[P004] Poster

1:00 PM - 1:50 PM JST(4:00 AM - 4:50 AM UTC)

[P02-242] A Novel Whole-Cell Biosensor for Pesticide Detection via OMV-Displayed and a Responsive Genetic Circuit

I-Chieh Wu¹, ○SHEN-LONG TSAI¹ (1. National Taiwan University of Science and Technology (Taiwan))

Keywords:

Organophosphorus biosensor,Outer membrane vesicles,Whole-cell biosensing,Paraoxon detection,Responsive Genetic Circuit

Organophosphorus compounds, such as paraoxon, are highly toxic inhibitors of acetylcholinesterase and pose serious risks to human health and environmental safety. However, the development of whole-cell biosensors for paraoxon detection remains challenging due to the absence of natural transcription factors that directly recognize this compound. To address this limitation, we engineered a modular Escherichia coli–based biosensing system that converts paraoxon into a detectable phenolic intermediate, enabling sensitive and cost-effective environmental monitoring. The system was constructed using the hyper-vesiculating strain E. coli JC8031 to establish a dual-functional microbial platform integrating extracellular degradation with intracellular signal transduction. To overcome mass-transfer constraints and enzyme inhibition associated with cytosolic expression, organophosphorus hydrolase (OPH) was displayed on outer membrane vesicles (OMVs). This surface localization provides a high surface-area-to-volume ratio, facilitating rapid hydrolysis of paraoxon in the extracellular environment. The hydrolysis product, p-nitrophenol (PNP), serves as a diffusible signaling molecule. Signal detection was achieved via a phenol-responsive transcriptional regulatory system. In the presence of PNP, a phenol-inducible transcription factor activates its cognate promoter, driving the expression of green fluorescent protein (GFP) as an optical reporter. This design effectively links extracellular enzymatic activity to intracellular gene expression, forming a “degradation-sensing” module. Experimental results demonstrate that OPH-displayed OMVs are efficiently secreted and localized extracellularly, resulting in significantly enhanced paraoxon degradation compared to traditional intracellular OPH expression. Upon paraoxon exposure, the system exhibited a clear, dose-dependent increase in GFP fluorescence. The biosensor showed high sensitivity and a strong signal-to-noise ratio, indicating effective coupling between paraoxon hydrolysis and reporter activation. Despite its performance, several considerations remain. The specificity of the phenol-inducible transcription factor may lead to cross-reactivity with other phenolic compounds present in environmental samples, potentially generating false-positive signals. In addition, the stability and functionality of OMVs under varying environmental conditions, including fluctuations in pH and temperature, require further evaluation to ensure reliability during field deployment. In summary, this study introduces a modular biosensing strategy that circumvents the lack of a direct paraoxon-responsive regulatory element by leveraging metabolic conversion to a detectable intermediate. By coupling extracellular enzymatic degradation with intracellular genetic reporting, this platform provides a practical approach for paraoxon detection and establishes a generalizable framework for sensing structurally complex toxins via their degradation products. This work highlights the potential of engineered microbial systems for environmental surveillance and biosensing applications.