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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 ;s. 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 changesWe 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 ;s. 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.show moreshow less

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Author:Avinash S. Kane, Armin S. Hoffmann, Peter Baumgärtel, Robert SecklerORCiDGND, Gerd Reichardt, David A. Horsley, Benjamin Schuler, Olgica Bakajin
URL:http://pubs.acs.org/doi/abs/10.1021/ac801764r
ISSN:0003-2700
Document Type:Article
Language:English
Year of first Publication:2008
Year of Completion:2008
Release Date:2017/03/25
Source:Analytical Chemistry . - ISSN 0003-2700. - 80 (2008), 24, S. 9534 - 9541
Organizational units:Mathematisch-Naturwissenschaftliche Fakultät / Institut für Biochemie und Biologie