物理化学学报 >> 2011, Vol. 27 >> Issue (08): 1990-1995.doi: 10.3866/PKU.WHXB20110804

生物物理化学 上一篇    下一篇

用于单分子动力学实验的微流控混合器

支泽勇1,3, 刘鹏程1,3, 黄岩谊2,3, 赵新生1,3   

  1. 1. 北京大学化学与分子工程学院化学生物学系, 北京分子科学国家实验室, 分子动态与稳态结构国家重点实验室, 北京 100871;
    2. 北京大学工学院, 北京 100871; 3北京大学生物动态光学成像中心, 北京 100871
  • 收稿日期:2011-04-27 修回日期:2011-06-01 发布日期:2011-07-19
  • 通讯作者: 赵新生 E-mail:zhaoxs@pku.edu.cn
  • 基金资助:

    国家自然科学基金(20733001, 20973015)和国家重点基础研究发展规划项目(973) (2006CB910300, 2010CB912302)资助

A Microfluidic Mixer for Single-Molecule Kinetics Experiments

ZHI Ze-Yong1,3, LIU Peng-Cheng1,3, HUANG Yan-Yi2,3, ZHAO Xin-Sheng1,3   

  1. 1. Beijing National Laboratory for Molecular Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, P. R. China;
    2. College of Engineering, Peking University, Beijing 100871, P. R. China;
    3. Biodynamic Optical Imaging Center, Peking University, Beijing 100871, P. R. China
  • Received:2011-04-27 Revised:2011-06-01 Published:2011-07-19
  • Contact: ZHAO Xin-Sheng E-mail:zhaoxs@pku.edu.cn
  • Supported by:

    The project was supported by the National Natural Science Foundation of China (20733001, 20973015) and National Key Basic Research Program of China (973) (2006CB910300, 2010CB912302).

摘要:

设计制作了用于单分子动力学实验的微流控混合器, 该混合器用聚二甲基硅氧烷(PDMS)芯片和石英载玻片密封而成, 具有低的荧光背景, 广泛的生物相容性, 结合激光共聚焦显微镜能够在非平衡态下进行单分子荧光探测. 我们设计的压力控制系统和进样流路方便而稳定, 保证了微流路中流形的长时间稳定, 从而实现了样品流速和流量的精准控制. 这些技术特点保证了单分子探测得到准确和高信噪比的结果. 利用蛋白质的塌缩过程远快于混合过程的特点,采用荧光标记的金黄色葡萄球菌核酸酶作为指示物,分辨出蛋白质变性态的特征峰,并利用变性态的荧光共振能量传递效率随时间的变化表征出混合器在适合于单分子探测条件下的混合时间为150 ms.

关键词: 微流控混合, 单分子探测, 荧光共振能量传递, 蛋白质折叠, 金黄色葡萄球菌核酸酶

Abstract:

We designed and built a microfluidic mixer based on the principle of hydrodynamic focusing governed by Navier-Stokes equation for single-molecule kinetics experiments. The mixer is a cast of poly(dimethylsiloxane) (PDMS) sealed with transparent fused-silica coverglass, which results in low fluorescence background and broad biological compatibility and this enables single-molecule fluorescence detection under nonequilibrium conditions. The pressure regulated sample delivery system is convenient for loading a sample and allows for precise and stable flow velocity control. The combination of microfluidic mixer and single-molecule fluorescence resonance energy transfer (smFRET) allows us to measure the time course of the distribution of the smFRET efficiency in protein folding. We used the fact that denatured protein collapses much faster than the mixing process to characterize the mixing time using donor and acceptor dyes labeled staphylococcal nuclease (SNase) as an smFRET efficiency indicator. By monitoring the smFRET efficiency of denatured SNase during the course of mixing, we determined that the mixing time was 150 ms under conditions suitable for single-molecule detection.

Key words: Microfluidic mixing, Single-molecule detection, Fluorescence resonance energy transfer, Protein folding, Staphylococcal nuclease

MSC2000: 

  • O643