Conjugated polyelectrolyte supported bead based assays for phospholipase A2 activity

Journal Article

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. © 2008 American Chemical Society.

Full Text

Duke Authors

Cited Authors

  • Chemburu, S; Ji, E; Casana, Y; Wu, Y; Buranda, T; Schanze, KS; Lopez, GP; Whitten, DG

Published Date

  • 2008

Published In

Volume / Issue

  • 112 / 46

Start / End Page

  • 14492 - 14499

International Standard Serial Number (ISSN)

  • 1520-6106

Digital Object Identifier (DOI)

  • 10.1021/jp803358j