Immobilization and surface characterization of NeutrAvidin biotin-binding protein on different hydrogel interlayers.
For a number of potential applications, it is desirable to immobilize avidin class molecules onto solid supports and exploit their ability to bind biotinylated molecules with high affinity. NeutrAvidin molecules were surface immobilized in various ways. In this study, NeutrAvidin was covalently attached by carbodiimide chemistry onto carboxyl groups of polyacrylic acid and carboxymethyl-dextran hydrogel interlayers. A third strategy involved the affinity "docking" of NeutrAvidin onto a biotinylated poly(ethylene glycol) interlayer. These three interlayers were selected for their low nonspecific binding of proteins, which was expected to minimize surface binding of NeutrAvidin by nonspecific interfacial adsorption. X-ray photoelectron spectroscopy (XPS) analyses allowed detailed characterization of the multilayer fabrication steps. An ELISA assay was used to measure NeutrAvidin activity, which varied with the surface immobilization route. Atomic force microcopy (AFM) force measurements showed that the hydrogel interlayer contributed to a repulsive force and verified the specific interaction between biotinylated AFM tips and the NeutrAvidin surfaces. When a solution of free biotin was injected into the AFM liquid cell, the force curve changed substantially and became identical to that recorded between surfaces carrying no NeutrAvidin, indicating that the free solution biotin had displaced NeutrAvidin proteins off the PEG-biotin layer.
Vermette
Patrick
P
CSIRO Molecular Science, Bag 10, Clayton South, Victoria 3169, Australia. patrick.vermette@usherbrooke.ca
Gengenbach
Thomas
T
Divisekera
Upulie
U
Kambouris
Peter A
PA
Griesser
Hans J
HJ
Meagher
Laurence
L
D001360:Avidin / Q000737:chemistry / Q000378:metabolism*; D001710:Biotin / Q000378:metabolism*; D004577:Electron Probe Microanalysis; D004797:Enzyme-Linked Immunosorbent Assay; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D018625:Microscopy, Atomic Force; D013499:Surface Properties
D001360:Avidin; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D001710:Biotin