On the use of aptamer microarrays as a platform for the exploration of human prothrombin/thrombin conversion.

Microarrays are particular biosensors with multiple grafted probes that are generally used for parallel and simultaneous detection of various targets. In this study, we used microarrays with aptamer probes in order to follow up the different biomolecular interactions of a single enzyme, the thrombin protein, involved in the complex coagulation cascade. More precisely, thanks to label-free surface plasmon resonance imaging, we were able to monitor in real time an important step in the firing of the coagulation cascade in situ-the enzymatic transformation of prothrombin into thrombin, catalyzed by factor Xa. We were also able to appraise the influence of other biochemical factors and their corresponding inhibiting or enhancing behaviors on thrombin activation. Our study opens the door for the development of a complete microarray-based platform not only for the whole coagulation cascade analysis but also for novel drug screening assays in pharmacology.

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.

Real time monitoring of thrombin interactions with its aptamers: insights into the sandwich complex formation.

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.

Glucose modulates [Ca2+]i oscillations in pancreatic islets via ionic and glycolytic mechanisms.

Pancreatic islets of Langerhans display complex intracellular calcium changes in response to glucose that include fast (seconds), slow ( approximately 5 min), and mixed fast/slow oscillations; the slow and mixed oscillations are likely responsible for the pulses of plasma insulin observed in vivo. To better understand the mechanisms underlying these diverse patterns, we systematically analyzed the effects of glucose on period, amplitude, and plateau fraction (the fraction of time spent in the active phase) of the various regimes of calcium oscillations. We found that in both fast and slow islets, increasing glucose had limited effects on amplitude and period, but increased plateau fraction. In some islets, however, glucose caused a major shift in the amplitude and period of oscillations, which we attribute to a conversion between ionic and glycolytic modes (i.e., regime change). Raising glucose increased the plateau fraction equally in fast, slow, and regime-changing islets. A mathematical model of the pancreatic islet consisting of an ionic subsystem interacting with a slower metabolic oscillatory subsystem can account for these complex islet calcium oscillations by modifying the relative contributions of oscillatory metabolism and oscillatory ionic mechanisms to electrical activity, with coupling occurring via K(ATP) channels.