Separation of phospholipids in microfluidic chip device
Sansan Lin, Anthony S. Fischl, Xiahui Bi, and Wally ParcePhospholipid molecules such as ceramide and phosphoinositides play crucial roles in signal transduction pathways. Lipid-modifying enzymes including sphingomyelinase and phosphoinositide kinases regulate the generation and degradation of these lipid-signaling molecules and are important therapeutic targets in drug discovery. We now report a sensitive and convenient method to separate these lipids using microfluidic chip-based technology. The method takes advantage of the high-separation power of the microchips that separate lipids based on micellar electrokinetic capillary chromatography (MEKC) and the high sensitivity of fluorescence detection.We further exploited the method to develop a homogenous assay to monitor activities of lipid-modifying enzymes. The assay format consists of two steps: an on-plate enzymatic reaction using fluorescently labeled substrates followed by an on-chip MEKC separation of the reaction products fromthe substrates. The utility of the assay format for high-throughput screening (HTS) is demonstrated using phospholipase A2 on the Caliper 250 HTS system: throughput of 80min per 384-well plate can be achieved with unattended running time of 5.4 h. This enabling technology for assaying lipid-modifying enzymes is ideal forHTS because it avoids the use of radioactive substrates and complicated separation/washing steps and detects both substrate and product simultaneously. 2003 Elsevier Science (USA). All rights reserved.
August 13, 2002
Analytical Biochemistry
Year: 2002. Volume: 314.