Comparison of different label-free imaging high-throughput biosensing systems for aptamer binding measurements using thrombin aptamers

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. •Comparison ofo 3 label-free high-throughput imaging systemso 2 label-free low-throughput non-imaging systems ando 1 in solution reference systemfor the determination of kinetic constants using thrombin aptamers.•Comparison of the influence of the analysis software used.•Comparison of the user influence.•High influence on the results by the user and from the assay conditions.•Recommendation to use at least two different systems and to analyze the kinetics best without prior knowledge of the assay and binding partners.