Gold Quenched Molecular Beacons

分子信标是DNA的发夹环，一端连接着荧光标记，另一端连接着猝灭剂。当结合到互补靶标时，信标打开，荧光团和猝灭剂分开，出现荧光。灵敏度取决于开放构象和闭合构象中荧光强度的“信噪比”。传统的dabcyl淬火信标的光强比高达100。然而，1.4纳米金团簇产生的强度比高达数千，由于它可以在广泛的波长范围内吸收，可以猝灭各种荧光团。将制备改进的金纳米颗粒淬火剂，使用更大的金颗粒通过表面等离子激元耦合实现更有效的淬火。这些标记的荧光猝灭性能将通过合成修饰配体来重现，这些修饰配体增加了He信标的溶解性、稳定性和生物兼容性，同时引入了荧光团的特定亲和力以将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。将开发纯化方法，并将金淬灭的DNA和PNA信标与常规类似物进行比较，以快速表征细菌。原型可重复使用的生物传感器将通过将金淬火的信标固定在光缆上来制造。拟议的商业应用：金猝灭剂有望将分子信标的信噪比提高一个数量级以上，从而能够更灵敏和快速地实时检测同质样本中的特定DNA序列。与快速DNA扩增方法相结合，金淬火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，用于对移植组织和器官进行基因筛查，并为更便携、侵入性更小的产前测试、筛查和基因图谱技术奠定基础。

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.