Polydiacetylene Liposomes Attached to Glass Fibers for Fluorescent Bioassays
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0942-W13-10
Polydiacetylene Liposomes Attached to Glass Fibers for Fluorescent Bioassays Mary A Reppy, and Bradford A Pindzola Analytical Biological Services Inc, 701-4 Cornell Business Park, Wilmington, DE, 19801
ABSTRACT Polydiacetylene (PDA) is a conjugated polymer that changes its fluorescent state in response to environmental changes and can act as a transducer to convert molecular interactions into a discernable output measurable in the macroscopic world. Energy transfer to fluorophores can further enhance the fluorescent signal. Diacetylene liposomes can be prepared with phospholipids and other cell membrane components in the liposomes, and photopolymerized. PDA liposomes have been used for absorbance based detection of biological targets; however, moving to fluorescence detection gives increased sensitivity and also allows sensing from PDA structures deposited on opaque membranes. Unfortunately, PDA liposomes are prone to aggregation, particularly in the presence of divalent cations. Many enzymes require divalent cations such as Mg2+, Mn2+, Ca2+, etc., as co-factors; the tendency of PDA liposomes to aggregate in the presence of these cations limits their use as a platform for detection of enzymatic activity. We have developed methods for attaching PDA liposomes to glass fiber membranes, via thiol-epoxide coupling chemistry, for use in bio-assays and have seen that these materials can be used in place of PDA liposome solutions. We present here the attachment of PDA liposomes to glass fiber membranes in 96-well format, cryogenic TEM scans of the attached liposomes and the use of these materials in fluorescence assays to detect the activity and inhibition of phospholipase A2. INTRODUCTION Chemical and biological sensors require a material component to act as a transducer from the molecular level event of interest to a discernable output measurable in the macroscopic world. One such material is polydiacetylene (PDA), a conjugated polymer whose optical properties change in response to environmental changes. Diacetylene surfactants will selfassemble in water to form liposomes; these can be photopolymerized to form PDA in situ. PDA liposomes have been prepared with phospholipids, sugars, and antibodies incorporated, for colorimetric detection of biological analytes [1-3]. We have shown that the fluorescence properties of PDA can be monitored in lieu of the color change to detect analytes of interest [4,5]. We have further amplified the fluorescent changes by incorporating fluorophores that can accept energy from the excited state of the PDA chains and fluoresce [6]. This FRET process appears to be competitive with excited state quenching mechanisms and leads to an overall improvement of the quantum yield of the PDA system. In our work on developing fluorescent enzymatic assays using PDA liposomes, we have observed that the liposomes often suffer from the disadvantage of aggregating during assays or during storage. Liposomes prepared from shorter chain diacetylenes (fewer than 22 carbons) will aggregate within hou
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