Crossflow Devices for Fast Mixing

Measurement of Rapid Conformational Changes of Proteins in a Fast Laminar Flow Mixing Device

Jonas Korlach*, Lisa Kwok*, Sally A Kim**, M. Neal Waxham**, Warren Zipfel*, Watt W Webb*, Lois Pollack*

*Cornell University, School of Applied Physics, Clark Hall, Ithaca, New York 14853

**University of Texas-Houston Health Science Center, 6431 Fannin St., Houston, Texas 77030

Measurements of rates of fast chemical reactions or rapid conformational changes of proteins are limited by mixing and measurement “dead” times of stopped-flow and other mixing devices. We report on the utilization of a fast laminar flow mixing device first described by Knight et al. (Phys. Rev. Lett. 80: 3863) in conjunction with sensitive fluorescence detection. We have characterized the dynamics of molecular mixing in the device with a combination of confocal and multiphoton imaging and fluorescence correlation spectroscopy. FCS is capable of accurately measuring fast flow velocities and sample composition in these microstructures with micrometer resolution, and can be used to map the three-dimensional flow profile inside the device. Because the reaction can be monitored prior to, during and after mixing, very fast transitions are measurable. Mixing times of around 10 ms were obtained, with a time resolution of ~100 ns in the device under effective measuring conditions. Its application is demonstrated by analyzing the fast conformational transition of calmodulin upon binding of calcium ions, using a fluorescently labeled calmodulin for which the fluorescence intensity is sensitive to the conformational state of the protein.