Characterization of Protein Aggregation by Solid-State Nanopore

Abstract

In this report, we use solid-state nanopore device to characterize native state aggregation of s-lactoglobulin variant A (βLGa) protein as a function of salt concentration, temperature, pH, and electric field strength. Silicon nitride nanopores used in this work, 5-20 nm in diameter, are fabricated by low energy ion beam sculpting method. The main component of a nanopore device is a nanopore supported by a silicon substrate which separates two PDMA chambers containing salt solution. When a charged protein molecule or a protein aggregate passes through a nanopore driven by an applied voltage across the nanopore chip, a protein aggregate that has larger volume than a single protein molecule,will generate larger current blockage, therefore a nanopore device can be used to characterize protein aggregation. We present data of βLGa self-association and aggregation at pH 4.6 as a function of temperature measured in 2M and 0.1M salt. The nanopore geometry is calibrated by a standard that has known geometry such as a long rod shaped dsDNA molecule. The volume of a translocating proteins or protein aggregates are estimated using the calibrated nanopore. We show that by using a reference dsDNA molecule, solid-state nanopore method is capable of measuring protein aggregation number and the aggregation number distribution in the conditions that is close to their native aqueous solution environment.