A solid-state membrane potentiometric microsensor for in situ sensing of NH₄⁺ in soybean root nodules

Soybean-rhizobia symbiotic nitrogen fixation, a process in which rhizobia mediate biological nitrogen fixation by converting inert atmospheric nitrogen (N₂) into biologically available forms (e.g., ammonium, NH₄⁺), has been extensively investigated. However, non-invasive, in situ monitoring methods for this process remain lacking. Herein, we report a solid-state membrane potentiometric ammonium ion-selective microelectrode (NH₄⁺-ISμE) for the in situ detection of NH₄⁺ in soybean root nodules. A Prussian blue analogue with ion channels, which enables the specific insertion/extraction of NH₄⁺ ions while excluding interfering cations, was electrodeposited on a carbon fiber to fabricate the microelectrode. The cation sorption capability and ion selectivity of the thin film were explored by modulating the intercalation/deintercalation process and reducing the interfering cations within the framework. The NH₄⁺-ISμE exhibits a Nernstian response to NH₄⁺ over the concentration range of 1.0 × 10^-6 to 1.0 × 10^-3 M, with a detection limit of 6.2 × 10^-7 M. This sensor enables in situ, real-time detection of NH₄⁺-the direct product of biological nitrogen fixation in the legume plant-rhizobium symbiotic system. The release of NH₄⁺ ions in soybean root nodules during nitrogen fixation was successfully monitored. Overall, this work provides a simple and versatile tool for studying and monitoring biological symbiotic nitrogen fixation processes.