Colony isolation of odorant receptor-expressing insect sensor cells in hollow hydrogel microparticles for enhanced...

Cell-based biohybrid sensors using insect olfactory receptors (ORs) have emerged as promising sensing platforms for the selective and sensitive detection of biomarkers. A single sensor cell provides a weak signal, so the readout of most biohybrid sensors is the accumulated response of a whole population of cells. Response heterogeneity within engineered sensor cell populations can limit sensor sensitivity because low-responding cells dilute the bulk-averaged signal. Selecting highly responsive subclones could improve sensor sensitivity. However, most insect cell lines proliferate poorly at low cell density, making it challenging to obtain expandable colonies from a single insect cell. Here, we present a colony-isolation workflow for insect sensor cells using hollow hydrogel microparticles (PicoShells). Insect sensor cells were encapsulated in PicoShells at limiting dilutions to physically isolate one or a few cells. The encapsulated insect sensor cells were co-cultured with non-encapsulated support cells in the same suspension culture to promote colony formation. Individual colony-containing PicoShells were isolated and expanded into subclonal cell lines. Functional screening using a portable fluorescence detector and a microwell array identified a highly responsive subclone. In a human urine sample spiked with acetophenone, a candidate volatile biomarker of disease, this subclone showed an approximately 2.4-fold higher response sensitivity than the parental cell line, and the limit of detection was reduced from 1.56 ± 0.17 μM to 0.93 ± 0.22 μM for this subclone. These results indicate that colony isolation using PicoShells, combined with functional screening, provides a practical strategy for improving the sensitivity of insect cell biohybrid sensors.