Aggregation and Oligomerization Characterization of ß-Lactoglobulin Protein Using a Solid-State Nanopore Sensor.

Protein aggregation is linked to many chronic and devastating neurodegenerative human diseases and is strongly associated with aging. This work demonstrates that protein aggregation and oligomerization can be evaluated by a solid-state nanopore method at the single molecule level. A silicon nitride nanopore sensor was used to characterize both the amyloidogenic and native-state oligomerization of a model protein ß-lactoglobulin variant A (ßLGa). The findings from the nanopore measurements are validated against atomic force microscopy (AFM) and dynamic light scattering (DLS) data, comparing ßLGa aggregation from the same samples at various stages. By calibrating with linear and circular dsDNA, this study estimates the amyloid fibrils' length and diameter, the quantity of the ßLGa aggregates, and their distribution. The nanopore results align with the DLS and AFM data and offer additional insight at the level of individual protein molecular assemblies. As a further demonstration of the nanopore technique, ßLGa self-association and aggregation at pH 4.6 as a function of temperature were measured at high (2 M KCl) and low (0.1 M KCl) ionic strength. This research highlights the advantages and limitations of using solid-state nanopore methods for analyzing protein aggregation.

Characterization of protein unfolding with solid-state nanopores.

In this work, we review the process of protein unfolding characterized by a solid-state nanopore based device. The occupied or excluded volume of a protein molecule in a nanopore depends on the protein's conformation or shape. A folded protein has a larger excluded volume in a nanopore thus it blocks more ionic current flow than its unfolded form and produces a greater current blockage amplitude. The time duration a protein stays in a pore also depends on the protein's folding state. We use Bovine Serum Albumin (BSA) as a model protein to discuss this current blockage amplitude and the time duration associated with the protein unfolding process. BSA molecules were measured in folded, partially unfolded, and completely unfolded conformations in solid-state nanopores. We discuss experimental results, data analysis, and theoretical considerations of BSA protein unfolding measured with silicon nitride nanopores. We show this nanopore method is capable of characterizing a protein's unfolding process at single molecule level. Problems and future studies in characterization of protein unfolding using a solid-state nanopore device will also be discussed.

Nanopore sculpting with noble gas ions.

We demonstrate that 3 keV ion beams, formed from the common noble gasses, He, Ne, Ar, Kr, and Xe, can controllably "sculpt" nanometer scale pores in silicon nitride films. Single nanometer control of structural dimensions in nanopores can be achieved with all ion species despite a very wide range of sputtering yields and surface energy depositions. Heavy ions shrink pores more efficiently and make thinner pores than lighter ions. The dynamics of nanopore closing is reported for each ion species and the results are fitted to an adatom diffusion model with excellent success. We also present an experimental method for profiling the thickness of the local membrane around the nanopore based on low temperature sputtering and data is presented that provides quantitative measurements of the thickness and its dependence on ion beam species.