Strategy of macromolecular grafting onto a gold substrate dedicated to protein-protein interaction measurements.
Many biotechnology applications use proteins immobilized on surface. For biosensor, the sensing layer is a key component interfacing the transducer and the sample. Strategies employed to activate the bidimensional surface act directly on the performance of the biosensor. In this paper we propose a novel strategy for engineered proteins self-assembly. Our original supramolecular structure allows a direct and fast covalent attachment of proteins onto bare gold substrate through a homobifunctional cross-linker, 1,4-di-([2'-pyridyldithio]propionamido)butane (DPDPB). In this work, engineered proteins and linker-protein complexes were synthesized and characterized by gel electrophoresis, chromatography and spectroscopy experiments. Macromolecular construction "DPDPB-GST tag-GEC1 protein" was conceived in order to guarantee a 2D architecture enhancing the capabilities of the target (tubulin) to recognize its partner (GEC1). Surface plasmon resonance measurements clearly showed potential of this particular self-assembled protein layer compared to a commercial immunosensor interface. At the concentrations tested, the recognition process occurs between tubulin and the immobilized GEC1; moreover enhanced binding was obtained with the home-made 2D sensing layer more than with 3D carboxymethyl dextran matrix.
Mansuy-Schlick
V
V
Equipe Estrogènes, Expression Génique et Pathologies du Système Nerveux Central, Université de Franche-Comté, U.F.R. Sciences et Techniques, IFR 133, 16 Route de Gray, 25030 Besançon Cedex, France.
Delage-Mourroux
R
R
Jouvenot
M
M
Boireau
W
W
D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D020099:Coated Materials, Biocompatible / Q000737:chemistry*; D003460:Crystallization / Q000379:methods*; D005240:Feasibility Studies; D006046:Gold / Q000737:chemistry*; D007118:Immunoassay / Q000379:methods*; D046911:Macromolecular Substances / Q000737:chemistry; D025941:Protein Interaction Mapping / Q000379:methods*; D020349:Surface Plasmon Resonance / Q000379:methods*
D020099:Coated Materials, Biocompatible; D046911:Macromolecular Substances; D006046:Gold