Tau and tubulin protein aggregation characterization by solid-state nanopore method and atomic force microscopy

In this study, a silicon nitride nanopore-based sensing system was used to measure tau and tubulin monomers and their aggregations in salt solution at a single molecule level. Nanopores (6–30 nm) were fabricated on silicon nitride membranes supported by silicon substrates using a combination of focused ion beam milling and ion beam sculpting. When a charged protein molecule in the salt solution passes through a nanopore driven by an applied voltage, the protein molecule increases pore resistivity, which induces an ionic current drop that can be measured. The current drop amplitude is directly proportional to the local excluded volume of the protein molecule in the nanopore. We measured the monomers and aggregations of tau and tubulin proteins at biased voltages from 60 to 210 mV in a solution of pH 7.0–10. Our results showed that (1) the nanopore method was able to differentiate tau and tubulin proteins in their monomer and aggregated forms by their excluded volumes; (2) the most probable aggregation form was dimer for α- and β-tubulin and pentamer for αβ tubulin plus tau under experimental conditions; (3) the protein excluded volumes measured by the nanopore method depended on the applied voltage, and this observation could be explained by the nonuniform charge distribution of proteins. The monomer and aggregated proteins were further analyzed using atomic force spectroscopy (AFM), and protein volumes estimated by AFM were consistent with nanopore results.