Witsanu Rapichai1, Sudarat Chaichalerm2, Jhirat Mearnchu3, Jesdawan Wichitwechkarn4. 1. Faculty of Engineering and Industrial Technology, Department of Biotechnology, Silpakorn University, Nakhon Pathom, 73000, Thailand. 2. Faculty of Science and Technology, Division of Science and Technology, Phetchaburi Rajabhat University, Phetchaburi, 76000, Thailand. 3. Faculty of Engineering and Industrial Technology, Department of Electrical Engineering, Silpakorn University, Nakhon Pathom, 73000, Thailand. 4. Faculty of Engineering and Industrial Technology, Department of Biotechnology, Silpakorn University, Nakhon Pathom, 73000, Thailand. wjesdawan@gmail.com.
Abstract
OBJECTIVE: To develop a convenient and efficient means for organophosphate (OP) insecticide detection, a simple, cost-effective, and easy-to-use absorbance-based sensing device was generated using methyl parathion hydrolase fused with glutathione-S-transferase (MPH-GST) covalently immobilized onto a chitosan film-coated microplate. RESULTS: With methyl parathion (MP) as a representative substrate, this MPH-GST sensing microplate had the detection limit of 0.1 µM and the linear range of 0.1-50 µM. Despite its highest stability at 4 °C, it was considerably stable at 25 °C with high activity for 30 days. It was also most stable at pH 8.0 and could be efficiently reused up to 100 rounds. The device revealed a high percentage of recovery for tap water spiked with a known concentration of MP, which was also comparable to the result obtained from gas chromatography-mass spectrometry. It also showed a high recovery of 82-100% with MP spiked agricultural products and satisfactory results with non-spiked samples. This immobilized enzyme sensing system was more sensitive and efficient than the whole cell system from our previous work. CONCLUSIONS: All of the advantages of the MPH-GST sensing microplate developed have rendered it suitable for rapid and convenient OP screening, and for being a bio-element for fabricating a potential optical biosensor in the future.
OBJECTIVE: To develop a convenient and efficient means for organophosphate (OP) insecticide detection, a simple, cost-effective, and easy-to-use absorbance-based sensing device was generated using methyl parathion hydrolase fused with glutathione-S-transferase (MPH-GST) covalently immobilized onto a chitosan film-coated microplate. RESULTS: With methyl parathion (MP) as a representative substrate, this MPH-GST sensing microplate had the detection limit of 0.1 µM and the linear range of 0.1-50 µM. Despite its highest stability at 4 °C, it was considerably stable at 25 °C with high activity for 30 days. It was also most stable at pH 8.0 and could be efficiently reused up to 100 rounds. The device revealed a high percentage of recovery for tap water spiked with a known concentration of MP, which was also comparable to the result obtained from gas chromatography-mass spectrometry. It also showed a high recovery of 82-100% with MP spiked agricultural products and satisfactory results with non-spiked samples. This immobilized enzyme sensing system was more sensitive and efficient than the whole cell system from our previous work. CONCLUSIONS: All of the advantages of the MPH-GST sensing microplate developed have rendered it suitable for rapid and convenient OP screening, and for being a bio-element for fabricating a potential optical biosensor in the future.