Barcoded Nanowires for Multiplexed Clinical Diagnostics

描述(由申请人提供)：我们建议开发快速多重检测方法来诊断临床样本中的呼吸道病原体。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，及时和准确地识别病原体对于患者的康复和公共卫生监测非常重要。目前的方法包括直接免疫荧光法和细胞培养法，这两种方法目前都需要对每种病原体进行单独测试。我们将使用条形码金属纳米线作为两种类型的多重诊断分析的编码载体，这将在临床样本上进行测试，并在拟议的工作过程中最终过渡到临床环境中。目的1.自猝灭纳米线信标。我们将利用金属表面附近的电磁效应(荧光寿命的猝灭、增强和改变)来生产纳米线信标探测器，使同一样品中的许多分析能够同时进行。由此产生的多路“闭管”核酸检测分析将不需要目标标记或清洗，也不需要传统荧光光学显微镜以外的任何特殊仪器，因此适用于接触点分析，如临床诊断。我们建议建立一种针对呼吸道病原体的17-plex检测方法。目的2.同时多重扩增固定化技术用于病原菌检测。对于水平非常低的病毒，必须进行扩增(通过在培养中生长或通过聚合酶链式反应)进行检测。我们将通过在条码纳米线(Main)上引入多重扩增和固定化来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，并将提供最小的空间位阻杂交和延伸，然后将利用这些进展，同时将所有感兴趣的聚合酶链式反应产物定向到不同的，视觉上可识别的纳米线载体上，用于敏感病原体的检测。

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.