Global Center for Medical Engineering and Informatics, Osaka University, Suita, Osaka 565-0871, Japan.
Institute for NanoScience Design, Osaka University, Toyonaka, Osaka 560-8531, Japan.
Ultrason Sonochem. 2021 May;73:105508. doi: 10.1016/j.ultsonch.2021.105508. Epub 2021 Mar 3.
Ultrasonication to supersaturated protein solutions forcibly forms amyloid fibrils, thereby allowing the early-stage diagnosis for amyloidoses. Previously, we constructed a high-throughput sonoreactor to investigate features of the amyloid-fibril nucleation. Although the instrument substantiated the ultrasonication efficacy, several challenges remain; the key is the precise control of the acoustic field in the reactor, which directly affects the fibril-formation reaction. In the present study, we develop the optimized sonoreactor for the amyloid-fibril assay, which improves the reproducibility and controllability of the fibril formation. Using β-microglobulin, we experimentally demonstrate that achieving identical acoustic conditions by controlling oscillation amplitude and frequency of each transducer results in identical fibril-formation behavior across 36 solutions. Moreover, we succeed in detecting the 100-fM seeds using the developed sonoreactor at an accelerated rate. Finally, we reveal that the acceleration of the fibril-formation reaction with the seeds is achieved by enhancing the primary nucleation and the fibril fragmentation through the analysis of the fibril-formation kinetics. These results demonstrate the efficacy of the developed sonoreactor for the diagnosis of amyloidoses owing to the accelerative seed detection and the possibility for further early-stage diagnosis even without seeds through the accelerated primary nucleation.
超声处理过饱和蛋白质溶液会强制形成淀粉样纤维，从而可以早期诊断淀粉样变性疾病。 先前，我们构建了高通量声反应器来研究淀粉样纤维成核的特征。 尽管该仪器证实了超声处理的功效，但仍存在一些挑战； 关键是要精确控制反应器中的声场，这直接影响纤维形成反应。 在本研究中，我们开发了用于淀粉样纤维测定的优化声反应器，从而提高了纤维形成的重现性和可控性。 使用β-微球蛋白，我们通过实验证明，通过控制每个换能器的振动幅度和频率来实现相同的声场条件，可使 36 种溶液中的纤维形成行为完全相同。 此外，我们成功地使用开发的声反应器以加速的速度检测到了 100fM 的种子。 最后，我们通过分析纤维形成动力学，揭示了通过增强初级成核和纤维碎裂，用种子加速纤维形成反应的机制。 这些结果表明，由于加速的种子检测以及即使没有种子也通过加速的初级成核实现进一步的早期诊断的可能性，所开发的声反应器可用于淀粉样变性疾病的诊断。
