Ultrasonic frequency and intensity dependence in β2-microglobulin amyloid seed detection: Balancing cavitation-induced nucleation, fragmentation, and radical formation.

Amyloid fibrils are aberrant proteinaceous aggregates involved in several intractable diseases called amyloidosis. Detecting fragments of amyloid fibrils, so-called seeds, in human biofluids is a promising approach for their early-stage diagnosis. Ultrasonication of amyloidogenic protein solutions has the potential to enhance the detection sensitivity of amyloid seeds in an accelerated manner through the effects of ultrasonic cavitation. However, the effects of acoustic frequency and intensity on ultrasonic seed detection have not been investigated. In this study, we explore the optimized acoustic conditions for rapid and sensitive amyloid-seed detection and sonochemical mechanisms behind it. Our results show that maximum detection performance is achieved at moderate acoustic intensities with frequencies below 109 kHz, where the balance between ultrasonic enhancement of nucleation and fragmentation pathways of amyloid formation is suitable for the detection of amyloid seeds. Under these conditions, the detection time for amyloid seeds is reduced fivefold while achieving detection sensitivity of 1 pM. We further find that excessively high-intensity ultrasonication is likely to cause fragmentation of protein monomers into smaller peptides at lower frequencies, while that at frequencies above 200 kHz introduces reactive radical species, hindering the ultrasonic seed detection assay. These results highlight the critical role of optimizing acoustic parameters for the application of ultrasonication in the amyloid-seed detection, offering a pathway for rapid diagnostic assays for amyloidosis.