Collision-Based Electrochemical Detection of Lysozyme Aggregation

Proteins are utilized across many biomedical and pharmaceutical industries; therefore, methods for rapid and accurate monitoring of protein aggregation are needed to ensure proper product quality. Although these processes have been previously studied, it is difficult to comprehensively evaluate protein folding and aggregation by traditional characterization techniques such as atomic force microscopy (AFM), electron microscopy, or X-ray diffraction, which require sample pre-treatment and do not represent native state proteins in solution. Herein, we report early tracking of lysozyme (Lyz) aggregation states by using single-particle collision electrochemistry (SPCE) of silver nanoparticle (AgNP) redox probes. The method relies on monitoring the rapid interaction of Lyz with AgNPs, which decreases the number of single AgNPs available for collisions and ultimately the frequency of oxidative impacts in the chronoamperometric profile. When Lyz is in a non-aggregated monomeric form, the protein forms a homogeneous coverage onto the surface of AgNPs, stabilizing the particles. When Lyz is aggregated, part of the AgNP surface remains uncoated, promoting the agglomeration of Lyz-AgNP conjugates. The frequency of AgNP impacts decreases with increasing aggregation time, providing a metric to track protein aggregation. Visualizations of integrated oxidation charge-transfer data displayed significant differences between the charge transfer per impact for AgNP samples alone and in the presence of non-aggregated and aggregated Lyz with 99% confidence using parametric ANOVA tests. Electrochemical results revealed meaningful associations with UV-vis, circular dichroism, and AFM, demonstrating that SPCE can be used as an alternative method for studying protein aggregation. This electrochemical technique could serve as a powerful tool to indirectly evaluate protein stability and screen protein samples for formation of aggregates.