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 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.
Minic
Jasmina
J
INRA, Neurobiologie de l'Olfaction et de la Prise Alimentaire, Equipe Récepteurs et Communication Chimique, Domaine de Vilvert, 78352 Jouy-en-Josas Cedex, France.
Grosclaude
Jeanne
J
Aioun
Josiane
J
Persuy
Marie-Annick
MA
Gorojankina
Tatiana
T
Salesse
Roland
R
Pajot-Augy
Edith
E
Hou
Yanxia
Y
Helali
Salwa
S
Jaffrezic-Renault
Nicole
N
Bessueille
François
F
Errachid
Abdelhamid
A
Gomila
Gabriel
G
Ruiz
Oscar
O
Samitier
Josep
J
D000818:Animals; D001666:Binding Sites, Antibody; D015374:Biosensing Techniques; D015153:Blotting, Western; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D018625:Microscopy, Atomic Force; D016624:Negative Staining; D011485:Protein Binding; D012243:Rhodopsin / Q000737:chemistry / Q000276:immunology / Q000648:ultrastructure*; D012374:Rod Cell Outer Segment / Q000737:chemistry / Q000648:ultrastructure; D012394:Rosaniline Dyes; D020349:Surface Plasmon Resonance
D012394:Rosaniline Dyes; C048139:Coomassie blue; D012243:Rhodopsin