Acta Phys. -Chim. Sin. ›› 2011, Vol. 27 ›› Issue (08): 1990-1995.doi: 10.3866/PKU.WHXB20110804

• BIOPHYSICAL CHEMISTRY • Previous Articles     Next Articles

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).

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