A Generalizable Screening Platform for Developing Functional Aptasensors

Aptamers are versatile sensing elements for the construction of biosensors. A common approach for signal generation in "aptasensors" involves the displacement of short complementary "probes" resulting from conformational changes upon aptamer-target binding. However, designing strands that rapidly and completely displace when the target binds is nontrivial. Typically, probes are discovered through a lengthy process of screening several potential sequences. Here, we explored properties governing probe displacement efficiency using a well-characterized aptamer for the agricultural contaminant ochratoxin A (OTA). Surprisingly, the length, probe affinity, and melting temperature did not correlate with probe displacement efficiency. We therefore developed a novel surface plasmon resonance (SPR) assay to rapidly measure target-induced displacement of probes from aptamers. Fitted displacement results from the SPR assay were correlated with fast proportional fluorescence recovery from quencher-labeled probe displacement. This new method allows for the rapid distinction of efficient probes, resulting in sensitive biosensing of OTA. Finally, we demonstrated our new method is adaptable to diverse aptamers, offering a generally applicable method to improve probe design and accelerate aptasensor development.