KD k trans Ki (k2) E + I E•I E-I• Idrossilammina E-I E + I- Idrossilammina Idrossilammina Paraoxon inhibition

  6. Paraoxon inhibition Febbraio et al., Extremophiles, 2008

  7. Paraoxon detection Febbraio et al., Analitical Chemistry, 2011

  8. New assay using fluorescent substrate Excitation wavelength: 365 nm 4-Methylumbelliferyl butyrate 4-Methylumbelliferone Emission wavelength: 445 nm Changes in enzyme specificity by site-directed mutagenesis

  9. Automated approach with Robotic Station

  10. Characterization of new EST2 mutants Binding with fluorescent probes Assay on nerveagents Development of principal component analysis software for OP mixture detection

  11. Collaborations People Paola Carullo Giovanni P. Cetrangolo Carla Gori Giuseppe Manco Luigi Mandrich GRANTS Industries

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