Amperometric Detection of Parathion and Methyl Parathion with a Microhole-ITIES Md. Mokarrom Hossain, a Chang Sup Kim, b Hyung Joon Cha, b Hye Jin Lee* a a Department of Chemistry and Green-Nano Materials Research Center, Kyungpook National University, 1370 Sankyuk-dong, Buk-gu, Daegu, 702-701 Korea b Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790–784, Korea *e-mail: hyejinlee@knu.ac.kr Received: March 31, 2011; & Accepted: May 5, 2011 Abstract An amperometric sensor featuring a microhole-liquid/gel interface for the detection of both parathion and methyl parathion is developed on the basis of their different kinetics behavior when interacting with the enzyme organo- phosphorus hydrolase (OPH). OPH hydrolyzes parathion and methyl parathion producing a common product of para-nitrophenol and either diethylthio- or dimethylthio- phosphoric acid, respectively, of which all can release pro- tons depending upon their pKa values. The detection method for both organophosphate (OP) compounds is de- signed to measure the current associated with the transfer of protons released from the products of OPH hydrolysis across a polarized microhole-water/polyvinylchloride-nitrophenyloctylether (PVC-NOPE) gel interface. The selec- tive transfer of protons across the interface is tailored by the use of a proton selective ligand, ETH 1778, in the gel layer. A disposable proton selective sensor that can quantitatively analyze the OP compounds is also fabricated using simple polydimethylsiloxane microfabrication. Cyclic voltammetry and differential pulse stripping voltamme- try are first utilized to characterize the transfer of protons across the microhole-water/PVC-NPOE gel interface ini- tiated by the OPH reaction with parathion and methyl parathion and to establish a detection limit for each OP compound. In order to sequentially detect parathion and methyl parathion using a single proton selective strip- sensor, a novel time-resolved detection methodology is developed based on the different catalytic kinetics of OPH with each OP analyte; the maximum peak current for the preconcentrated protons transferring back from the or- ganic to water phase assisted by ETH 1778 increases proportionally to the concentration of each OP agent. Since the maximum peak currents for both OP analytes are observed at different reaction times it was possible to demon- strate the multiplexed analysis of both parathion and methyl parathion down to 0.5 mM using a single sensor. Keywords: Parathion, Methyl parathion, Enzyme catalysis, Microhole-liquid/gel interface, Sensors DOI: 10.1002/elan.201100190 1 Introduction Parathion (O,O-diethyl-O(4-nitrophenyl) thiophosphate) and methyl parathion (O,O-dimethyl-O(4-nitrophenyl) thiophosphate) are known to be one of the most effective insecticide agents that have been used for pest control in agricultural fields. For instance, they have been widely used to control diseases affecting various crops and vege- tables due to their low cost and high insecticidal activity against numerous pest species [1]. However, they irrever- sibly inhibit the activity of acetylcholinesterase (AChE) and cause the failure of nervous systems, behavioral defi- cits, respiratory paralysis and even death to humans and other animals [2, 3]. Also distinctive properties of these organophosphate (OP) compounds such as persistence, bioaccumulation and rapid adsorption through skin may cause even severe health hazard to living species as well as environmental damages [4, 5]. Therefore, a rapid and reliable quantification method for OP agents is of tre- mendous importance and there is a growing interest in the development of simple and inexpensive field deploya- ble devices. Over the past few decades, various analytical method- ologies, mainly gas or high-performance liquid chroma- tography and mass spectroscopy, have been developed for the determination of parathion and methyl parathion in environmental and contaminated food samples [6–8]. They are highly sensitive and accurate but also expensive, tedious, time consuming, labor-intensive as well as requir- ing pretreatment of samples. These drawbacks make them less amenable for field or on-site monitoring appli- cations. Alternatively, electroanalytical biosensors can offer an excellent alternative due to their diverse advan- tages including compact nature, rapid response, low cost, low power consumption and easy handling in field trials. In spite of the superb reduction-oxidation activities of Electroanalysis 2011, 23, No. 9, 2049 – 2056  2011 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim 2049 Full Paper