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

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Specialty Chemistry
Date 2022 Dec 2
PMID 36457283
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Abstract

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.

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References
1.
Ouldali H, Sarthak K, Ensslen T, Piguet F, Manivet P, Pelta J . Electrical recognition of the twenty proteinogenic amino acids using an aerolysin nanopore. Nat Biotechnol. 2019; 38(2):176-181. PMC: 7008938. DOI: 10.1038/s41587-019-0345-2. View

2.
Zhang S, Huang G, Abraham Versloot R, Bruininks B, de Souza P, Marrink S . Bottom-up fabrication of a proteasome-nanopore that unravels and processes single proteins. Nat Chem. 2021; 13(12):1192-1199. PMC: 7612055. DOI: 10.1038/s41557-021-00824-w. View

3.
Duan L, Yobas L . Label-Free Multiplexed Electrical Detection of Cancer Markers on a Microchip Featuring an Integrated Fluidic Diode Nanopore Array. ACS Nano. 2018; 12(8):7892-7900. DOI: 10.1021/acsnano.8b02260. View

4.
Palla M, Punthambaker S, Stranges B, Vigneault F, Nivala J, Wiegand D . Multiplex Single-Molecule Kinetics of Nanopore-Coupled Polymerases. ACS Nano. 2020; 15(1):489-502. DOI: 10.1021/acsnano.0c05226. View

5.
Fahie M, Yang B, Mullis M, Holden M, Chen M . Selective Detection of Protein Homologues in Serum Using an OmpG Nanopore. Anal Chem. 2015; 87(21):11143-9. PMC: 5065927. DOI: 10.1021/acs.analchem.5b03350. View