Abstract

Picoliter Thin Layer Chromatography (pTLC) for Assay of Lipid Signaling in Single Cells

Nancy Allbritton, Frank and Julie Jungers Dean of the College of Engineering and Professor of Bioengineering, University of Washington in Seattle

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 many compounds are now in clinical trials to target 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. These and other advances in biomedical microdevices are paving the way for rapid discoveries in basic and pharmaceutical sciences, as well as personalized medicine.