Engineering a Monomeric Nanopore Scaffold for Multiplexed Protein Detection.

The complexity of biological samples often necessitates specialized processing and instrumentation to enable the assessment of disease-associated biomarkers, highlighting the need for simple and robust methods of multiplex detection. Nanopore biosensors represent a promising technology to address this challenge, with biological nanopores emerging as versatile scaffolds for sensor development. In particular, monomeric outer membrane proteins (OMPs) provide an accessible platform for site-specific engineering with reproducible, atomically precise fabrication. Outer membrane protein G (OmpG), a monomeric Escherichia coli porin possessing seven flexible extracellular loops that can be functionalized with affinity ligands, has been utilized as a highly selective sensor through a distinctive gating-pattern detection mode arising from loop-mediated sampling of the molecular surface of bound analytes. Here, we present a general protocol for the molecular grafting of two peptide affinity motifs onto OmpG, followed by purification and characterization of the engineered construct, to generate a single-channel multiplex sensor.