title: Kinetics and thermodynamics of biotinylated oligonucleotide probe binding to particle-immobilized avidin and implications for multiplexing applications.
abstract: In this work, the kinetics and dissociation constant for the binding of a biotin-modified oligonucleotide to microparticle-immobilized avidin were measured. Avidin has been immobilized by both covalent coupling and bioaffinity capture to a surface prefunctionalized with biotin. The measured rate and equilibrium dissociation constants of avidin immobilized by these different methods have been compared with those for nonimmobilized avidin. We found that immobilization resulted in both a decrease in the rate of binding and an increase in the rate of dissociation leading to immobilized complexes having equilibrium dissociation constants of 7 ± 3 × 10(-12) M, higher than the value measured for the complex between biotin-modified oligonucleotide and nonimmobilized avidin and approximately 4 orders of magnitude larger than values for the wild-type avidin-biotin complex. Immobilized complex half-lives were found to be reduced to 5 days, which resulted in biotin ligands migrating between protein attached to different particles. Different immobilization methods showed little variation in complex stability but differed in total binding and nonspecific biotin-modified oligonucleotide binding. These findings are critical for the design of multiplexed assays where probe molecules are immobilized to biosensors via the avidin-biotin interaction.
authors.affiliation: School of Chemistry, University of Southampton, Southampton SO17 1BJ, U.K.
authors.forename: Graham R
authors.initials: GR
authors.lastname: Broder
authors.forename: Rohan T
authors.initials: RT
authors.lastname: Ranasinghe
authors.forename: Cameron
authors.initials: C
authors.lastname: Neylon
authors.forename: Hywel
authors.initials: H
authors.lastname: Morgan
authors.forename: Peter L
authors.initials: PL
authors.lastname: Roach
mesh_terms: D001360:Avidin / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D015374:Biosensing Techniques; D001710:Biotin / Q000378:metabolism; D019921:Biotinylation; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D015345:Oligonucleotide Probes / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D013816:Thermodynamics
chemical_list: D055767:Immobilized Proteins; D015345:Oligonucleotide Probes; D001360:Avidin; D001710:Biotin