Microfluidic mixers for the investigation of rapid protein folding kinetics using synchrotron radiation circular dichroism spectroscopy.
We have developed a microfluidic mixer optimized for rapid measurements of protein folding kinetics using synchrotron radiation circular dichroism (SRCD) spectroscopy. The combination of fabrication in fused silica and synchrotron radiation allows measurements at wavelengths below 220 nm, the typical limit of commercial instrumentation. At these wavelengths, the discrimination between the different types of protein secondary structure increases sharply. The device was optimized for rapid mixing at moderate sample consumption by employing a serpentine channel design, resulting in a dead time of less than 200 micros. Here, we discuss the design and fabrication of the mixer and quantify the mixing efficiency using wide-field and confocal epi-fluorescence microscopy. We demonstrate the performance of the device in SRCD measurements of the folding kinetics of cytochrome c, a small, fast-folding protein. Our results show that the combination of SRCD with microfluidic mixing opens new possibilities for investigating rapid conformational changes in biological macromolecules that have previously been inaccessible.
Kane
Avinash S
AS
Chemistry, Materials, Life and Earth Sciences Directorate, Lawrence Livermore National Laboratory, 7000 East Avenue, L-233, Livermore, California 94550, USA.
Hoffmann
Armin
A
Baumgärtel
Peter
P
Seckler
Robert
R
Reichardt
Gerd
G
Horsley
David A
DA
Schuler
Benjamin
B
Bakajin
Olgica
O
D000818:Animals; D002942:Circular Dichroism / Q000379:methods*; D045304:Cytochromes c / Q000737:chemistry*; D006321:Heart; D006736:Horses; D007700:Kinetics; D044085:Microfluidics / Q000295:instrumentation* / Q000379:methods; D017510:Protein Folding; D017356:Synchrotrons
D045304:Cytochromes c