Fast kinetic studies of protein folding shed important insights into the mechanisms of macromolecular self-assembly. However rapid fluid mixing experiments have been limited by the dead time (~1 ms) of conventional mixers. We demonstrate a microfluidic mixer capable of kinetic measurements on microsecond time scales. The 5-inlet port device facilitates rapid and uniform mixing by decoupling diffusive mixing process from hydrodynamic focusing of laminar flow. The mixer is fabricated from optical-grade plastic by hot imprinting method. Large quantities of disposable devices with identical characteristics can be produced with low cost and short turn-around times with the fabrication method. The three-dimensional flow profile within the mixer is obtained with Fluorescence Correlation Spectroscopy (FCS). The diffusive mixing is characterized by monitoring collisional quenching of fluorescence by iodide ions. We demonstrate the utility of our microfluidic mixer by performing kinetic studies of conformational changes of calmodulin ( CaM ) upon calcium binding with multiphoton microscopy. Vertebrate CaM is labeled with acrylodan (6-acryloyl-2-dimethylaminonaphthalene), whose emission intensity and spectrum is sensitive to the polarity of its environments. We identify two kinetic events of different time constants, which we attribute to binding of two calcium ions on each globular domain of CaM .

Hye Yoon Park is a Ph.D. candidate in Applied Physics at Cornell University . She received her B.S. degree in Physics from the Seoul National University ( Seoul , KOREA ) and her M.S. degree in Applied Physics from Cornell University . For her M.S. degree, Hye Yoon performed research in Prof. Harold Craighead’s group to develop fabrication processes of polymeric microfluidic devices. Hye Yoon is currently working towards her Ph.D. degree in Prof. Lois Pollack’s laboratory. Her Ph.D. thesis work is focused on development of a novel laminar flow mixer and its application for kinetic studies of protein and RNA folding on a microsecond time scale.