ORIGINAL ABSTRACT

Glyphosate is the most widely used herbicide worldwide, but many current detection methods rely on lab-based chromatography, requiring costly equipment and expert users. Here, we describe a low-cost, field-deployable fluorescence biosensing platform for glyphosate detection in water and soil. An engineered variant of the Escherichia coli periplasmic binding protein PhnD was optimized through strategic fluorophore placement to produce a robust fluorescence signal increase upon glyphosate binding. The biosensor was integrated into a self-contained, 3D-printed device that functions as a miniature fluorometer, providing a simple yes-or-no output for non-expert users while retaining access to raw fluorescence data. The device exhibits nanomolar fluorescence sensitivity with results comparable to a benchtop fluorometer. Using this platform, glyphosate was reliably detected in buffered solutions, commercial herbicides, tap water, and soil extracts. To mitigate false positives arising from phosphate interference, we developed a dual-sensor strategy incorporating an independent phosphate biosensor and a second-generation device capable of multi-wavelength fluorescence detection. Together, these results demonstrate an affordable and versatile biosensing platform with strong potential for field-based environmental monitoring.