Spatiotemporally Resolved Protein Detection in Live Cells Using Nanopore Biosensors

Spatiotemporal detection of proteins in living cells is a persistent challenge but is the key to understanding their cellular biology and developing theranostic technologies. We develop a dual-nanopore biosensor using affinity-tunable peptide probes, which enables label-free and spatiotemporal monitoring of protein abundance and its concentration change in single live cells. We demonstrate that by screening for peptide probes with tunable affinities, the nanopore modified with a medium-affinity peptide allowed reversible and sensitive detection of the protein kinase A (PKA) catalytic subunit with a detection limit of 0.04 nM. The sensor is shown to have the ability to effectively eliminate interferences from cell membrane resistance and coexisting species in live cell detection. Moreover, our sensor is successfully implemented in monitoring of dynamic PKA activity changes (PKA catalytic subunit dynamic concentration changes) under different stimulations in single live cells. Our design may provide a paradigm for developing nanopore biosensors for spatiotemporally resolved protein analysis in live cells.