© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 565 Biotechnol. J. 2011, 6, 565–571 DOI 10.1002/biot.201000348 www.biotechnology-journal.com 1 Introduction Methyl parathion (MPT) (O,O-dimethyl O-4-nitro- phenyl phosphorothioate) is an organophosphate (OP) insecticide that has caused many health prob- lems, particularly in developing countries since it was introduced to the market in the early 1950s [1]. WHO classifies MPT as a class Ia ‘extremely haz- ardous’ pesticide, highly toxic by inhalation and in- gestion, and moderately toxic by dermal adsorption [2].The use of MPT is banned or restricted in many countries and its import is illegal in some countries. Uptake and absorption of MPT in plants is fairly rapid and occurs within 4 days after application [3]. WHO still recommends that a range of fruits and vegetables be monitored for the residue regularly. The LC, GC or GC-MS are normally used for qual- itative and quantitative determination of OPs in- cluding MPT [4, 5]; however, a drawback to these methods is that they require expensive instrumen- tation and are not suitable for on-site use. For this reason, there has been interest in the detection of OPs by biosensors, as one possible strategy to de- velop simple hand-held detectors. Reported meth- ods have involved the use of acetylcholinesterase (AChE) inhibition-based amperometric detection and optical techniques [6-8]; however, these meth- ods have a limitation in selectivity, since AChE is inhibited by various neurotoxins including OPs, carbamates, and many other compounds [9]. To provide better selectivity,either organophosphorus hydrolase (OPH), or methyl parathion hydrolase (MPH), which hydrolyze parathion into p-nitro- phenol and diethyl thiophosphate can be used to develop amperometric, potentiometric, and optical Research Article A colorimetric assay for determination of methyl parathion using recombinant methyl parathion hydrolase Dau Hung Anh 1 , Kritsananporn Cheunrungsikul 1 , Jesdawan Wichitwechkarn 2 and Werasak Surareungchai 3 1 Pilot Plant Development and Training Institute, King Mongkut’s University of Technology Thonburi, Bangkok, Thailand 2 Department of Biotechnology, Faculty of Engineering and Industrial Technology, Silpakorn University, Amphor Muang, Nakhon Pathom, Thailand 3 School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi, Bangkok, Thailand A simple, rapid and sensitive colorimetric dipstick assay for the detection of the organophospho- rous insecticide methyl parathion (MPT) residue in vegetables was developed. The assay was based on the hydrolysis of MPT by a recombinant methyl parathion hydrolase (recMPH), the en- coding gene of which was isolated from Burkholderia cepacia, a soil bacterium indigenous to Thai- land. This reaction generates protons leading to a change in pH that correlates with the amount of MPH present. Hence, the pH indicator bromothymol blue was used to monitor the MPH hydrolysis as the associated color changes can be observed by the naked eye. The recMPH was immobilized on a PVDF membrane to establish a dipstick assay format. The assays could detect MPT residues in spiked vegetable samples at the concentration of 1 mg/L without using analytical instrumentation. The test is reusable and stable for up to 3 months in the absence of any pre- servatives. Keywords: Biosensors · Bromothymol blue · Colorimetric assay · Methyl parathion · Recombinant methyl parathion hydrolase Correspondence: Dr. Werasak Surareungchai, School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi, Bangkhuntien-Chaitalay Rd., Bangkok 10150, Thailand Fax: +66-2-452-3455 E-mail: werasak.sur@kmutt.ac.th Abbreviations: AChE, acetylcholinesterase; BTB, bromothymol blue; EPN, O-ethyl O-(4-nitrophenyl) phenylphosphonothioate; KPi, potassium phos- phate buffer; MPT, methyl parathion; MPH, methyl parathion hydrolase; OP, organophosphate; OPH, organophosphorus hydrolase; recMPH, recombinant methyl parathion hydrolase Received 14 October 2010 Revised 22 December 2010 Accepted 24 January 2011