Synergistic improvement in the performance of insect odorant receptor based biosensors in the presence of Orco.

Insect odorant receptors (ORs) are believed to be a complex of an odorant binding subunit, OrX, and an ion channel forming subunit, Orco. In our previous study, we showed that the OrX subunit on its own in liposomes could detect volatile organic compounds (VOCs) ultrasensitively using Electrochemical Impedance Spectroscopy (EIS). In this study, we investigated the effect of the presence of Orco on the response of the OrX subunit to detect the VOCs. The OrXs - Or10a, Or22a, Or35a and Or71a, together with Orco, were recombinantly expressed, purified and integrated into liposomes. These OrX/Orco liposomes were covalently attached to a gold surface modified with N-hydroxysuccinimide/1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) (NHS/EDC)-activated self-assembled monolayers (SAMs) of 6-mercaptohexanoic acid (MHA). It was demonstrated that the OrX/Orco liposomes could sensitively and selectively detect their ligands by monitoring a change in frequency and impedance signal upon binding with both Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) and EIS. Using EIS, three OrXs (Or10a, Or22a and Or35a) showed a shift in their dose-response curves when Orco was co-integrated, reflecting an increase in ligand sensitivity and a decrease in limit of detection (LOD). Or71a in the presence of Orco did not show any improvement in ligand sensitivity as this is a highly tuned receptor which may be already at the sensitivity limit for EIS. The observed enhancement in sensor performance is believed to be an effect of Orco which is stabilizing the OrX in a more active conformation and amplifying charge transfer to result in a greater reduction in impedance.