Real-time observation of affinity reactions using grating couplers: determination of the detection limit and calculation of kinetic rate constants.
The use of integrated optical grating couplers for the analysis of bioaffinity reactions in order to calculate kinetic rate constants was investigated. The specificity of the sensor surface was determined by adsorptive or covalent attachment of the specific ligands. As an evanescent field sensor, the specific interaction of the corresponding ligand could be observed in real time and without labels. The detection limit in terms of the molecular weight of the analyte was studied by the specific binding of biotinylated proteins of different molecular weights to avidin-loaded sensors. It was shown that grating coupler sensors allowed detection of compounds of at least 2000 daltons using high-affinity receptors, while the direct sensing of low molecular analytes, such as biotin, could not be significantly achieved. Association rate constants were calculated for the interaction of the different biotinylated proteins to avidin-covered sensors from single binding curves. Due to the strong binding between avidin and biotin, the dissociation of the formed complex could not be observed. Kinetic rate constants and equilibrium constants were determined by studying the interaction of human immunoglobulin with the immobilized receptor, protein G. For the four human immunoglobulin subclasses a high affinity to protein G was determined with affinity constants ranging from 3.3 to 8.4 x 10(8) M-1.
Polzius
R
R
Gesellschaft für Biotechnologische Forschung mbH, Enzymologie, Braunschweig, Germany.
Diessel
E
E
Bier
F F
FF
Bilitewski
U
U
D000915:Antibody Affinity; D001360:Avidin; D015374:Biosensing Techniques; D001710:Biotin; D006801:Humans; D007074:Immunoglobulin G / Q000145:classification / Q000378:metabolism; D007158:Immunologic Techniques; D066298:In Vitro Techniques; D007700:Kinetics; D008024:Ligands; D055095:Optics and Photonics; D017452:Receptors, IgG / Q000276:immunology / Q000378:metabolism
D007074:Immunoglobulin G; D008024:Ligands; D017452:Receptors, IgG; D001360:Avidin; D001710:Biotin