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.