Paul Debreczeny Distinguished Professor
Department of Chemistry
Ph.D., Medical Physics/Medical Engineering
- Microanalytical separation techniques to study cell signaling
- Development of synthetic chemical reporters of enzyme activity
- Microfabricated platforms for cell manipulation and analysis
- Microfluidic systems for cell analysis
Profiling cellular signal transduction networks
A major focus of research addresses quantitative measurements of the enzymatic activity of signal transduction proteins in cells. A powerful chemical cytometry method for biochemical assays has been pioneered by the laboratory that enables the activity of one or more enzymes to be measured simultaneously in a single cell. This technology is unique in both enabling the measurement of multiple enzymes and its applicability to primary cells obtained directly from organisms and patients. An important aspect of the system is the development of sampling techniques that enable single mammalian cells of picoliter volumes to be rapidly lysed and efficiently loaded into the analysis device. Cell lysis in millisecond timescales has been achieved by both laser and electrical means. This feature is required in many assays to immediately terminate biochemical reactions, thus preventing measurement artifacts resulting from the sampling process. The assay strategy is applicable to a broad range of intracellular enzymes, including kinases, phosphatases, proteases, and lipid metabolic enzymes. A significant ongoing effort in this regard is the design and validation of enzyme substrates that can be taken up by cells or expressed, and act as faithful intracellular indicators of the enzyme activity of interest.
Microfluidic technologies for biochemical assays require solutions to fundamental issues in analytic chemistry and micro-systems engineering to successfully carry out analyses of intracellular biochemical reactions using this “lab-on-a-chip” technology. Challenges related to sampling human tumor cells and performing high-throughput chemical separations on glass-based devices are being met through a long-standing collaboration with Dr. J. Michael Ramsey here at UNC. This work has been funded by the NIH since 1998 to develop a device for profiling human tumor cells for upregulated kinase activity. Applications of these glass microfluidic devices are expected to be in areas requiring high-throughput, high quality separations, for example, screening large numbers of cells for inhibition of enzymatic activity in response to candidate pharmaceutical compounds. A collaborative effort with investigators at UC Irvine is targeted towards the development of polymer-based devices and integrated micro-systems. One aspect of this work is focused on the development of an inexpensive, disposable device for clinical applications, for example profiling drug sensitivities of a patient's tumor cells to the new generation of kinase-inhibiting, chemotherapeutic agents.
Micropallet Arrays for Cell Sorting
Another line of research is the development of integrated platforms for positive selection and isolation of adherent cells and for high-throughput biochemical assays of adherent cells. A new technology for cell sorting has been developed that is comprised of a transparent array formed from pedestals of micron dimensions, termed micropallets, on which cells are cultured. A distinctive feature is that the individual micropallets and their attached cells can be released from the array using a laser and collected with very high rates of cell viability. By means of the micropallet array, cells can be selected based on a large number of attributes including morphology, fluorescence, dynamic traits (e.g., growth rate) and the transmission of properties to daughter cells. The cell arrays have been used for a variety of purposes including single-cell and colony sorting and rapid cloning of stably transfected cells. A number of collaborations are ongoing in the further development and application of this new cell sorting technique.
Click above for PubMed publications.
- Phillips, K.S., Kottegoda, S., Kang, K.M., Sims, C.E., Allbritton, N.L. (2009) Separations in poly(dimethylsiloxane) microchips coated with supported bilayer membranes. Anal Chem 80: 9756–9762 [epub ahead of print]. Abstract
- Shadpour, H., Sims, C.E., Thresher, R.J., Allbritton, N.L. (2009) Sorting and expansion of murine embryonic stem cell colonies using micropallet arrays. Cytometry A 75A:121-129 [epub ahead of print]. Abstract
- Lai, H.-H, Quinto-Su, .PA., Sims, C.E., Bachman, M., Li, G.P., Venugopalan, V., Allbritton, N.L. (2008) Characterization and use of laser-based lysis for cell analysis on-chip. J R Soc Interface 5: Suppl 2: S113-21. Abstract
- Quinto-Su, P.A., Salazar, G.T., Sims, C.E., Allbritton, N.L., Venugopalan, V. (2008) Mechanism of Pulsed Laser Microbeam Release of SU-8 Polymer Micropallets for the Collection and Separation of Adherent Cells. Anal Chem 80: 4675-4679. Abstract
- Borland, L.M., Allbritton, N.L. (2008) Use of micellar electrokinetic chromatography to measure palmitoylation of a peptide. J Chromatogr B Analyt Technol Biomed Life Sci 875: 451-458. Abstract
- Wang, Y., Salazar, G., Pai, J.-H., Sims, C.E., Allbritton, N.L. (2008) Micropallet arrays with poly(ethylene glycol) walls. Lab Chip 8: 734-740. Abstract
- Kottegoda, S., Aoto, P.C., Sims, C.E., Allbritton, N.L. (2008) The Biarsenical-Tetracysteine Motif as a Fluorescent Tag for Detection in Capillary Electrophoresis. Anal Chem 80: 5358-5366. Abstract
- Marc, P.J., Sims, C.E., Bachman, M., Li, G.P., Allbritton, N.L. (2008) Fast-lysis cell traps for chemical cytometry. Lab Chip 8: 710-716. Abstract
- Salazar, G., Wang, Y., Bachman, M., Sims, C.E., Li, G.P., Allbritton, N.L. (2008) Characterization of the laser-based release of micropallets from arrays. J Biomed Optics 13: 034007. Abstract
- Lee, K.J., Mwongela, S.M., Kottegoda, S. Borland, L., Nelson, A.R., Sims, C.E., Allbritton, N.L. (2008) Determination of sphingosine kinase activity for cellular signaling studies. Anal Chem 80: 1620-1627. Abstract
- Quinto-Su, P.A., Lai, H.-H., Yoon, H.H., Sims, C.E., Allbritton, N.L., Venugopalan, V. (2008) Examination of laser microbeam cell lysis in a PDMS microfluidic channel using time-resolved imaging. Lab Chip 8: 408-414. Abstract
- Nelson, A.R., Borland, L., Allbritton, N.L., Sims, C.E. (2007) Myristoyl-based transport of peptides into living cells. Biochemistry 46: 14771-14781. Abstract
- Fernandes, N., Bailey, D.E., Vanvranken, D.L., Allbritton, N.L. (2007) Use of docking peptides to design modular substrates with high efficiency for mitogen-activated protein kinase extracellular signal-regulated kinase. ACS Chem Biol 2: 665-73. Abstract
- Marc, P.J., Sims, C.E., Allbritton, N.L. (2007) Coaxial flow system for chemical cytometry. Anal Chem 79: 9054-9059. Abstract