Lee Joon Seok, Um Eujin, Park Je-Kyun, Park Chan Beum
Department of Materials Science and Engineering, and Department of Bio and Brain Engineering, KAIST, 335 Gwahangno, Daejeon 305-701, Republic of Korea.
Langmuir. 2008 Jul 15;24(14):7068-71. doi: 10.1021/la800907c. Epub 2008 Jun 13.
We report the self-assembly of insulin monomers into amyloid fibrils within microchannels. To demonstrate the microfluidic amyloid formation and fibril growth on a solid surface, we seeded the internal surfaces of the microchannels with insulin monomers via N-hydroxysuccinimide ester activation and continuously flushed a fresh insulin solution through the microchannels. According to our analysis using optical and fluorescence microscopy, insulin amyloid preferentially formed in the center of the microchannels and, after reaching a certain density, spread to the side walls of the microchannels. By using ex situ atomic force microscopy, we observed the growth of amyloid fibrils inside the microchannels, which occurred at a much higher rate than that in bulk systems. After 12 h of incubation, insulin formed amyloid spherulites having "Maltese cross" extinction patterns within the microchannels according to the polarized microscopic analysis. Microfluidic amyloid formation enabled low consumption of reagents, reduction of incubation time, and simultaneous observation of amyloid formation under different conditions. This work will contribute to the rapid analysis of amyloid formation associated with many protein misfolding diseases.
我们报道了胰岛素单体在微通道内自组装成淀粉样纤维的过程。为了证明微流控条件下淀粉样蛋白的形成以及在固体表面上纤维的生长，我们通过N - 羟基琥珀酰亚胺酯活化，将胰岛素单体接种到微通道的内表面，并持续用新鲜的胰岛素溶液冲洗微通道。根据我们使用光学显微镜和荧光显微镜的分析，胰岛素淀粉样蛋白优先在微通道的中心形成，达到一定密度后扩散到微通道的侧壁。通过使用非原位原子力显微镜，我们观察到微通道内淀粉样纤维的生长，其生长速率比在本体系统中高得多。孵育12小时后，根据偏振显微镜分析，胰岛素在微通道内形成了具有“马耳他十字”消光图案的淀粉样球晶。微流控淀粉样蛋白形成能够实现试剂的低消耗、孵育时间的缩短以及在不同条件下对淀粉样蛋白形成的同时观察。这项工作将有助于快速分析与许多蛋白质错误折叠疾病相关的淀粉样蛋白形成。
