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Quantification of receptor targeting aptamer binding characteristics using single-molecule spectroscopy

摘要:

This experimental design presents a single molecule approach based on fluorescence correlation spectroscopy (FCS) for the quantification of outer membrane proteins which are receptors to an aptamer specifically designed to target the surface receptors of live Salmonella typhimurium. By using correlation analysis, we also show that it is possible to determine the associated binding kinetics of these aptamers on live single cells. Aptamers are specific oligonucleotides designed to recognize conserved sequences that bind to receptors with high affinity, and therefore can be integrated into selective biosensor platforms. In our experiments, aptamers were constructed to bind to outer membrane proteins of S. typhimurium and were assessed for specificity against Escherichia coli. By fluorescently labeling aptamer probes and applying FCS, we were able to study the diffusion dynamics of bound and unbound aptamers and compare them to determine the dissociation constants and receptor densities of the bacteria for each aptamer at single molecule sensitivity. The dissociation constants for these aptamer probes calculated from autocorrelation data were 0.1285 and 0.3772nM and the respective receptor densities were 42.27 receptors per μm~2 and 49.82 receptors per μm~2. This study provides ample evidence that the number of surface receptors is sufficient for binding and that both aptamers have a high-binding affinity and can therefore be used in detection processes. The methods developed here are unique and can be generalized to examine surface binding kinetics and receptor quantification in live bacteria at single molecule sensitivity levels. The impact of this study is broad because our approach can provide a methodology for biosensor construction and calculation of live single cell receptor-ligand kinetics in a variety of environmental and biological applications.

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作者: Book, B. [1] ; Chen, J. [1] ; Irudayaraj, J. [1]
作者单位: Purdue University, Bindley Bioscience and Birck Nanotechnology Center, 215 ABE Building, 225 South University Street, West Lafayette, IN 47907, United States [1]
DOI: 10.1016/j.aca.2005.08.026
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