Department of Biotechnology and Bioinformatics, Korea University, Sejong 30019, Korea.
Interdisciplinary Graduate Program for Artificial Intelligence Smart Convergence Technology, Korea University, Sejong 30019, Korea.
Int J Mol Sci. 2022 May 13;23(10):5480. doi: 10.3390/ijms23105480.
Processes that monitor the nucleation of amyloids and characterize the formation of amyloid fibrils are vital to medicine and pharmacology. In this study, we observe the nucleation and formation of lysozyme amyloid fibrils using a facile microfluidic system to generate nanoliter droplets that can control the flow rate and movement of monomer-in-oil emulsion droplets in a T-junction microchannel. Using a fluorescence assay, we monitor the nucleation and growth process of amyloids based on the volume of droplets. Using the microfluidic system, we demonstrate that the lag phase, which is vital to amyloid nucleation and growth, is reduced at a lower droplet volume. Furthermore, we report a peculiar phenomenon of high amyloid formation at the edge of a bullet-shaped droplet, which is likely due to the high local monomer concentration. Moreover, we discovered that amyloid fibrils synthesized in the nanoliter droplets are shorter and thicker than fibrils synthesized from a bulk solution via the conventional heating method. Herein, a facile procedure to observe and characterize the nucleation and growth of amyloid fibrils using nanoliter droplets is presented, which is beneficial for investigating new features of amyloid fibril formation as an unconventional synthetic method for amyloid fibrils.
监测淀粉样蛋白成核并描述淀粉样纤维形成的过程对医学和药理学至关重要。在这项研究中，我们使用简单的微流控系统观察溶菌酶淀粉样纤维的成核和形成，该系统可生成纳升级别的液滴，从而控制 T 型微通道中单体油乳液液滴的流速和运动。我们使用荧光分析，根据液滴体积监测淀粉样蛋白的成核和生长过程。通过微流控系统，我们证明在较低的液滴体积下，对淀粉样蛋白成核和生长至关重要的滞后期会缩短。此外，我们报告了在子弹形液滴边缘处高淀粉样蛋白形成的奇特现象，这可能是由于局部单体浓度较高所致。此外，我们发现，通过纳升级别的液滴合成的淀粉样纤维比通过传统加热方法从体相溶液中合成的纤维更短、更厚。本文提出了一种使用纳升级别液滴观察和描述淀粉样纤维成核和生长的简单方法，这有助于研究淀粉样纤维形成的新特征，作为淀粉样纤维的一种非常规合成方法。
