Ultrasensitive and regenerable nanopore sensing based on target induced aptamer dissociation

For ionic current rectification (ICR) based sensing, nanopore functionalizations are mostly designed for directly binding target molecules to generate detectable signals from surface charge variation. However, this strategy is highly dependent on the charge difference between the captured molecules and surface functionalization layers, which will increase the nanopore design difficulty and subsequently limit the nanopore applicability. Another key challenge for ICR based sensing is the nanopore regenerability that is critical if online monitoring or repeated determination needs to be performed with one sensor. Though some types of nanopore regeneration have been realized on some specific targets or with harsh conditions, it is still highly favored to develop a regenerability using mild conditions for various targets. To address these two challenges, we developed a novel and universal sensing strategy for aptamer-functionalized nanopore that can be easily regenerated after each usage without any harsh conditions and independent of target molecule charge or size for ICR based nanopore sensing. Ochratoxin A (OTA) was used as a model analyte and its corresponding aptamer partially hybridized with the pre-immobilized complementary DNA (cDNA) onto the nanopore inner surface. We demonstrated that the recognition and conjugation of OTA with its aptamer resulted in rectified ionic current variations due to the dissociation between the OTA aptamer and its partially paired cDNA. The performance of this nanopore sensor including sensitivity, selectivity, regenerability, and applicability was characterized using rectified ionic current. This nanopore sensing strategy will provide a promising platform for extensive targets and online sensing applications.