Barcoded Nanowires for Multiplexed Clinical Diagnostics

描述（由申请人提供）：我们建议开发用于临床样品中呼吸道病原体诊断的快速多重检测方法。许多病毒和细菌感染引起的呼吸道疾病症状非常相似，因此，及时准确地识别病原体对患者康复和公共卫生监测至关重要。目前的方法包括直接免疫荧光和细胞培养，这两种方法目前都需要对每种病原体进行单独的测试。我们将使用条形码金属纳米线作为两种多路诊断分析的编码支持，这些检测将在临床样品上进行测试，并在拟议的工作过程中最终过渡到临床环境中。目的1。自淬纳米线信标。我们将利用金属表面附近的电磁效应（猝灭、增强和荧光寿命的改变），生产纳米线信标探针，使许多分析能够在同一样品中同时进行。由此产生的可重复使用的“封闭管”核酸检测分析将不需要标靶或清洗，也不需要除传统荧光光学显微镜之外的任何特殊仪器，因此适用于临床诊断等接触点分析。我们在此建议开发一种17-plexed检测呼吸道病原体的方法。目标2。同时多路扩增和固定化用于病原体检测。对于极低水平的病毒，必须进行扩增（通过培养或PCR）才能进行检测。我们将通过引入多路扩增和固定在条形码纳米线（MAIN）上，克服现有方法检测病毒的局限性。为了实现这一目标，我们将开发强大的表面附着化学，它可以承受热循环，并将提供最小的杂交和扩展的空间障碍，然后将利用这些进步，同时将所有感兴趣的PCR产物引导到不同的，视觉上可识别的纳米线载体上，用于敏感的病原体检测。

DESCRIPTION (provided by applicant): We propose to develop rapid multiplexed assays for the diagnosis of respiratory pathogens in clinical samples. Many viral and bacterial infections cause respiratory illnesses with very similar symptoms, thus, timely and accurate pathogen identification is important for patient recovery and public health monitoring. Current methods include direct immunofluorescence and cell culture, both of which currently require individual tests for each pathogen. We will use bar-coded metallic nanowires as encoded supports for two types of multiplexed diagnostic assays, which will be tested on clinical samples and ultimately transitioned into a clinical setting during the course of the proposed work. Aim 1. Self quenching nanowire beacons. We will take advantage of the electromagnetic effects near metal surfaces (quenching, enhancement, and alteration of fluorescence lifetimes), to produce nanowire beacon probes that enable many assays to be conducted simultaneously in the same sample. The resulting multiplexible, "closed tube" nucleic acid detection assays will require no target labeling or washing, nor any special instrumentation beyond a conventional fluorescence optical microscope, and are therefore amenable to point-of-contact assays such as clinical diagnostics. We propose here to develop a 17-plexed assay for respiratory pathogens. Aim 2. Simultaneous multiplexed amplification and immobilization for pathogen detection. For viruses shed at very low levels, amplification (by growing in culture or by PCR) is necessary for detection. We will overcome current limitations of viral detection by existing methods by introducing multiplexed amplification and immobilization onto bar-coded nanowires (MAIN). To accomplish this we will develop robust surface attachment chemistry that can withstand thermo cycling and will provide for minimal steric hindrance of hybridization and extension, and will then use these advances to simultaneously direct all PCR products of interest onto different, visually identifiable nanowire supports for sensitive pathogen detection.