Investigating Fluorescence Resonance Energy Transfer in Conjugated Liposomes

描述（由申请人提供）：荧光共振能量转移（FRET）已被开发为活细胞中基因组学、蛋白质组学和其它过程中的分子传感的选择性、特异性和灵敏性机制。对于这些应用，FRET依赖于供体-受体分离距离的显著效率。然而，迄今为止，很少有研究工作涉及Forster方程中的其他因素，如光谱重叠（J）、给体-受体偶极取向和给体的量子产率，用于传感机制。该R15提案申请的总体目标是研究使用J和受体量子产率（Q）的调制作为传感机制的可行性。 双层脂质体由丹磺酰（作为供体）、聚联乙炔（PDA，作为受体）和受体（如抗体）组成。附着于脂质体的受体和抗原（在微生物表面上）之间的相互作用将在缀合的PDA骨架中诱导应力，这导致其吸收光谱中的蓝色光谱偏移。PDA吸收位移的结果导致供体发射和受体吸收之间的光谱重叠（J）的变化，这改变了从供体到受体的FRET效率。总的效果是荧光强度的受控调制。也就是说，抗体-抗原相互作用将通过FRET测量来间接地感测。我们预计拟定检测试剂盒的检测限在纳摩尔范围内;通过仔细优化，有可能检测皮摩尔浓度范围内的分析物。 拟议工作的三个具体目标是： (1)不同二乙炔和丹磺酰标记二乙炔单体的设计、合成和表征。(2)含荧光团和受体的脂质体的合成与表征。(3)E. coli和革兰氏阳性菌S.金黄色葡萄球菌，使用所提出的基于FRET的脂质体系统在溶液相中。 该研究为构建高选择性、高灵敏度的脂质体提供了一种新的光学驱动机制，可用于生物分子和颗粒的快速传感。

DESCRIPTION (provided by applicant): 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.