Single Cell Assays of Lipid Signaling Using Ultra-Small Volume Thin Layer Chromatography (Picoliter TLC)

Description:
Lipid signaling pathways regulate a multitude of cellular behaviors in humans including cell proliferation, death, migration, and many other attributes. These pathways are implicated in a host of diseases and as such a large number of compounds are now in clinical trials to target the critical enzymes within these signaling cascades. However, most existing technologies for assay of cellular lipids possess poor sensitivity and require large sample volumes making them unsuitable for the assay of mammalian cells which are 1 pL in volume and may possess as little as 10-20 moles of key regulatory lipids. To address this challenge, an array of open microchannels filled with a monolithic silica was developed using microfabrication and sol-gel chemistry. When ultra-small volumes (<1 nL) of fluorescent lipids including [phosphatidylinositol 4,5-bisphosphate, phosphatidylinositol 3,4,5-trisphosphate, phosphatidylcholine (PC), phosphatidylethanolamine (PE), 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, sphingosine, sphingosine-1-phosphate (S1P), and ceramide] were spotted at the channel inlet followed by separation, the lipids were detectable with excellent sensitivity (10-20 moles). To demonstrate the power of pTLC, leukemic cells were loaded with fluorescent PC, PE or sphingosine and a single cell printed at the inlet of a pTLC channel. PE and PC within a single cell were separated by pTLC in less than 1 min with excellent resolution and detection limits. When cells were loaded with a fluorescent sphingosine, the conversion of sphingosine to S1P was also detected and the reaction blocked by sphingosine kinase inhibitors. These novel biomedical microdevices can be fabricated in large scale arrays for fully automated, high-throughput assay of ultra-small-sized samples as well as for screening of therapeutic modulators of lipid signaling. Additionally pTLC enables assay of the diversity of signaling activity of key lipid pathways amongst single cells.

Speaker: Nancy Allbritton - University of Washington
Nancy Allbritton is the Frank & Julie Jungers Dean of Engineering and Professor of Bioengineering at the University of Washington in Seattle (2019- current). Her research focuses on the development of novel technologies for applications in single-cell analysis, micro-arrays and fluidics, and organ-on-chip and has resulted in >200 full-length journal publications and patents and led to >15 commercial products. Five companies have been formed based on her research discoveries: Protein Simple (acquired by Bio-Techne in 2014), Intellego, Cell Microsystems (www.cellmicrosystems.com), Altis Biosystems (www.altisbiosystems.com) and Piccolo Biossystems. Dr. Allbritton is a Fellow of the American Association for the Advancement of Science, the American Institute for Medical & Biological Engineering, and the National Academy of Inventors. She obtained her B.S. in physics from LSU, M.D. from Johns Hopkins University, and Ph.D. in Medical Physics/Medical Engineering from the MIT.