Cell-based biohybrid sensing of a volatile aggregation pheromone component associated with the invasive red palm weevil

The red palm weevil (Rhynchophorus ferrugineus, RPW) is a highly destructive invasive pest of palm trees, causing severe agricultural and economic losses worldwide. Adult males release an aggregation pheromone, primarily (4RS,5RS)-4-methylnonan-5-ol (ferrugineol), which mediates colony formation and infestation within palm trunks. Because all life stages of this weevil are hidden inside the tree and remain undetected until fatal damage occurs, rapid and sensitive detection of pheromone emissions from the weevil colony is crucial for early detection and monitoring. However, practical sensor technologies capable of detecting this pheromone have not yet been established. Here, we report a cell-based biohybrid sensor capable of detecting pheromones in the vapor phase. This sensor employs HEK293 cells transiently co-expressing the RPW pheromone receptor RferOR1, its co-receptor RferOrco, and the genetically encoded fluorescent calcium indicator GCaMP. The specificity and sensitivity of these cells were first validated for ferrugineol in aqueous solution (0.1-10 μM), showing decreased responses at 100 μM indicative of non-monotonic behavior. The cells were then encapsulated in hydrogel matrices and integrated into a microwell array. We found that the resulting cell-based sensor exhibited a monotonic fluorescence response to ferrugineol across a broader concentration range (0.1-100 μM), likely due to moderated diffusion of ferrugineol within the hydrogel. Furthermore, the sensor successfully detected ferrugineol in the vapor phase at sub-ppm concentrations (0.1-100 ppm). These findings demonstrate that the developed sensor provides a technological basis for pheromone-detection systems for RPW monitoring, thereby extending the applicability of biohybrid sensing to ecologically relevant odorants.