An Engineered OmpG Nanopore with Displayed Peptide Motifs for Single‐Molecule Multiplex Protein Detection

• Published in: Angewandte Chemie International Edition Vol. 62; no. 7; pp. e202214566 - n/a
• Main Authors: Foster, Joshua C., Pham, Bach, Pham, Ryan, Kim, Minji, Moore, Matthew D., Chen, Min
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 06.02.2023
• Edition: International ed. in English
• Subjects: Avidity; Bacterial Outer Membrane Proteins - metabolism; Flow cytometry; Membrane proteins; Multiplex; Multiplexing; Nanopore; Nanopores; Peptides - metabolism; Porins - metabolism; Protein G; Proteins; Sensitivity; Sensors; Single-Molecule Detection; Proteins; Nanopore; Sensors; Single-Molecule Detection; Multiplex
• ISSN: 1433-7851; 1521-3773; 1521-3773
• DOI: 10.1002/anie.202214566

Molecular detection via nanopore, achieved by monitoring changes in ionic current arising from analyte interaction with the sensor pore, is a promising technology for multiplex sensing development. Outer Membrane Protein G (OmpG), a monomeric porin possessing seven functionalizable loops, has been reported as an effective sensing platform for selective protein detection. Using flow cytometry to screen unfavorable constructs, we identified two OmpG nanopores with unique peptide motifs displayed in either loop 3 or 6, which also exhibited distinct analyte signals in single‐channel current recordings. We exploited these motif‐displaying loops concurrently to facilitate single‐molecule multiplex protein detection in a mixture. We additionally report a strategy to increase sensor sensitivity via avidity motif display. These sensing schemes may be expanded to more sophisticated designs utilizing additional loops to increase multiplicity and sensitivity. The Outer Membrane Protein G (OmpG) nanopore has been demonstrated as a highly selective platform for protein sensing. By simultaneously functionalizing two of the porin's seven loops with peptide motifs we achieve single‐molecule multiplex protein detection.