) Microfabricated fluidics devices originally designed for the Human Genome Project will be developed as a means to screen single tumor cells for aberrantly regulated kinase activity. Kinases regulate nearly all cellular functions including those critical for oncogenesis. The long-term goal of this work is to determine the number, identities, and degree of activation of kinases in individual cells within a malignancy. This information will aid kinase antagonist development, enhance patient prognosis, and identify potential therapeutic regimens. The investigators have established a collaboration to combine their respective talents to develop this new technology using a concerted, stepwise approach. The Allbritton Lab is highly experienced with the use of capillary electrophoresis for single-cell measurements of signal transduction molecules and kinase activity, and the Ramsey Lab has pioneered the development of these """"""""lab-on-a-chip"""""""" microfabricated devices. The devices to be developed will measure the activity of multiple kinases in a single tumor cell by clectrophoretic identification and quantification of phosphorylated and nonphosphorylated fluorescently-labeled, kinase-substrate peptides. This general approach will be applicable to single-cell measurements of the activities of a broad range of enzymes. The microchip format has the capability for single-cell measurements by virtue of exquisite sensitivity, extemely high separation efficiency, and miniaturization. This chip-based approach also lends itself to a contained, monolithic, cost-effective device for widespread dissemination to research and clinical labs alike.
The specific aims of this proposal are directed at method development and validation for physiologic measurements in single cells.
These aims are to define the separation conditions and detection limits for the substrate peptides on the chip, to optimize the cellular manipulations needed for the measurement, to determine the biologic properties of the peptide indicators, and to measure the activities of multiple kinases in a single tumor cell. When fully developed, this integrated microchip will represent a new paradigm in single-cell measurement techniques.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA078858-04
Application #
6377214
Study Section
Special Emphasis Panel (ZCA1-RLB-Y (M1))
Project Start
1998-09-30
Project End
2003-07-31
Budget Start
2001-08-01
Budget End
2003-07-31
Support Year
4
Fiscal Year
2001
Total Cost
$358,340
Indirect Cost
Name
University of California Irvine
Department
Physiology
Type
Schools of Medicine
DUNS #
161202122
City
Irvine
State
CA
Country
United States
Zip Code
92697
Poulsen, Claus R; Culbertson, Christopher T; Jacobson, Stephen C et al. (2005) Static and dynamic acute cytotoxicity assays on microfluidic devices. Anal Chem 77:667-72
McClain, Maxine A; Culbertson, Christopher T; Jacobson, Stephen C et al. (2003) Microfluidic devices for the high-throughput chemical analysis of cells. Anal Chem 75:5646-55
Hu, Shuwen; Ren, Xueqin; Bachman, Mark et al. (2002) Surface modification of poly(dimethylsiloxane) microfluidic devices by ultraviolet polymer grafting. Anal Chem 74:4117-23
McClain, M A; Culbertson, C T; Jacobson, S C et al. (2001) Flow cytometry of Escherichia coli on microfluidic devices. Anal Chem 73:5334-8
Ren, X; Bachman, M; Sims, C et al. (2001) Electroosmotic properties of microfluidic channels composed of poly(dimethylsiloxane). J Chromatogr B Biomed Sci Appl 762:117-25
Li, H; Wu, H Y; Wang, Y et al. (2001) Improved capillary electrophoresis conditions for the separation of kinase substrates by the laser micropipet system. J Chromatogr B Biomed Sci Appl 757:79-88
Meredith, G D; Sims, C E; Soughayer, J S et al. (2000) Measurement of kinase activation in single mammalian cells. Nat Biotechnol 18:309-12
Soughayer, J S; Krasieva, T; Jacobson, S C et al. (2000) Characterization of cellular optoporation with distance. Anal Chem 72:1342-7