Application of microanalytical techniques for chemical separations has had a profound impact on biological investigation, and recent advances for chemical analysis of single cells hold great promise for understanding cellular physiology in health and disease. An emerging area in the molecular analysis of cellular function is the study of the activation of signal transduction proteins within individual cells. The current grant proposes to develop a platform that will greatly enhance the throughput for measurements of enzyme activity in single, adherent cells. State-of-the-art microfabrication methods and mass-produced, printed-circuit technology will be integrated to develop arrays of electrically addressable cell positions. This array in conjunction with capillary electrophoresis (CE) will provide a unique approach for subsecond cell lysis and sampling. This rapid sampling component will be combined with a new buffer-delivery strategy for CE and with electronics control systems to produce a semi-automated platform enabling rapid, serial sampling and chemical separation of the contents of individual, adherent, mammalian cells. The system will be used to perform quantitative biochemical assays of the activation of signal transducing kinases in single cells. Three kinases (protein kinase C, protein kinase B, and calcium/calmodulin-activated kinase II) which regulate a diverse array of processes will be targeted; however, the device and method are applicable to most other cellular kinases. A multidisciplinary team of investigators with expertise in analytical chemistry, microfabrication, and cell biology will collaborate to produce and validate the device which will mount on the stage of a microscope so that cells can be studied prior to measurement of their kinase activation status. A critical part of the development process will be the conduct of careful controls to insure cell health and viability prior to cell sampling. The integrated device and method will enable analyses of adherent cells in the numbers needed to quantitatively characterize signaling responses after physiologic stimulation. In the final phase of the grant, the assays will be performed on populations of single cells to test a fundamentally important concept in the field of cellular signal transduction: the existence of kinase substates during signal transduction in mammalian cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Research Project (R01)
Project #
1R01EB004597-01
Application #
6857902
Study Section
Special Emphasis Panel (ZRG1-BECM (01))
Program Officer
Korte, Brenda
Project Start
2005-04-01
Project End
2009-01-31
Budget Start
2005-04-01
Budget End
2006-01-31
Support Year
1
Fiscal Year
2005
Total Cost
$358,800
Indirect Cost
Name
University of California Irvine
Department
Physiology
Type
Schools of Medicine
DUNS #
046705849
City
Irvine
State
CA
Country
United States
Zip Code
92697
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Borland, Laura M; Allbritton, Nancy L (2008) Use of micellar electrokinetic chromatography to measure palmitoylation of a peptide. J Chromatogr B Analyt Technol Biomed Life Sci 875:451-8
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Marc, Paul J; Sims, Christopher E; Allbritton, Nancy L (2007) Coaxial flow system for chemical cytometry. Anal Chem 79:9054-9
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Pai, Jeng-Hao; Wang, Yuli; Salazar, Gina To'A et al. (2007) Photoresist with low fluorescence for bioanalytical applications. Anal Chem 79:8774-80

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