Journal
JOURNAL OF PHYSICAL CHEMISTRY B
Volume 112, Issue 46, Pages 14492-14499Publisher
AMER CHEMICAL SOC
DOI: 10.1021/jp803358j
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Funding
- Center for Biomedical Engineering (CBME) at the University of New Mexico
- National Science Foundation (NSF) through the NSF SENSORS [CTS0332315, DMR-0611616]
- Defense Threat Reduction Agency (DTRA) [W911NF-07-1-0079]
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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.
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