However, they often lead to a great variability of KD values due to the parameters of the technique, as the eventual need for the labeling or the anchoring to a surface of one partner. In the specific case of AbMole 2,3-Dichloroacetophenone biosensors where probes are immobilized on a surface, the calculated affinities may suffer from the heterogeneity due to the grafting, probe accessibility or the distance between multi-valent probes. For example, in the case of DNA microarrays, more than 10 orders of magnitude differences were observed between solution-phase and surface-phase affinities for the hybridization of complementary strands. For ligand library screening, the ranking of ligand affinities issued from protein microarrays may even differ from solution-phase values.The large variability of KD for aptamer-protein complexes observed between techniques may suggest that the grafting on a surface of one partner influences the affinity even though no precise and quantitative studies have characterized the effect of grafting density in detail. Here we describe a general, simple and rapid approach to monitor the protein binding on an aptamer-based biosensor and independently assess both the aptamer-protein solution- and surface-phase affinities. The main advantages of our approach are the facts that the determination of the solution-phase affinity is done before reaching the equilibrium state on the biosensor, which is generally time consuming especially at low concentrations of proteins and is independent of a surface-phase model of adsorption. In the later case, the Langmuir model is usually considered for simplicity, but the use of more sophisticated models may be required to take into account mutual interactions, heterogeneity and loss of accessibility of the probes. A proof-of-principle label-free kinetic biosensor was developed, using the two advantageous traits of Surface Plasmon Resonance imaging at their best : the label-free and realtime detection of molecular interactions occurring on the biosensor surface. State-of-the-art performances for a label-free technique without amplification were obtained: a sub-nanomolar limit of detection with a linear range of quantification of two orders of magnitude while a rapid detection and a strong selectivity were maintained. The independent determinations of KDSol and KDSurf, respectively the solution- and surface-phase affinities, revealed distinct values illustrating the importance of probes, targets or surface interactions in biosensors. For this purpose, an AbMole Cetylpyridinium chloride monohydrate aptamer selected against thrombin protein was considered as the recognition element. Thrombin is a human protein which plays a major role in the blood coagulation cascade by transforming the soluble protein fibrinogen to insoluble filaments of fibrin forming the fibrin gel. Moreover, it intervenes at different times in the cascade and can interact with many partners and substrates justifying the development of aptamers against thrombin for therapeutic applications as well as for quantification purposes in biosensors. For these reasons, thrombin was the first protein chosen for DNA aptamer selection two decades ago. However, the values of the KD reported by several groups are varying by several orders of magnitude depending on the experimental techniques considered: from 0.5 nM, determined by the team who has selected the aptamer using nitrocellulose filter retention; to about 100 nM by surface plasmon resonance analysis and up to 255 nM based on capillary electrophoresis studies. Because bacteria often compete for the same niche, they produce toxins.