Aptamer-based QCM-D platform for monitoring biofertilizers in soil

The increasing use of bioinoculants to enhance soil quality and crop productivity requires efficient sensor platforms for fast monitoring. EU and national registration demand analytical methods to evaluate environmental fate and long-term effectiveness. Addressing these challenges requires evaluating bioinoculant persistence and impact on microbial communities, emphasizing the need for reliable detection methods in soil. This study presents an innovative aptamer-based sensing platform using Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) to detect microbial inoculants in soil extract, establishing the basis for portable sensors. The QCM-D platform is functionalized with single-stranded DNA (ssDNA) aptamers specifically designed to detect Bacillus subtilis strains. Specificity and selectivity are evaluated using inactivated B. subtilis in buffer and compared with negative controls ( P.protegens and mixed samples). One aptamer exhibits outstanding specificity, producing a statistically significant distinction between positive and negative controls ( p = 0.019) with a limit of detection (LoD) between 10 5 and 10 6 CFU/ml. To validate performance in real matrices, the platform is applied to inoculated soils. Two distinct soils are tested: sandy soil from Poland (neutral pH) and sandy loam from Germany (sub-alkaline pH). An optimized bacterial extraction method yields QCM-D-compatible samples, revealing an average persistence rate of approximately 72 %. In Polish soil, B. subtilis generates a response significantly different from controls ( p = 0.0002), while in German soil differences between B. subtilis and P. protegens are also significant ( p = 0.04). QCM-D analysis demonstrates significant specificity in both soils, as confirmed by triplicate experiments, supporting the aptasensor's robustness and potential for on-site monitoring of microbial inoculants in complex soil matrices. This study presents a novel QCM-D aptasensor for detecting Bacillus subtilis in soil, utilizing ssDNA aptamers for high specificity and sensitivity. Tested on Polish and German soils, the sensor maintained robust performance despite complex matrices, achieving a detection limit between 10 5 and 10 6 CFU/ml. The platform's adaptability makes it promising for real-time biofertilizer monitoring and agricultural applications. • QCM-D aptasensor detects B. subtilis with high specificity in clean buffer. • Aptamer selection based on best performance for complex matrix applications. • Optimized bacterial extraction from soil ensures QCM-D compatibility. • Reliable detection demonstrated in sandy soil from Poland and loam soil from Germany. • Lays the groundwork for practical on-site biofertilizer monitoring.