RESEARCH PAPER Quantum dot–NBD–liposome luminescent probes for monitoring phospholipase A 2 activity Venkata R. Kethineedi & Georgeta Crivat & Matthew A. Tarr & Zeev Rosenzweig Received: 16 April 2013 /Revised: 30 July 2013 /Accepted: 7 October 2013 /Published online: 31 October 2013 # Springer-Verlag Berlin Heidelberg 2013 Abstract In this paper we describe the fabrication and characterization of new liposome encapsulated quantum dot–fluorescence resonance energy transfer (FRET)-based probes for monitoring the enzymatic activity of phospholipase A 2. To fabricate the probes, luminescent CdSe/ZnS quantum dots capped with trioctylphosphine oxide (TOPO) ligands were incorporated into the lipid bilayer of unilamellar liposomes with an average diameter of approximately 100 nm. Incorporating TOPO capped quantum dots in liposomes enabled their use in aqueous solution while maintaining their hydrophobicity and excellent photophysical properties. The phospholipid bilayer was labeled with the fluorophore NBD C 6 -HPC (2-(6-(7- nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexa decanoyl-sn -glycero-3-phosphocholine). The luminescent quantum dots acted as FRET donors and the NBD dye molecules acted as FRET acceptors. The probe response was based on FRET interactions between the quantum dots and the NBD dye molecules. The NBD dye molecules were cleaved and released to the solution in the presence of the enzyme phospholipase A 2. This led to an increase of the luminescence of the quantum dots and to a corresponding decrease in the fluorescence of the NBD molecules, because of a decrease in FRET efficiency between the quantum dots and the NBD dye molecules. Because the quantum dots were not attached covalently to the phospholipids, they did not hinder the enzyme activity as a result of steric effects. The probes were able to detect amounts of phospholipase A 2 as low as 0.0075 U mL -1 and to monitor enzyme activity in real time. The probes were also used to screen phospholipase A 2 inhibitors. For example, we found that the inhibition efficiency of MJ33 (1-hexadecyl-3-(trifluoroethyl)-sn - glycero-2-phosphomethanol) was higher than that of OBAA (3-(4-octadecyl)benzoylacrylic acid). Keywords Quantum dots . Liposomes . FRET . Phospholipase A 2 Introduction Luminescent quantum dot (QD)-based probes have been extensively utilized in the last decade for optical imaging of cells and tissues, and in bioassays [1, 2]. Luminescent QD are regarded as viable alternatives to organic fluorophores because of their unique photophysical properties, including high emission quantum yield, high photostability, broad excitation wavelength range, narrow emission spectra, and size-dependent tunability [ 3–8]. Luminescent QD are typically synthesized in organic solvents, and are capped with hydrophobic ligands, which makes them insoluble in aqueous solution [ 9, 10]. QD were recently incorporated into phospholipid vesicles, liposomes, to enable their application in aqueous and biological systems without surface modification [11–13]. QD-containing liposomes have been used in bio-imaging applications in vitro and in vivo [14] and have been used as fluorescent markers in immunoassays [15]. QD–liposomes have also been used for targeting and imaging tumor cells via receptor-mediated endocytosis [16]. V. R. Kethineedi : M. A. Tarr Department of Chemistry and the Advanced Materials Research Institute, University of New Orleans, New Orleans, LA 70148, USA G. Crivat Department of Entomology and Program in Cell and Molecular Biology, University of Maryland, College Park, MD 20742, USA Z. Rosenzweig (*) Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, MD 21250, USA e-mail: zrosenzw@umbc.edu e-mail: zrosenzw@nsf.gov e-mail: zrosenzw@uno.edu Anal Bioanal Chem (2013) 405:9729–9737 DOI 10.1007/s00216-013-7422-z