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)已被用作基因组学、蛋白质组学和活细胞中其他过程中的分子传感的选择性、特异性和敏感性机制。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，到目前为止，很少有研究工作涉及Forster方程中的其他因素，如光谱重叠(J)、施主-受体偶极取向和施主的量子产额，用于传感机制。R15方案应用的总体目标是研究使用J和受体量子产额(Q)的调制作为传感机制的可行性。 双层脂质体将由丹酚(作为供体)、聚二乙炔(PDA，作为受体)和受体(如抗体)组成。结合在脂质体上的受体与(微生物表面的)抗原之间的相互作用会在结合的PDA主干上产生应力，从而导致其吸收光谱的蓝移。PDA吸收位移的结果是改变了供体发射和受体吸收之间的光谱重叠(J)，从而改变了FRET效率从供体到受体。总体效果是对荧光强度的可控调制。也就是说，抗体与抗原的相互作用将通过FRET测量间接感受到。我们预计，建议的检测下限在纳摩尔范围内；通过仔细的优化，将有可能检测到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的二乙炔单体的设计、合成和表征。(2)含荧光载体和受体的脂质体的合成与表征。(3)以FRET为基础的脂质体体系在溶液相中对大肠杆菌和金黄色葡萄球菌的检测。 最终，提出的工作将提供一种新的光学驱动机制，以构建高选择性和高灵敏度的脂质体，用于快速检测生物分子和颗粒。

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.