Solution-phase vs surface-phase aptamer-protein affinity from a label-free kinetic biosensor.
Aptamers are selected DNA ligands that target biomolecules such as proteins. In recent years, they are showing an increasing interest as potential therapeutic agents or recognition elements in biosensor applications. In both cases, the need for characterizing the mating between the target and the aptamer either in solution or immobilized on a surface, is pressing. In this context, we have developed a kinetic biosensor made of micro-arrayed anti-thrombin aptamers to assess the kinetic parameters of this interaction. The binding of label-free thrombin on the biosensor was monitored in real-time by Surface Plasmon Resonance imaging. Remarkable performances were obtained for the quantification of thrombin without amplification (sub-nanomolar limit of detection and linear range of quantification to two orders of magnitude). The independent determinations of both the solution- and surface-phase affinities, respectively KD(Sol) and KD(Surf), revealed distinct values illustrating the importance of probes, targets or surface interactions in biosensors. Interestingly, KD(Surf) values depend on the aptamer grafting density and linearly extrapolate towards KD(Sol) for highly diluted probes. This suggests a lesser impact of the surface compared to the probe or target cooperativity interactions since the latter decrease with a reduced grafting density.
Laboratoire Structure et Propriétés d'Architectures Moléculaires, UMR 5819 CEA/CNRS/UJF, Institut Nanosciences et Cryogénie, Grenoble, France.
Camille
C
Daniel
Yoann
Y
Roupioz
Didier
D
Gasparutto
Thierry
T
Livache
Arnaud
A
Buhot
D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000378:metabolism; D015374:Biosensing Techniques; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D015203:Reproducibility of Results; D012996:Solutions; D020349:Surface Plasmon Resonance / Q000379:methods; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism
D052157:Aptamers, Nucleotide; D011506:Proteins; D012996:Solutions; C081676:thrombin aptamer; D013917:Thrombin