Nanopores are currently
utilized as powerful tools for
single-molecule
protein sensing. The reporting signal typically requires protein analytes
to enter the nanopore interior, yet a class of these sensors has emerged
that allows targeted detection free in solution. This tactic eliminates
the spatial limitation of nanopore confinement. However, probing proteins
outside the nanopore implies numerous challenges associated with transducing
the physical interactions in the aqueous phase into a reliable electrical
signature. Hence, it necessitates extensive engineering and tedious
optimization routes. These obstacles have prevented the widespread
adoption of these sensors. Here, we provide an experimental strategy
by developing and validating single-polypeptide-chain nanopores amenable
to single-molecule and bulk-phase protein detection approaches. We
utilize protein engineering, as well as nanopore and nanodisc technologies,
to create nanopore sensors that can be integrated with an optical
platform in addition to traditional electrical recordings. Using the
optical modality over an ensemble of detectors accelerates these sensors’
optimization process for a specific task. It also provides insights
into how the construction of these single-molecule nanopore sensors
influences their performance. These outcomes form a basis for evaluating
engineered nanopores beyond the fundamental limits of the resistive-pulse
technique.