Assays of Lipid Signaling In Single Cells Of Clinical Samples

Lipids play a crucial role in cell function by forming membranes, acting as energy reserves, and functioning as molecular messengers. Dysfunction in lipid metabolism has been linked to various diseases including cancer, neurodegenerative disorders, and viral infections. Assessing the activity of lipid-modifying enzymes is of particular importance since the mis-regulation of these enzymes often plays a role in the pathophysiology of disease states. While strategies now exist for the assay of DNA, RNA and proteins within single cells, measurement of critical lipid second messengers in key signaling pathways has remained quite challenging. A major reason is the small cell volume (~1 pL) coupled with the low concentration of key signaling lipids. For example, sphingosine 1-phosphate and phosphatidylinositol (3,4,5)-trisphosphate are estimated to be ~0.1-1 attomole [1-10 exp(-19) moles] per cell. These scarce lipids are also present amid high levels of structural lipids further challenging their quantification in single cells. To address this challenge, an array of open microchannels filled with a monolithic silica was developed using microfabrication and sol-gel chemistry. The microdevice with large numbers of parallel silica microbands supports the separation of fluorescently labelled or clickable lipids from ultrasmall volumes (<1 pL) including single cells or cell lysates. This new technology termed picoliter thin layer chromatography (pTLC) enables separation of a wide array of lipids including sphingolipids, phospholipids, fatty acids, and others with high sensitivity detection [~10 exp(-20) moles]. Assays of single cells from small-sized primary clinical samples will be demonstrated. These and other advances in biomedical microdevices are paving the way for rapid discoveries in basic and pharmaceutical sciences, as well as personalized medicine.