Protein Aggregation Characterization by Nanopore technology*

Protein aggregation is one of the leading causes of many neurodegenerative diseases such as Alzheimer’s and Parkinson’s. In this talk, we report our study on characterizing protein aggregation with a solid-state nanopore device, together with AFM and DLS techniques. Our model proteins used in these studies are ß-lactoglobulin variant A (bLGa) and neuronal Tau and tubulin proteins. The main component of a nanopore device is a nanometer size pore fabricated in free-standing silicon nitride membrane supported by a silicon substrate which separates two PDMA chambers containing salt solution. A stable ionic current is established by applying a biased voltage on a pair of silver chloride (AgCl) electrodes across the nanopore membrane. When charged protein molecules are added to the grounded chamber, applying a correct biase potential to the other chamber drives a protein molecule into the nanopore which partially blocks ionic flow that can be measured as a transient current drop signal. A protein aggregate which has larger volume than a single protein molecule would block larger amount of current or generate a greater current drop amplitude, therefore a nanopore device can be used to characterize protein aggregation at single molecule level. The volume of translocating protein aggregates are estimated using a calibrated nanopore by a standard that has known geometry such as a dsDNA molecule. We show that solid state nanopore method is capable of measuring protein aggregation number and the aggregation number distribution in the conditions close to their native aqueous environment. The nanopore experiments were performed under applied voltages from 60-210 mV at different pH, temperature, and salt concentrations. We present data of bLga, and Tau and tubulin aggregations measured by nanopore method, and compare them with the results from AFM and DLS.