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.

Daniel C ，Mélaïne F ，Roupioz Y ，Livache T ，Buhot A ... -  《-》

Surface plasmon resonance spectroscopy study of interfacial binding of thrombin to antithrombin DNA aptamers.

Tang Q ，Su X ，Loh KP 《JOURNAL OF COLLOID AND INTERFACE SCIENCE》

Piezoelectric biosensors for aptamer-protein interaction.

Tombelli S ，Bini A ，Minunni M ，Mascini M ... -  《-》

Aptamer/thrombin/aptamer-AuNPs sandwich enhanced surface plasmon resonance sensor for the detection of subnanomolar thrombin.

A sensitive and selective aptamer/thrombin/aptamer-AuNPs sandwich enhanced surface plasmon resonance (SPR) sensor has been developed for real-time detection of subnanomolar thrombin. In this protocol, one thiol-modified thrombin aptamer (TBA29) was immobilized on gold nanoparticles (AuNPs) via Au-S bonding. The other biotinylated thrombin aptamer (TBA15) was grafted onto streptavidin pretreated SPR gold film through biotin-streptavidin recognition. The presence of thrombin would then induce the formation of a double aptamer sandwich structure on the SPR gold film and results in obvious enhancement of SPR signal, which was proportional to the concentration of thrombin. This proposed assay took advantage of sandwich binding of two affinity aptamers for increased specificity, AuNPs for signal enhancement, as well as SPR signal readout for real-time detection. The SPR signal had a good linear relationship with thrombin concentration in the range of 0.1-75nM, and the detection limit for thrombin was determined to be as low as 0.1nM. It was found that aptamer functionalized AuNPs enhanced the signal of SPR response and thus increased the limit of detection 4-fold and 5-fold compared to direct detection format without AuNPs. This sensor also showed good selectivity for thrombin without being affected by some other proteins, such as BSA and lysozyme. Furthermore, this proposed SPR sensing platform was successfully applied to thrombin analysis in diluted human serum samples.

Bai Y ，Feng F ，Zhao L ，Wang C ，Wang H ，Tian M ，Qin J ，Duan Y ，He X ... -  《-》

Monitoring complex formation in the blood-coagulation cascade using aptamer-coated SAW sensors.

Gronewold TM ，Glass S ，Quandt E ，Famulok M ... -  《-》