Figure 4.

Multiplex detection of two protein analytes in a simple mixture by OmpG^{L3-FLAG/L6-SA1.1} nanopore a) Schematic view of OmpG^{L3-FLAG/L6-SA1.1} sensor. b-d) Representative single-channel current recordings of OmpG^{L3-FLAG/L6-SA1.1} b) basal behavior and recapitulation of c) FG4R (20 nM) and d) streptavidin (800 nM) binding signals. e-f) Representative single-channel current recording traces showing recapitulation of individual analyte signals when analytes are present in a simple mixture g) Concurrent observation of both binding signals. h-i) Concentration dependence of streptavidin 1/τ_On and 1/τ_Off. Error bars represent standard deviation from three independent pores. j-k) Gaussian fit of τ_On and τ_Off values from log transformed millisecond values representing quantification of streptavidin binding. Light gray and blue represent when only streptavidin is present (N = 3, n = 813), dark gray and blue represent concurrent streptavidin and FG4R quantification (N = 3, n = 594). Experiments were performed in 50 mM Na₂HPO₄ pH 6.0 buffer containing 300 mM KCl at an applied potential of ±50 mV. Traces were filtered using a 500 Hz lowpass digital gaussian filter.