title: Kinetics of bioconjugate nanoparticle label binding in a sandwich-type immunoassay.
abstract: Nanoparticle labels have enhanced the performance of diagnostic, screening, and other measurement applications and hold further promise for more sensitive, precise, and cost-effective assay technologies. Nevertheless, a clear view of the biomolecular interactions on the molecular level is missing. Controlling the ratio of molecular recognition over undesired nonspecific adhesion is the key to improve biosensing with nanoparticles. To improve this ratio with an aim to disallow nonspecific binding, a more detailed perspective into the kinetic differences between the cases is needed. We present the application of two novel methods to determine complex binding kinetics of bioconjugate nanoparticles, interferometry, and force spectroscopy. Force spectroscopy is an atomic force microscopy technique and optical interferometry is a direct method to monitor reaction kinetics in second-hour timescale, both having steadily increasing importance in nanomedicine. The combination is perfectly suited for this purpose, due to the high sensitivity to detect binding events and the ability to investigate biological samples under physiological conditions. We have attached a single biofunctionalized nanoparticle to the outer tip apex and studied the binding behavior of the nanoparticle in a sandwich-type immunoassay using dynamic force spectroscopy in millisecond timescale. Utilization of the two novel methods allowed characterization of binding kinetics in a time range spanning from 50 ms to 4 h. These experiments allowed detection and demonstration of differences between specific and nonspecific binding. Most importantly, nonspecific binding of a nanoparticle was reduced at contact times below 100 ms with the solid-phase surface.
authors.affiliation: Laboratory of Biophysics, Institute of Biomedicine and Medicity Research Laboratories, University of Turku, Tykistökatu 6A, 20520, Turku, Finland, tuonar@utu.fi.
authors.forename: Tuomas
authors.initials: T
authors.lastname: Näreoja
authors.forename: Andreas
authors.initials: A
authors.lastname: Ebner
authors.forename: Hermann J
authors.initials: HJ
authors.lastname: Gruber
authors.forename: Barbara
authors.initials: B
authors.lastname: Taskinen
authors.forename: Ferry
authors.initials: F
authors.lastname: Kienberger
authors.forename: Pekka E
authors.initials: PE
authors.lastname: Hänninen
authors.forename: Vesa P
authors.initials: VP
authors.lastname: Hytönen
authors.forename: Peter
authors.initials: P
authors.lastname: Hinterdorfer
authors.forename: Harri
authors.initials: H
authors.lastname: Härmä
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D002417:Cattle; D005063:Europium / Q000737:chemistry; D006801:Humans; D007118:Immunoassay; D007368:Interferometry; D007700:Kinetics; D008027:Light; D053768:Metal Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D018625:Microscopy, Atomic Force; D011137:Polystyrenes / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000737:chemistry; D013972:Thyrotropin / Q000032:analysis*; D013997:Time Factors
chemical_list: D000911:Antibodies, Monoclonal; D011137:Polystyrenes; D012710:Serum Albumin, Bovine; D005063:Europium; D013972:Thyrotropin