Conventional nanopore platforms rely on rigid nanopore structures that produce a steady open pore current. Decrease of the ionic current indicates the detection of an analyte, which may be translocation-based or non-translocation based. Since these detection schemes are based on the decrease of the ionic current, analytes with a similar size and properties may generate similar current changes, limiting specificity in addition to size limitations.<br/><br/>Outer membrane protein G (OmpG) is a 32kDa monomeric beta-barrel porin with 7 flexible extracellular loops, the most flexible being loop 6. Contrary to rigid nanopores, these flexible loops generate an indigenous pattern of current fluctuations, which we call gating. We exploit OmpG’s loops by introducing a cysteine into loop 6, allowing an analyte-specific ligand to be tethered to OmpG, generating a nontranslocation-based OmpG nanopore sensor.<br/><br/>Using this approach, we found that binding of an analyte changes the dynamic behavior of the loops, thus changing the gating pattern of the OmpG nanopore. Using a biotin ligand tethered to OmpG, we successfully detected and discriminated between biotin-binding homologues due to each analyte’s ability to generate a different and distinguishable gating pattern when bound to the ligand on OmpG. We’ve also detected two human carbonic anhydrase isoforms using a benezenesulfonamide inhibitor ligand. These analytes range from 30kDa to over 150kDa in size, illustrating the OmpG nanopore’s ability to detect a wide range of analytes independent of size. Furthermore, OmpG retains its discriminatory ability in a sample mixture of fetal bovine serum. In addition to OmpG’s sensing capabilities, we are able to extract single molecule kinetic data, revealing differences in affinity among homologues. Future works include expanding the OmpG nanopore analyte library and investigating the effects of a double-ligand on kinetics.

Selective detection of protein homologues by outer membrane protein G nanopore

253rd National Meeting (2017)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.