Abstract ED08-02: Designing Life Chips and their impact for the prevention and early detection of cancer

Abstract

Abstract ED08-02 Microelectrophoretic methods can make direct measurements of the activity of normal and oncogenic kinases in tumor cells. Issues of heterogeneity within cell populations, both in terms of cell type and biochemical behavior, often mandate single-cell studies for accurate determination of kinase activity in tumor cells. Chemical cytometry, the application of high-sensitivity chemical separations to characterize the contents of single or small numbers of cells, is emerging as an important approach for such studies. Electrophoretic separations within a glass capillary tube or a microfluidic channel combined with laser-induced fluorescence detection (LIF) are the most sensitive among these methods. Numerous groups have used these separation methods for a variety of biochemical studies in single cells. The Allbritton group has been a leader in using chemical cytometry to develop single-cell assays relevant to the field of signal transduction, particularly kinase assays. The method utilizes an integrated platform comprised of an inverted microscope, pulsed laser, and capillary electrophoresis (CE) system. For the assay, cells are loaded with fluorescently-labeled kinase substrates which function as reporters of the activity of the targeted kinase. For kinases, the substrate is typically a fluorescently labeled peptide, protein, or lipid. At the desired time after reporter loading, a cell is lysed within 10 microseconds by a focused 5 nansecond laser pulse. Dilution of cytoplasmic molecules by diffusion and turbulent mixing terminates cellular reactions with sufficient rapidity to prevent measurement artifacts from cell lysis. Following lysis, the cellular contents are immediately loaded into an overlying capillary or adjacent microfluidic channel and separated by electrophoresis. Due to their different chemical properties, the substrate and product forms of the reporter are readily separated, detected using LIF, and identified by their characteristic migration times. The ratio of the peak areas of the substrate and product are then used as a measure of the targeted enzyme's activation. Routine detection limits for fluorescein-labeled peptide substrates obtained from single mammalian cells are 10 zeptomoles, corresponding to an intracellular substrate concentration of ~10 nM in mammalian cells (~1 pl). The ability to detect the enzyme substrate present in nanomolar to low micromolar concentrations in the cell is an important feature of this assay as the exogenous substrate concentrations are at or below that of endogenous substrates so that competition between endogenous and exogenous substrate (reporter) is minimal. Fluorescent peptides, proteins, and lipids have been used successfully as substrates for kinases. An additional advantage to this approach is the ability to measure the activation of multiple enzymes simultaneously in a single cell by loading the cell with reporters for a variety of enzymes. At present, three kinases have been assayed at the same time in single cells, although the very high separation efficiency of microelectrophoretic methods makes it likely that a much larger number is possible. A second advantage of this microelectrophoresis-based method is that it is fully compatible with light microscopy so that the cells can be monitored using a variety of optical tools prior to electrophoretic analysis. Citation Information: Cancer Prev Res 2008;1(7 Suppl):ED08-02.