Barcoded Nanowires for Multiplexed Clinical Diagnostics

我们建议开发快速多重检测方法用于临床呼吸道病原体的诊断。 样本。许多病毒和细菌感染引起的呼吸道疾病具有非常相似的症状，因此， 及时准确的病原体识别对患者康复和公共卫生监测具有重要意义。 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检测。我们将使用条形码金属纳米线作为两个 多种类型的诊断分析，将在临床样本上进行测试，并最终过渡 在拟议的工作过程中进入临床环境。 目的1.自猝灭纳米线信标。我们将利用金属附近的电磁效应 表面(猝灭、增强和改变荧光寿命)，以产生纳米线信标 能够在同一样本中同时进行多项分析的探头。由此产生的 多重的、“封闭的”核酸检测分析将不需要标记物或清洗，也不需要 特殊的仪器，超越了传统的荧光光学显微镜，因此是服从的 到接触点分析，如临床诊断。我们建议在这里建立一种17-plex的检测方法 呼吸道病原体。 目的2.同时多重扩增固定化技术用于病原菌检测。对于病毒的脱落 在很低的水平下，检测需要扩增(通过在培养中生长或通过聚合酶链式反应)。我们会 引入多重扩增克服现有病毒检测方法的局限性 以及固定在条形码纳米线上(主要)。为了实现这一点，我们将开发健壮的曲面 可以承受热循环的附着化学，并将提供最小的空间位阻 杂交和延伸，然后将使用这些进展同时指导所有的 对不同的、视觉上可识别的纳米线载体感兴趣，用于敏感病原体检测。

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 barcoded 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 barcoded nanowires (MAIN). To accomplish this we will develop robust surface attachment chemistry that can withstand thermocycling 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.