title: Resolved single-molecule detection of individual species within a mixture of anti-biotin antibodies using an engineered monomeric nanopore.
abstract: Oligomeric protein nanopores with rigid structures have been engineered for the purpose of sensing a wide range of analytes including small molecules and biological species such as proteins and DNA. We chose a monomeric β-barrel porin, OmpG, as the platform from which to derive the nanopore sensor. OmpG is decorated with seven flexible loops that move dynamically to create a distinct gating pattern when ionic current passes through the pore. Biotin was chemically tethered to the most flexible one of these loops. The gating characteristic of the loop's movement in and out of the porin was substantially altered by analyte protein binding. The gating characteristics of the pore with bound targets were remarkably sensitive to molecular identity, even providing the ability to distinguish between homologues within an antibody mixture. A total of five gating parameters were analyzed for each analyte to create a unique fingerprint for each biotin-binding protein. Our exploitation of gating noise as a molecular identifier may allow more sophisticated sensor design, while OmpG's monomeric structure greatly simplifies nanopore production.
authors.affiliation: Molecular and Cellular Biology Program and Department of Chemistry, University of Massachusetts , Amherst, Massachusetts 01003, United States.
authors.forename: Monifa
authors.initials: M
authors.lastname: Fahie
authors.forename: Christina
authors.initials: C
authors.lastname: Chisholm
authors.forename: Min
authors.initials: M
authors.lastname: Chen
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D015374:Biosensing Techniques / Q000379:methods*; D001710:Biotin / Q000276:immunology*; D004560:Electricity; D004738:Engineering; D029968:Escherichia coli Proteins / Q000737:chemistry*; D008024:Ligands; D008958:Models, Molecular; D058608:Nanopores; D036103:Nanotechnology / Q000379:methods*; D011092:Polyethylene Glycols / Q000737:chemistry; D018272:Porins / Q000737:chemistry*; D017433:Protein Structure, Secondary; D019809:Streptavidin / Q000032:analysis
chemical_list: D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D008024:Ligands; C114347:OmpG protein, E coli; D018272:Porins; D011092:Polyethylene Glycols; D001710:Biotin; D019809:Streptavidin
keywords: OmpG; nanopore; protein sensor; single-molecule detection