Fluorescence resonance energy transfer (FRET) has been exploited as a selective, specific, and sensitive mechanism for molecular sensing in genomics, proteomics, and other processes in living cells. For these applications, FRET relies on the remarkable efficiency of the donor-acceptor separation distance. However, to date, little research work has addressed the use of the other factors in the Forster equation, such as spectral overlap (J), donor-acceptor dipole orientation, and quantum yield of donor, for sensing mechanism. The overall objective of this R15 proposal application is to investigate the feasibility of using modulation of J and acceptor quantum yield (Q) as a sensing mechanism. The bilayered liposome will be composed of dansyl (act as donor), polydiacetylene (PDA, act as acceptor), and receptors (such as antibodies). The interactions between receptors attached to liposomes and antigens (on the surface of a microbe) will induce stress in the conjugated PDA backbone which results in a blue spectral shift in its absorption spectrum. The consequence of PDA absorption shift results in changes in the spectral overlap (J) between donor emission and acceptor absorption which changes the FRET efficiency from donor to acceptor. The overall effect is controlled modulation of the fluorescence intensity. That is, the antibody-antigen interactions will be indirectly sensed through FRET measurements. We expect a detection limit of the proposed assay in the nanomolar range;with careful optimization, it would be possible to detect analytes in picomolar concentration range.
Three specific aims for the proposed work are: (1) Design, synthesis and characterization of different diacetylene and dansyl-tagged diacetylene monomers. (2) Synthesis and characterization of fluorophores and receptors containing liposomes. (3) Detection of E. coli and S. aureus using proposed FRET-based liposome system in solution phase. Ultimately, the proposed work would provide a novel optical actuating mechanism to construct highly selective and sensitive liposomes for rapid sensing of biological molecules and particles.
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