Detection of Tubulin and TAU Proteins Aggregations using Solid-State Nanopore and Atomic Force Microscopy (AFM)

Abstract

Aggregations of tau and tubulin proteins are one of the leading causes of many neurodegenerative diseases, such as Pick's, Alzheimer's, and Parkinson's. In this work, protein aggregations are analyzed in salt solution using a solid-state nanopore sensing system. A varieties of fabrication techniques, such as photolithography, chemical vapor deposition, focused ion beam milling and ion beam sculpting are used to fabricate 8 - 30 nm nanopores in free-standing silicon nitride membrane on silicon. The nanopore is placed in between two polydimethylsiloxane chambers with microfluidic channels filled with 1 M KCl solution, and a pair of silver chloride electrodes are embedded in the channels. After a stable ionic current is established by applying bias voltage across the electrodes, charged protein molecules are added to the top chamber (ground), and driven to the bottom chamber by the applied electric field. The passing protein molecules partially block the ion flow in the nanopore and increase the pore resistivity, as a result the ionic current drops. The amplitude of the current drops is proportional to the size of the protein molecules. In this work, the aggregations of tau and tubulin proteins are detected at bias voltages ranges from 60 - 210 mV and pH ranges from 7.4 - 12.0. Monomer proteins were measured with 8 - 10 nm nanopores, aggregated proteins were measured using 15 - 30 nm nanopores. Finally, the amplitude of current drops for monomer and aggregated proteins are compared. Our results show that the tau and tubulin proteins are aggregated to dimer, trimer and multimers. The nanopore results are verified by AFM scanning of dried protein samples on mica surface. The AFM results are found to be consistent with the nanopore measurements.