1 - Published on Web A Microfluidic Device for Kinetic...

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A Microfluidic Device for Kinetic Optimization of Protein Crystallization and In Situ Structure Determination Carl L. Hansen, ² Scott Classen, ² James M. Berger, ² and Stephen R. Quake* Department of Applied Physics, California Institute of Technology, 1200 East California Boule V ard, Pasadena, California 91125 Received November 10, 2005; E-mail: [email protected] Recently microfluidic technologies have emerged as viable platforms for nano-volume protein crystallization screening. 1 - 4 In particular, screening in nanoliter volume free interface diffusion (FID) reactors has been instrumental in the crystallization of a broad range of targets that had proven to be intractable by conventional screening techniques and has been successfully incorporated into academic and industrial structural biology efforts. 5,6 However, crystals grown in nanoliter volume reactors may be of insufficient size for diffraction studies, and scale-up usually depends on growth kinetics that vary with reactor details. 3,5 Moreover, previously reported microfluidic crystallization devices do not allow the post- crystallization addition of cryoprotectant necessary for diffraction studies at cryogenic temperatures. 11 In this paper, we report a microfluidic device which provides a link between chip-based nanoliter volume crystallization screening and structure analysis through “kinetic optimization” of crystal- lization reactions and in situ structure determination. Kinetic optimization of mixing rates through systematic variation of reactor geometry and actuation of micromechanical valves is used to screen a large ensemble of kinetic trajectories that are not practical with conventional techniques. Using this device, we demonstrate control over crystal quality, reliable scale-up from nanoliter volume reactions, facile harvesting and cryoprotectant screening, and protein structure determination at atomic resolution from data collected in- chip. The basic structure of the kinetic optimization device implements five parallel, FID reaction chambers 7 encased beneath a semiperme- able membrane at the bottom of a macroscopic fluid reservoir. The FID reactors equilibrate not only with themselves via diffusion but also with reservoir solutions through the membrane, which defines an osmotic bath that controls both the rate and extent of vapor transport to and from the reactors. The combined FID - vapor diffusion motif (Figure 1) is repeated 20 times on the chip; each version has different channel lengths and volumes. The device has channel lengths ranging from 300 to 2400 μ m, thereby allowing the characteristic mixing time to be varied by a factor of 8. 7 The present device further allows control over the rate and extent of water vapor transport through the permeable poly(dimethyl- siloxane) (PDMS) membrane.
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This note was uploaded on 07/07/2010 for the course CHBE 471 taught by Professor Kraft during the Spring '08 term at University of Illinois at Urbana–Champaign.

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1 - Published on Web A Microfluidic Device for Kinetic...

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