The goal is to investigate a new class of materials that has to potential to provide a significant improvement in protein electrophoresis, compared to polymer gels. Today's gels give distorted, tailing zones of irreproducible position due to inhomogeneities in the gel, and they hamper the separation of hydrophobic proteins by their poor compatibility with organic solvents. The proposed work is to investigate materials comprised by ordered arrays of silica nanospheres having nanometer-scale polymer chains tethered to their surfaces. The interstitial volume serves as a sieving medium, and the polymer chains resist protein adsorption. The structural definition imposed by the silica nanospheres promises high separation efficiency and reproducibility in protein electrophoresis. The proposed work entails three parts. First, the controlled deposition of crystalline films of silica nanospheres onto plates and into channels of microfluidic chips is to be investigated. The chemical compositions of polymer coating and the nanosphere will be varied to learn how to maximize separation efficiency for proteins and base stability for the polymer and substrate. Second, the sieving transport of proteins through these mesoporous media will be studied. The migration rates and zone profiles for molecular weight standards and for fluorescence-labeled proteins will be characterized to develop a model for transport through the ordered medium. The adsorptive properties of the material will be probed by fluorescence correlation spectroscopy to determine the mobile and immobile fractions of proteins, and the rate constants for any desorption processes. Third, isoelectric focusing will be investigated to understand the roles of polymer functional group, carrier solvent, and position of the immobiline, in promoting protein solubility. Fluorescence correlation spectroscopy will probe mobile and immobile fraction. Two-dimensional electrophoretic separations will be investigated, primarily sieving and isoelectric focusing for comparison with gels, and also sieving and pore-size gradients, and electrochromatography with electrophoresis. The proposed work addresses the central issues in protein transport through crystalline mesoporous media to lay the groundwork for development of powerful new protein separations.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
7R01GM065980-02
Application #
6895680
Study Section
Special Emphasis Panel (ZRG1-BECM (03))
Program Officer
Edmonds, Charles G
Project Start
2003-06-01
Project End
2007-05-31
Budget Start
2004-02-01
Budget End
2004-05-31
Support Year
2
Fiscal Year
2003
Total Cost
$138,261
Indirect Cost
Name
University of Arizona
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
806345617
City
Tucson
State
AZ
Country
United States
Zip Code
85721
Birdsall, Robert E; Koshel, Brooke M; Hua, Yimin et al. (2013) Modeling of protein electrophoresis in silica colloidal crystals having brush layers of polyacrylamide. Electrophoresis 34:753-60
Koshel, Brooke M; Wirth, Mary J (2012) Trajectory of isoelectric focusing from gels to capillaries to immobilized gradients in capillaries. Proteomics 12:2918-26
Wei, Bingchuan; Rogers, Benjamin J; Wirth, Mary J (2012) Slip flow in colloidal crystals for ultraefficient chromatography. J Am Chem Soc 134:10780-2
Zhang, Zhaorui; Ratnayaka, Saliya N; Wirth, Mary J (2011) Protein UTLC-MALDI-MS using thin films of submicrometer silica particles. J Chromatogr A 1218:7196-202
Malkin, Douglas S; Wei, Bingchuan; Fogiel, Arthur J et al. (2010) Submicrometer plate heights for capillaries packed with silica colloidal crystals. Anal Chem 82:2175-7
Wei, Bingchuan; Malkin, Douglas S; Wirth, Mary J (2010) Plate heights below 50 nm for protein electrochromatography using silica colloidal crystals. Anal Chem 82:10216-21
Hua, Yimin; Koshel, Brooke M; Wirth, Mary J (2010) Field-free remobilization of proteins after isoelectric focusing in packed capillaries. Anal Chem 82:8910-5
Egas, David A; Wirth, Mary J (2008) Fundamentals of protein separations: 50 years of nanotechnology, and growing. Annu Rev Anal Chem (Palo Alto Calif) 1:833-55
Ross, Eric E; Wirth, Mary J (2008) Silica colloidal crystals as three-dimensional scaffolds for supported lipid films. Langmuir 24:1629-34
Wirth, Mary J (2007) Mass transport in sub-2-microm high-performance liquid chromatography. J Chromatogr A 1148:128-30

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