Kinetics and thermodynamics of biotinylated oligonucleotide probe binding to particle-immobilized avidin and implications for multiplexing applications
Broder, Graham R., Ranasinghe, Rohan T., Neylon, Cameron, Morgan, Hywel and Roach, Peter L. (2011) Kinetics and thermodynamics of biotinylated oligonucleotide probe binding to particle-immobilized avidin and implications for multiplexing applications. Analytical Chemistry, 83, (6), 2005-2011. (doi:10.1021/ac102762q). (PMID:21291180).
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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.
|Digital Object Identifier (DOI):||doi:10.1021/ac102762q|
|Subjects:||Q Science > QD Chemistry
Q Science > QR Microbiology > QR355 Virology
|Divisions:||University Structure - Pre August 2011 > School of Chemistry
|Date Deposited:||31 Mar 2011 10:59|
|Last Modified:||28 Mar 2014 15:17|
Basic Technology: 4 Billion Bases a Day - Practical Individual Genome Sequencing
Funded by: EPSRC (GR/S23513/01)
1 October 2003 to 30 September 2008
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