Rapid and selective multiplex detection of independent biomarkers could prove useful for disease diagnosis. Nanopore biosensors can serve as such a platform; they use ion flow through a pore as a readout for analyte detection and quantification. Although nanopores have been used for multiplex detection of peptides and proteins, their reliance on antibodies for mediating analyte capture limits the potential to offer high specificity in complex biological and clinical samples.

To address these limitations, Foster et al. engineered the existing outer membrane protein G (OmpG) nanopore — a monomeric porin from Escherichia coli with seven flexible extracellular loops — to host distinct affinity reagents at its loops for independent multiplex detection. Using flow cytometry, they evaluated integration of the FLAG motif across OmpG’s seven loops for display efficiency via detection with an antibody to FLAG. Loop 3 was the sole candidate that translated FLAG detection into a discernible signal in current recordings. The researchers applied the same flow-cytometry and current-recording workflow to streptavidin detection to find a top candidate (SA1.1) from four unique streptavidin-binding motifs integrated into OmpG loop 6. Given that both motifs exhibited distinct analyte signals in single-channel current recordings, they generated an OmpG with both FLAG and SA1.1 that was capable of single-molecule multiplex protein detection from a mixture.

Furthermore, leveraging avidity and motif display by appending SA1.1 to loops 3 and 6 of OmpG led to an increase in sensor sensitivity to streptavidin detection. Kinetic analysis of the binding event(s) and affinities indicated that two SA1.1 motifs on a single OmpG may simultaneously bind to the streptavidin tetramer, which undergoes repeated cycles of release and re-capture by either SA1.1. These engineering efforts on OmpG highlight its potential utility for multiplex detection schemes.

Original reference: Angew. Chem. Int. Ed. e202214566 (2022)