Development of an OmpG Multiplex Biosensor

The rapid and selective detection of disease biomarkers is integral for improving patient outcomes. However, commonly employed diagnostic techniques exhibit drawbacks related to selective biomarker detection from complex samples while maintaining rapid assay time. Nanopore sensing offers one solution to address these limitations, achieving highly selective, single-molecule protein detection by monitoring ionic current fluctuations through an engineered protein pore. Outer membrane protein G (OmpG) is a promising scaffold for the development of these sensors as each of OmpG’s seven flexible extracellular loops can function as a display platform for affinity reagents, either tethered small molecules, or genetically encoded peptide motifs. Upon target-interaction with the corresponding loop-displayed affinity reagent recognizable current patterns termed “gating” are generated, allowing for biomarker detection and quantitation. Prior OmpG sensors have focused on the use of a single loop, predominantly loop 6 (L6), as a display platform which limited assay-throughput and multiplex potential. Here we aimed to expand the utility of this sensing method by simultaneously displaying peptide motifs on OmpG loops 3 and 6. Using this method, we achieved a next generation multiplex OmpG sensor capable of detection and discrimination of two protein analytes, an IgG antibody and streptavidin, simultaneously. We additionally attempted to improve detection sensitivity through the simultaneous display of multiple motif copies, which enhanced the avidity of analyte binding, but did not result in a lower limit of detection. In summary, this sensor design represents an advance in nanopore sensing that can be applied for multiplex biomarker detection in diagnostics.