Quantitative Measurements of Protein Volume and Concentration using Hydrogel-Backed Nanopores.

Accurate identification and quantification of proteins in solution using nanopores is technically challenging in part because of the large fraction of missed translocation events due to short event times and limitations of conventional current amplifiers. Previously, we have shown that a nanopore interfaced with a poly(ethylene glycol)-dimethacrylate hydrogel with an average mesh size of 3.1 nm significantly enhances the protein residence time within the pore, reducing the number of missed events. We used hydrogel-backed nanopores to sense unlabeled proteins as small as 5.5 kDa in size and 10 fM in concentration. We show that the frequency of protein translocation events linearly scales with bulk concentration over a wide range of concentrations and that unknown protein concentrations can be determined from an interpolation of the frequency-concentration curve with less than 10% error. Further, we show an iterative method to determine a protein volume accurately from measurement data for proteins with a diameter comparable to a nanopore diameter. Our measurements and analysis also suggest several competing mechanisms for the detection enhancement enabled by the presence of the hydrogel.