Gold Quenched Molecular Beacons

分子信标是DNA的发夹环，一端与荧光标记结合，另一端与猝灭剂结合。与互补靶结合后，信标打开，荧光团和猝灭剂分开，荧光出现。灵敏度取决于“信噪比”的荧光强度在开放和封闭构象。传统的达布尔淬灭信标的强度比高达100。然而，1.4 nM的纳米金团簇产生的强度比高达几千，并且由于它吸收的波长范围很广，可以熄灭各种荧光团。将制备改进的金纳米粒子猝灭剂，使用更大的金粒子通过表面等离子体耦合实现更有效的猝灭。这些标记的荧光猝灭特性将通过合成修饰的配体来重现，这些配体增加了信标的溶解度、稳定性和生物相容性，同时为荧光团引入了特定的亲和力，使金颗粒和猝灭剂更紧密地结合在一起，并在封闭构型中增加猝灭。纯化方法将被开发，金淬灭的DNA和PNA信标将与传统的类似物进行比较，以快速表征细菌。可重复使用的生物传感器原型将通过在光纤电缆上固定金淬信标来制造。建议的商业应用：金淬灭剂有望将分子信标的信噪比提高一个数量级以上，从而能够更灵敏、更快速地实时检测均匀标本中的特定DNA序列。与快速DNA扩增方法相结合，金淬灭信标将用于病毒和细菌分型、传染病诊断和癌症检测。作为光纤生物传感器的组成部分，它们将用于研究单个细胞中的事件，组装成束，这些将用于移植组织和器官的遗传筛选，并形成更便携，侵入性更小的产前测试，筛选和遗传分析技术的基础。

Molecular beacons are hairpin loops of DNA, conjugated to a fluorescent label at one end and a quencher at the other. Upon binding to a complementary target, the beacon opens, the fluorophore and quencher move apart, and fluorescence appears. Sensitivity is dependent upon the "signal-to-noise ratio" of fluorescence intensities in the open and closed conformations. Conventional dabcyl-quenched beacons give intensity ratios up to 100. However, the 1.4 nM Nanogold cluster yields intensity ratios up to several thousand, and since it absorbs over a wide range of wavelengths, can quench a variety of fluorophores. Improved gold nanoparticle quenchers will be prepared, using larger gold particles to achieve more effective quenching through surface plasmon coupling. Fluorescence quenching properties of these labels will be made reproducible through the synthesis of modified ligands which increase the solubility, stability and biological compatibility of he beacon, while introducing specific affinities for fluorophores to hold the gold particle and quencher closer together and increase quenching in the closed configuration. Purification methods will be developed and gold quenched DNA and PNA beacons will be compared with conventional analogs for rapid bacterial characterization. Prototype reusable biosensors will be fabricated by immobilizing gold-quenched beacons on fiber optic cables. PROPOSED COMMERCIAL APPLICATIONS: Gold quenchers are expected to improve the signal-to-noise ratio of molecular beacons by more than a order of magnitude, enabling more sensitive and rapid real-time detection of specific DNA sequences in homogeneous specimens. Combined with rapid DNA amplification methods, gold quenched beacons would be used for viral and bacterial typing, diagnosis of infectious disease, and detection of cancer. As components of fiber optic biosensors, they would be used to study events in individual cells, assembled into bundles, these would be used for the genetic screening of tissues and organs for transplantation, and form the basis for more portable, less invasive prenatal testing, screening and genetic profiling technology.