Sensitive Detection of Nitrogen-Fixing Soil Bacteria by Impedimetric Immunosensing

Rhizobium bacteria convert atmospheric nitrogen into ammonia that leguminous plants can use as a nitrogen source, reducing the need for synthetic nitrogen fertilizers. Bradyrhizobium sp. SUTN9–2 is an effective biofertilizer suitable for legume-rice rotational cropping systems that promotes growth and stress resistance. Deploying this biofertilizer for sustainable agriculture requires a rapid and cost-effective method for monitoring bacterial concentrations in the soil. In this work, we developed and optimized an antibody-based biosensor for the detection of Bradyrhizobium sp. SUTN9–2 via electrochemical impedance spectroscopy (EIS). The immunosensor was fabricated by covalently immobilizing a recombinant antibody specific to SUTN9–2 onto a dithiobis-succinimidyl propionate (DSP) self-assembled monolayer on a gold electrode. The resulting immunosensor effectively captures Bradyrhizobium sp. SUTN9–2 cells are present in samples, leading to changes in electrode impedance. The immunosensor demonstrated excellent specificity, a wide linear detection range of 1 × 10³ to 1 × 10⁷ CFU/mL (R² = 0.992), and a limit of detection approaching the single-cell detection limit. The compatibility of the sensor with a soil sample was also demonstrated. The platform holds great promise for the portable monitoring of soil biofertilizer levels and could also be adapted to other strains of interest.