Toward protein fingerprinting with a solid-state nanopore.

Abstract

Solid-state nanopores have shown great potential for single-molecule protein identification in recent years. As a first step toward single-molecule protein fingerprinting using a solid-state nanopore device, thorough characterization of the passage of proteins under the different conformational states is required. For this purpose, we use green fluorescent protein (GFP), antibiotin immunoglobulin G (IgG), and cytochrome c (cyt c) as model proteins, and show results from field-driven translocations through nanopores with diameters ranging from 2 to 10 nm in SiN membranes of thicknesses from 10 to 20 nm. As expected, the distinguishable current blockades observed during individual translocations allow the identification of different protein conformations adopted throughout its passage, ranging from partially unfolded to completely unfolded conformations. Here we show how different temperatures and voltages facilitate protein unfolding and alter their folding kinetics. This is achieved by varying voltages from 0.1 V to 1 V and by varying the temperature from room temperature to 60 oC while maintaining low-noise ionic current recording at 1 MHz bandwidth using a custom instrument with thermal control offering ±0.2 oC precision and ±1 oC repeatability. These results will serve to better understand the accessible states in the protein energy landscape and to benchmark future studies furthering protein fingerprinting.