Separation of phospholipids in microfluidic chip device: application to high-throughput screening assays for lipid-modifying enzymes.
Phospholipid 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 from the substrates. The utility of the assay format for high-throughput screening (HTS) is demonstrated using phospholipase A(2) on the Caliper 250 HTS system: throughput of 80min per 384-well plate can be achieved with unattended running time of 5.4h. This enabling technology for assaying lipid-modifying enzymes is ideal for HTS because it avoids the use of radioactive substrates and complicated separation/washing steps and detects both substrate and product simultaneously.
Lin
Sansan
S
Caliper Technologies Corp., Mountain View, CA 94043, USA. sansan.lin@calipertech.com
Fischl
Anthony S
AS
Bi
Xiahui
X
Parce
Wally
W
D004798:Enzymes / Q000378:metabolism*; D008832:Microchemistry / Q000295:instrumentation* / Q000379:methods*; D008904:Miniaturization / Q000295:instrumentation / Q000379:methods; D010741:Phospholipases A / Q000378:metabolism; D010743:Phospholipids / Q000302:isolation & purification* / Q000378:metabolism*; D015203:Reproducibility of Results
D004798:Enzymes; D010743:Phospholipids; D010741:Phospholipases A