Gold Surface Functionalization and Patterning for Specific Immobilization of Olfactory Receptors Carried by Nanosomes

Abstract

There is substantial interest in engineering solid supports to achieve functional immobilization of membrane receptors both for investigation of their biological function and for the development of novel biosensors. Three simple and practical strategies for immobilization of a human olfactory receptor carried by nanosomes are presented. The basis of the functionalization of solid gold surfaces is a self-assembled monolayer (SAM) containing biotinyl groups. Biotinyl groups are subsequently used to attach neutravidin and then biotinylated monoclonal antibody directed against the receptor to allow its specific grafting. Surface plasmon resonance technique is implemented for real-time monitoring of step-by-step surface functionalization and, in addition, for testing the functional response of immobilized olfactory receptors. We show that OR1740 is functional when immobilized via a tag attached to its C-terminus, but not via its N-terminus. Finally, we demonstrate that gold surfaces can be patterned by the SAMs tested using microcontact printing. AFM images of immobilized nanosomes onto a patterned surface suggest that small nanosomes flatten and fuse into larger vesicles but do not merge into a continuous layer. The whole study emphasizes the outstanding performances of the BAT/PEGAT SAM, which could be useful for developing on-a-chip sensor formats for membrane receptor investigations and use.

Bioelectronic Noses Based on Olfactory Receptors

“…They can be obtained by cell disruption, purification of membrane microsomes and then by size homogenization and miniaturization of microsomes by extensive sonication (Vidic et al, 2006;Casuso et al, 2008). Each of this nanometric vesicular structure was shown to contain one or few receptors of the given type (Casuso et al, 2008;Vidic et al, 2007). The maintenance of nanosome homogeneity in the solution suggested that they can be used as a recognition part in an elementary nanobioelectronic sensor (Casuso et al, 2008;Vidic et al, 2006;Gabriel et al, 2006).…”

“…In order to optimize nanosome functional immobilization on them, several immobilization methods were checked on miliand micrometric scale. Functional responses were observed when nanosomes were simply adsorbed on the hydrophobic surface (Hou et al, 2006;Vidic et al, 2006;Benilova et al, 2008;Vidic et al, 2007) or when the receptor was captured by its specific antibody previously grafted to the sensor surface (Hou et al, 2006;Benilova et al, 2008). To further determinate optimal surface orientation of the receptor, various tagged OR17-40 were produced in yeast.…”

“…Thus, in the final device configuration, nanosomes bearing olfactory receptors tagged on their C-terminal were specifically immobilized onto conducting substrates via a self assembled monolayer containing biotinyl groups. Biotinyl groups were used to attach neutravidin and specific anti-tag antibodies to allow receptor specific grafting (Hou et al, 2006;Vidic et al, 2007;Benilova et al, 2008). The process was optimized by microcontact printing, and the anchored nanovesicles were visualized by Atomic Force Microscopy , Casuso et al, 2008.…”

Nanobiosensors Based on Individual Olfactory Receptors

“…Membrane proteins are clearly observed [4]. C -Electron microscopy of an immunogold-labeled nanosome : gold grains indicate the presence of the receptor [5].…”

“…Figure 5 : Specific immobilization of nanosomes on gold surface functionalized by a mixed Self Assembled Monolayer [6]. Schematic view of the two modes of nanosomes immobilization: nonspecific "adsorption" onto SAM, and "capture" by grafting via a specific antibody [5].…”