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.

Improved Measurement of Proteins Using a Solid-State Nanopore Coupled with a Hydrogel.

Although resistive pulse sensing using solid-state nanopores is capable of single-molecule sensitivity, previous work has shown that nanoparticles, such as proteins, pass through nanopores too quickly for accurate detection with typical measurement apparatus. As a result, nanopore measurements of these particles significantly deviate from theoretically estimated current amplitudes and detection rates. Here, we show that a hydrogel placed on the distal side of a nanopore can increase the residence time of nanoparticles within the nanopore, significantly increasing the detection rate and allowing improved resolution of blockage currents. The method is simple and inexpensive to implement while being label-free and applicable to a wide range of nanoparticle targets. Using hydrogel-backed nanopores, we detected the protein IgG with event frequencies several orders of magnitude higher than those in the absence of the hydrogel and with larger measured currents that agree well with theoretical models. We also show that the improved measurement also enables discrimination of IgG and bovine serum albumin in a mixed solution. Finally, we show that measurements of IgG with the hydrogel-backed nanopores can also yield current amplitude distributions that can be analyzed to infer its approximate shape.