Odorant receptors are an excellent example of natural superiority in specifically binding specific, small and hydrophobic
molecules. They are of particular interest in the development of a sensor platform for G protein-coupled receptors
(GPCRs). Odorant receptors (OR5) of Rattus norvegicus were incorporated into model membranes by in vitro synthesis
and vectorial incorporation for achieving natural receptor function. The vectorial insertion of OR5 into the planar membrane
and their lateral distribution, their interactions and their mobility within the membrane are of great importance for
ligand-receptor interaction. We applied total internal reflection fluorescence (TIRF) microscopy and image analysis to
assess the insertion and the OR5 distribution as well as the lateral mobility of these receptors at the single molecule level.
The vectorial incorporation of OR5 into planar lipid membranes was investigated with TIRF microscopy and image segmentation.
With increasing expression time, the OR5 incorporation density and aggregation increased linearly by about
0.02μm-2min-1. The expression and incorporations of single OR5s were completed within about 8 minutes. The mobility
of the incorporated receptors was measured with fluorescence correlation spectroscopy (FCS) and fluorescence recovery
after photo-bleaching (FRAP). These measurements revealed that the incorporated receptors were immobilized with this
class of lipid membranes.
This contribution summarizes some of our efforts in designing, assembling and functionally characterizing supramolecular interfacial architectures for bio-affinity studies and for biosensor development. All the surface interaction studies will be based on the recently introduced novel sensor platforms involving surface plasmon fluorescence spectroscopy (SPFS) and -microscopy (SPFM). Emphasis will be put on documenting the distance-dependence of fluorescence intensity at the metal-dielectric interface and utilizing this principle to optimize the conformation/orientation of the interfacial supra-molecular sensor coatings. This is exemplified by a number of examples, including a layer-by-layer assembly system, antibody-antigen interactions, oligonucleotide-oligonucleotide, and oligonucleotide-PCR amplicon hybridization. For practical sensing purposes, a three-dimensionally extended surface coating is then employed to overcome the fluorescence quenching problem on a planar matrix. A commercial dextran layer is shown to be an optimized matrix for SPFS, with an example of a protein-binding study.