title: Real time monitoring of thrombin interactions with its aptamers: insights into the sandwich complex formation.
abstract: Aptamers are raising an increasing interest for biosensor applications as replacements for antibodies due to their high stability and low cost. Thrombin, a key enzyme in the coagulation cascade, is an archetypical target against which two different aptamers, binding to two different exosites, have been selected. Recent studies dedicated to thrombin monitoring applications of biosensors have taken advantage of a potential sandwich-like structure between thrombin and these two aptamers for amplification purposes. However, in most cases, only end-point analysis was observed as a result of labeling requirements, thus preventing access to the kinetics of the complex formation. By using Surface Plasmon Resonance (SPR) imaging of aptamer-functionalized biosensors, we followed the binding of thrombin on the sensor and its interaction with a second reporter aptamer in real-time and in a label-free manner. Surprisingly, we showed that the injection of a second unlabeled-aptamer following the previous thrombin injection destabilized the thrombin-aptamer complex formed on the sensor surface, thus limiting any further amplification. However, the direct co-injection of thrombin, pre-complexed with a biotinylated aptamer bound to streptavidin efficiently increased the SPR signal by comparison to single thrombin detection. The various injection sequences performed may be rationalized considering a poor selectivity of one of the aptamers towards its exosite and a further negative allosteric effect upon sandwich complexation of the thrombin with its aptamers.
authors.affiliation: SPrAM (UMR 5819: CEA, CNRS, UJF), INAC, CEA Grenoble, 17 rue des Martyrs, 38054 Grenoble cedex 9, France.
authors.forename: Camille
authors.initials: C
authors.lastname: Daniel
authors.forename: Feriel
authors.initials: F
authors.lastname: Mélaïne
authors.forename: Yoann
authors.initials: Y
authors.lastname: Roupioz
authors.forename: Thierry
authors.initials: T
authors.lastname: Livache
authors.forename: Arnaud
authors.initials: A
authors.lastname: Buhot
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000235:genetics; D015374:Biosensing Techniques / Q000295:instrumentation*; D003199:Computer Systems; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D025941:Protein Interaction Mapping / Q000295:instrumentation*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D020349:Surface Plasmon Resonance / Q000295:instrumentation*; D013917:Thrombin / Q000032:analysis* / Q000737:chemistry / Q000235:genetics
chemical_list: D052157:Aptamers, Nucleotide; D013917:Thrombin