Conjugated polyelectrolyte supported bead based assays for phospholipase A2 activity.
A fluorescence based assay for human serum-derived phospholipase activity has been developed in which cationic conjugated polyelectrolytes are supported on silica microspheres. The polymer-coated beads are overcoated with an anionic phospholipid (1,2-dimyristoyl-sn-glycero-3-[phospho- rac-(1-glycerol)) (DMPG) to provide "lipobeads" that serve as a sensor for PLA2. The lipid serves a dual role as a substrate for PLA2 and an agent to attenuate quenching of the polymer fluorescence by the external electron transfer quencher 9,10-anthraquinone-2,6-disulfonic acid (AQS). In this case quenching of the polymer fluorescence by AQS increases as the PLA2 digests the lipid. The lipid can also be used itself as a quencher and substrate by employing a small amount of energy transfer quencher substituted lipid in the DMPG. In this case the fluorescence of the polymer is quenched when the lipid layer is intact; as the enzyme digests the lipid, the fluorescence of the polymer is restored. The sensing of PLA2 activity has been studied both by monitoring fluorescence changes in a multiwell plate reader and by flow cytometry. The assay exhibits good sensitivity with EC50 values in the nanomolar range.
Chemburu
Sireesha
S
Department of Chemical and Nuclear Engineering, University of New Mexico, Albuquerque, New Mexico 87131, USA.
Ji
Eunkyung
E
Casana
Yosune
Y
Wu
Yang
Y
Buranda
Tione
T
Schanze
Kirk S
KS
Lopez
Gabriel P
GP
Whitten
David G
DG
D000880:Anthraquinones; D004789:Enzyme Activation; D005453:Fluorescence; D006801:Humans; D008855:Microscopy, Electron, Scanning; D008863:Microspheres; D010715:Phosphatidylglycerols / Q000378:metabolism; D054467:Phospholipases A2 / Q000032:analysis* / Q000097:blood; D011108:Polymers; D012822:Silicon Dioxide
D000880:Anthraquinones; D010715:Phosphatidylglycerols; D011108:Polymers; D012822:Silicon Dioxide; C002773:dimyristoylphosphatidylglycerol; D054467:Phospholipases A2