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.

Characterizing respiratory aerosol emissions during sustained phonation.

OBJECTIVE: To elucidate the role of phonation frequency (i.e., pitch) and intensity of speech on respiratory aerosol emissions during sustained phonations. METHODS: Respiratory aerosol emissions are measured in 40 (24 males and 16 females) healthy, non-trained singers phonating the phoneme /a/ at seven specific frequencies at varying vocal intensity levels. RESULTS: Increasing frequency of phonation was positively correlated with particle production (r = 0.28, p < 0.001). Particle production rate was also positively correlated (r = 0.37, p < 0.001) with the vocal intensity of phonation, confirming previously reported findings. The primary mode (particle diameter ~0.6 µm) and width of the particle number size distribution were independent of frequency and vocal intensity. Regression models of the particle production rate using frequency, vocal intensity, and the individual subject as predictor variables only produced goodness of fit of adjusted R2 = 40% (p < 0.001). Finally, it is proposed that superemitters be defined as statistical outliers, which resulted in the identification of one superemitter in the sample of 40 participants. SIGNIFICANCE: The results suggest there remain unexplored effects (e.g., biomechanical, environmental, behavioral, etc.) that contribute to the high variability in respiratory particle production rates, which ranged from 0.2 particles/s to 142 particles/s across all trials. This is evidenced as well by changes in the distribution of participant particle production that transitions to a more bimodal distribution (second mode at particle diameter ~2 µm) at higher frequencies and vocal intensity levels.