Development of electrochemical biosensors using insect odorant receptors

Abstract

Natural olfactory systems of insects achieve remarkable sensitivity and accuracy through a repertoire of odorant receptors (OR) with distinct specificities. To harness insects’ extraordinary sense of smell, biosensors for odorant detection must employ insect ORs as biorecognition elements. Sensor approaches have used insect ORs expressed in cells, or purified insect ORs reconstituted into liposomes, lipid bilayers and nanodisc formats. The nanodiscs have emerged as a prominent display format for their superior sensitivity, stability and nano-scale dimensions suitable for achieving a high surface coverage on miniaturized devices. This thesis reports research for developing an electrochemical biosensor for odorant detection using insect ORs in nanodiscs. The odorant sensing methodology employs electrochemical impedance spectroscopy (EIS) to evaluate conducting polymer and gold substrates for sensor development. A recombinantly expressed and purified OR from the common fruit fly (DmOr22a, Drosophila melanogaster) was reconstituted in nanodiscs and used as the model receptor-display format system for this research. Sensor development was first explored with a conducting polymer substrate which featured an optimised polymer deposition to yield rough, conductive and stable surfaces of poly(pyrrole-co-pyrrole-3-carboxylic acid). Odorant sensing performance was evaluated with DmOR22a nanodiscs immobilised via multiple modes including covalent attachment, entrapment during electro polymerisation and physical adsorption. The developed sensor exhibited non-specific signals for empty nanodiscs and indistinguishable responses to target and control ligands for DmOr22a. The report discusses potential factors for the observed sensor performance and challenges of the trialled methodology. Subsequently, a simplified gold substrate for further sensor development is reported. DmOR22a nanodiscs covalently immobilized on a gold substrate were characterized to study surface modification and understand the underlying mechanisms associated with ligand binding. The developed EIS sensor validated DmOr22a activity by exhibiting a high sensitivity and specificity towards ethyl hexanoate with a detection limit of 5.5 fM. Furthermore, neutron reflectometry studies provided evidence to support conformational changes in the receptor upon ligand binding. Following the successful development of a facile odorant sensing methodology, the next step was to attempt to improve signal variation and sensitivity. Nanodisc preparations were investigated by a multitude of characterisation techniques to develop a deeper understanding of the sample. Nanodisc sample and sensor surface characterisations were performed using atomic force microscopy (AFM), quartz crystal microbalance with dissipation monitoring (QCM-D), transmission electron microscopy (TEM), neutron reflectometry and dynamic light scattering (DLS). The research reports discovery of amorphous structures and aggregates of various dimensions in nanodisc preparations prompting further purification of the sample. Nickel nitrilotriacetic acid (Ni-NTA) purification was carried out to isolate an active and homogeneous fraction. EIS sensing experiments showed the purified fraction to be functional, however, a reduced sensitivity is observed compared to the DmOr22a nanodisc preparations. Potential factors are discussed in this report with proposals for further investigations to achieve superior sensor performance. The findings in this study significantly contribute towards understanding fundamental aspects of odorant sensing mechanism and nature of the DmOr22a nanodisc preparations. The outcomes of this study have the potential to lead to sensor design and methodology improvements for insect OR nanodisc based biosensors.