Tuning the Selectivity and Sensitivity of an OmpG Nanopore Sensor by Adjusting Ligand Tether Length

Abstract

We have previously shown that a biotin ligand tethered to the rim of an OmpG nanopore can be used to detect biotin-binding proteins. Here, we investigate the effect of the length of the polyethylene glycol tether on the nanopore's sensitivity and selectivity. When the tether length was increased from 2 to 45 ethylene repeats, sensitivity decreased substantially for a neutral protein streptavidin and slightly for a positively charged protein (avidin). In addition, we found that two distinct avidin binding conformations were possible when using a long tether. These conformations were sensitive to the salt concentration and applied voltage. Finally, a longer tether resulted in reduced sensitivity due to slower association for a monoclonal anti-biotin antibody. Our results highlight the importance of electrostatic, electroosmotic and electrophoretic forces on nanopore binding kinetics and sensor readout.

Mechanism of OmpG pH-Dependent Gating from Loop Ensemble and Single Channel Studies

“…14,18–20 Our recent works have demonstrated the use of OmpG gating behavior for highly specific protein homologue sensing. 19,21,22 Thus, understanding of OmpG gating may also guide the design of OmpG-based nanopore sensors for new applications.…”

Nanopore Sensing

“…123 Site-directed mutagenesis has been used on OmpG pores to minimize gating behaviors, and the resultant “quieter” OmpG pore has been used for traditional stochastic current sensing experiments. 124 Another interesting approach demonstrated by Chen and co-workers directly exploits the gating behavior of OmpG and uses the characteristic signal generated from specific interactions between the flexible loops and analyte molecules (i.e., loop dynamics) for analyte identification 43, 44, 125, 126 .…”

The Effects of Tetramethylammonium Cation on Oligonucleotide Analysis with Aerolysin Nanopore

“…According to this principle, single-strand DNA could be sequenced as each base sequentially traverses nanopore. [8] In addition to the widely used αhemolysin, varieties of membrane proteins have been used as biological nanopore for single-molecule analysis, such as mycobacterium smegmatis porin A (MspA), [9][10] Outer membrane protein G (OmpG), [11][12] cytolysin A (ClyA) [13][14] and Aerolysin. [15][16] Aerolysin is a β-pore-forming toxin from Aeromonas hydrophila, [17] which could spontaneously oligomerize and insert into the lipid bilayer forming a nanopore, which narrowest diameter is only 1.0 nm and possess a long and uniform βbarrel.…”