Kinetics and Thermodynamics of Biotinylated Oligonucleotide Probe Binding to Particle-Immobilized Avidin and Implications for Multiplexing Applications

摘要

In this work, the kinetics and dissociation constantnfor the binding of a biotin-modified oligonucleotide to microparticle-nimmobilized avidin were measured. Avidin has beennimmobilized by both covalent coupling and bioaffinity capturento a surface prefunctionalized with biotin. The measured ratenand equilibrium dissociation constants of avidin immobilizednby these different methods have been compared with those fornnonimmobilized avidin. We found that immobilization resultednin both a decrease in the rate of binding and an increase in thenrate of dissociation leading to immobilized complexes having equilibrium dissociation constants of 7(3u000110-12 M, higher than the valuenmeasured for the complex between biotin-modified oligonucleotide and nonimmobilized avidin and approximately 4 orders of magnitudenlarger than values for the wild-type avidin-biotin complex. Immobilized complex half-lives were found to be reduced to 5 days, whichnresulted in biotin ligands migrating between protein attached to different particles. Different immobilization methods showed littlenvariation in complex stability but differed in total binding and nonspecific biotin-modified oligonucleotide binding. These findings arencritical for the design of multiplexed assays where probe molecules are immobilized to biosensors via the avidin-biotin interaction.