Investigating Fluorescence Resonance Energy Transfer in Conjugated Liposomes

摘要 荧光共振能量转移(FRET)已被开发为一种选择性、特异性和 在基因组学、蛋白质组学和活细胞的其他过程中的分子传感的敏感机制。 对于这些应用，FRET依赖于供体-受体分离的显著效率 距离。然而，到目前为止，很少有研究工作涉及F中其他因素的使用 方程式，如光谱重叠(J)，施主-受体偶极取向，和施主的量子产额， 感应机制。此R15提案应用程序的总体目标是调查 利用J和受主量子产额(Q)的调制作为传感机制的可行性。 该双层脂质体将由丹酚(作为供体)、聚二乙炔(PDA)作为供体 受体)和受体(如抗体)。脂质体上受体间的相互作用 而(微生物表面的)抗原会在结合的PDA主干上产生压力，这会 导致其吸收光谱发生蓝移。PDA吸收漂移结果的后果 在施主发射和受主吸收之间的光谱重叠(J)的变化中，这改变了 从捐赠者到接受者的焦虑效率。总体效果是对荧光的受控调制 强度。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；经过仔细的优化，它 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化合物的设计、合成与表征 二乙炔单体。 (2)含荧光载体和受体的脂质体的合成与表征。 (3)应用基于FRET的脂质体系统检测大肠埃希菌和金黄色葡萄球菌 解决方案阶段。 最终，提出的工作将提供一种新的光学驱动机构来构建 高选择性和高灵敏度的脂质体，用于生物分子和颗粒的快速传感。 1

Summary 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 F¿rster 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 compose 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. 1