- Chemburu, Sireesha and Ji, Eunkyung and Casana, Yosune and Wu, Yang and Buranda, Tione and Schanze, Kirk S. and Lopez, Gabriel P. and Whitten, David G., Conjugated Polyelectrolyte Supported Bead Based Assays for Phospholipase A(2) Activity,
JOURNAL OF PHYSICAL CHEMISTRY B, vol. 112 no. 46
pp. 14492--14499 [doi] .
(last updated on 2010/02/08)
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 PLA(2). The lipid serves a dual role as a substrate for PLA(2) 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 PLA(2) 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 PLA(2) 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.