Immobilization of native membrane-bound rhodopsin on biosensor surfaces

In this paper, we evaluated the grafting of G-protein-coupled receptors (GPCRs) onto functionalized surfaces, which is a primary requirement to elaborate receptor-based biosensors, or to develop novel GPCR assays. Bovine rhodopsin, a prototypical GPCR, was used in the form of receptor-enriched membr...

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Published in:Biochimica et biophysica acta 2005-08, Vol.1724 (3), p.324-332
Main Authors: Minic, Jasmina, Grosclaude, Jeanne, Aioun, Josiane, Persuy, Marie-Annick, Gorojankina, Tatiana, Salesse, Roland, Pajot-Augy, Edith, Hou, Yanxia, Helali, Salwa, Jaffrezic-Renault, Nicole, Bessueille, François, Errachid, Abdelhamid, Gomila, Gabriel, Ruiz, Oscar, Samitier, Josep
Format: Article
Language:English
Subjects:
Animals
Atomic force microscopy
Binding Sites, Antibody
Biosensing Techniques
Blotting, Western
Cattle
Cell Membrane - chemistry
Cell Membrane - ultrastructure
Chemical Sciences
Electrochemical impedance spectroscopy
Electrophoresis, Polyacrylamide Gel
Functional immobilization
G-protein-coupled receptor
Membrane
Microscopy, Atomic Force
Negative Staining
Protein Binding
Rhodopsin - chemistry
Rhodopsin - immunology
Rhodopsin - ultrastructure
Rod Cell Outer Segment - chemistry
Rod Cell Outer Segment - ultrastructure
Rosaniline Dyes
Surface Plasmon Resonance
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Summary:In this paper, we evaluated the grafting of G-protein-coupled receptors (GPCRs) onto functionalized surfaces, which is a primary requirement to elaborate receptor-based biosensors, or to develop novel GPCR assays. Bovine rhodopsin, a prototypical GPCR, was used in the form of receptor-enriched membrane fraction. Quantitative immobilization of the membrane-bound rhodopsin either non-specifically on a carboxylated dextran surface grafted with long alkyl groups, or specifically on a surface coated with anti-rhodopsin antibody was demonstrated by surface plasmon resonance. In addition, a new substrate based on mixed self-assembled multilayer that anchors specific anti-receptor antibodies was developed. Electrochemical impedance spectroscopy performed upon deposition of membrane-bound rhodopsin of increasing concentration exhibited a significant change, until a saturation level was reached, indicating optimum receptor immobilization on the substrate. The structures obtained with this new immobilization procedure of the rhodopsin in its native membrane environment are stable, with a controlled density of specific anchoring sites. Therefore, such receptor immobilization method is attractive for a range of applications, especially in the field of GPCR biosensors.
ISSN:0304-4165
0006-3002
1872-8006
DOI:10.1016/j.bbagen.2005.04.017