Abstract
To enable the analysis of several hundreds to thousands of interactions in parallel, high-throughput systems were developed. We used established thrombin aptamer assays to compare three such high-throughput imaging systems as well as analysis software and user influence. In addition to our own iRIf-system, we applied bscreen and IBIS-MX96. As non-imaging reference systems we used Octet-RED96, Biacore3000, and Monolith-NT.115. In this study we measured 1378 data points. Our results show that all systems are suitable for analyzing binding kinetics, but the kinetic constants as well as the ranking of the selected aptamers depend significantly on the applied system and user. We provide an insight into the signal generation principles, the systems and the results generated for thrombin aptamers. It should contribute to the awareness that binding constants cannot be determined as easily as other constants. Since many parameters like surface chemistry, biosensor type and buffer composition may change binding behavior, the experimenter should be aware that a system and assay dependent KD is determined. Frequently, certain conditions that are best suited for a given biosensing system cannot be transferred to other systems. Therefore, we strongly recommend using at least two different systems in parallel to achieve meaningful results.