A simple three-dimensional-focusing, continuous-flow mixer for the study of fast protein dynamics
Abstract
We present a simple, yet flexible microfluidic mixer with a demonstrated mixing time as short as 80 μs that is widely accessible because it is made of commercially available parts. To simplify the study of fast protein dynamics, we have developed an inexpensive continuous-flow microfluidic mixer, requiring no specialized equipment or techniques. The mixer uses three-dimensional, hydrodynamic focusing of a protein sample stream by a surrounding sheath solution to achieve rapid diffusional mixing between the sample and sheath. Mixing initiates the reaction of interest. Reactions can be spatially observed by fluorescence or absorbance spectroscopy. We characterized the pixel-to-time calibration and diffusional mixing experimentally. We achieved a mixing time as short as 80 μs. We studied the kinetics of horse apomyoglobin (apoMb) unfolding from the intermediate (I) state to its completely unfolded (U) state, induced by a pH jump from the initial pH of 4.5 in the sample stream to a final pH of 2.0 in the sheath solution. The reaction time was probed using the fluorescence of 1-anilinonaphthalene-8-sulfonate (1,8-ANS) bound to the folded protein. We observed unfolding of apoMb within 760 μs, without populating additional intermediate states under these conditions. We also studied the reaction kinetics of the conversion of pyruvate to lactate catalyzed by lactate dehydrogenase using the intrinsic tryptophan emission of the enzyme. We observe sub-millisecond kinetics that we attribute to Michaelis complex formation and loop domain closure. These results demonstrate the utility of the three-dimensional focusing mixer for biophysical studies of protein dynamics.
Additional Information
© 2013 The Royal Society of Chemistry. Received 19th April 2013, Accepted 5th June 2013. First published online 05 Jun 2013. This work was supported by NIH grants GM068036 and GM053640 (RBD) and by an NSF graduate fellowship DGE-0940903 (MJR).Attached Files
Published - c3lc50497b.pdf
Supplemental Material - c3lc50497b_supp.pdf
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Additional details
- PMCID
- PMC3733270
- Eprint ID
- 39879
- Resolver ID
- CaltechAUTHORS:20130812-145228325
- GM068036
- NIH
- GM053640
- NIH
- DGE-0940903
- NSF Graduate Research Fellowship
- Created
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2013-08-12Created from EPrint's datestamp field
- Updated
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2021-11-09Created from EPrint's last_modified field