Dual Colorimetric and Fluorometric Biosensing Assays for Organophosphate Pesticide Detection Using Monomeric Teal Fluorescent Protein (mTFP1)-Organophosphate Degrading Enzyme Fusion Proteins Expressed in <i>E. coli</i>

Organophosphate compounds (OPs) are widely used chemicals generally employed as insecticides, pesticides, and chemical warfare agents. They bioaccumulate and cause toxicity to the environment and living organisms. To safeguard human health, it is important to develop a reliable analytical method for quantifying these chemicals in the environment. In recent years, enzymatic biosensors have gained popularity for their rapid, precise, specific, and continuous monitoring of the analyte. In this study, two fusion proteins, namely, organophosphate acid anhydrolase-FL variant-monomeric teal fluorescent protein (OPAA-FL-mTFP1) and Nus-Organophosphate hydrolase-monomeric teal fluorescent protein (Nus-OPH-mTFP1), were utilized for colorimetric and fluorescence biosensing of organophosphate insecticides and pesticides. These fusion proteins have been shown to exhibit both the OP hydrolyzing ability and pH-based fluorescence sensing ability simultaneously. The limits of detection (LOD) for fluorometric biosensing of ethyl paraoxon (EP) using OPAA-FL-mTFP1 and Nus-OPH-mTFP1 were 42 and 76 μM, respectively. Spike and recovery of EP in tap water showed recoveries in the range of 94–110%. This biosensing assay offers advantages in terms of sensitivity, simplicity, and rapid response times, positioning it as a promising tool for monitoring organophosphate contamination in the environment.