A Sensitive, Quantitative, Low-cost, Fluorescence Diagnostic System for Dengue

登革热是世界上传播最快的蚊媒病毒，目前每年感染3.9亿人。研究表明，现有的床旁检测灵敏度不足，无法识别许多感染结核分枝杆菌的个体。 登革热。没有一个有必要的定量能力，以帮助检测严重的登革热，特别是在继发性登革热感染。我们的方法将提供一个低成本但定量和敏感的系统。我们的侧流免疫测定（LFIA）测试将分析和定量登革热病毒非结构蛋白（DENV NS 1）的存在，这是一种经常用于确定登革热诊断的分析物。与现有的快速检测相比，荧光染料将用于提高检测限。智能手机用于成像、分析和自动报告;后一功能将简化疾病跟踪。长斯托克斯位移染料，R-藻红蛋白（PE）将被用作荧光报告，无论是单独或捆绑成一个集群。PE的光学性质允许使用廉价的照明设备; PE的最小非特异性结合导致具有宽动态范围的灵敏检测。通过设计用于解离免疫复合蛋白以改善供试试剂可及性的集成检测功能，将实现灵敏度的额外提高。在第一阶段，我们将：1）优化抗NS 1抗体上的PE报告基因（簇）; 2）开发智能手机应用程序以允许自动化分析; 3）开发LFIA条上的“解离区”以帮助分析复合的NS 1; 4）开发侧流条上的对照线的使用以允许NS 1的绝对定量; 5）设计、制造和测试低成本荧光系统的原型，所述荧光系统的材料成本低于35美元。6)通过将结果与使用患者样品的市售ELISA和LFIA测试进行比较来验证NS 1 LFIA。这些调查将证明我们的系统的检测和分析NS 1的性能优势。对于第二阶段，我们希望包括多种分析物，如DENV IgG和IgM，或多重发热性疾病测试条（例如疟疾和DENV）。

DESCRIPTION (provided by applicant): Dengue is the most rapidly spreading mosquito-borne virus in the world and currently infects 390 million people annually. Studies have shown that existing point-of-care tests have inadequate sensitivity to identify many individuals infected with dengue. None have the quantitative capability necessary to aid in the detection of severe dengue, especially in secondary dengue infections. Our approach will provide a low-cost yet quantitative and sensitive system. Our lateral flow immunoassay (LFIA) test will analyze and quantitate the presence of dengue virus non-structural protein (DENV NS1), an analyte that is frequently used to determine a diagnosis of dengue. Fluorescent dyes will be used to improve the limit of detection compared to existing rapid tests. A smart phone is utilized for imaging, analysis and automated reporting; the latter feature will simplify disease tracking. A long Stokes shift dye, R-phycoerythrin (PE) will be used as the fluorescent reporter, either singly or bundled into a cluster. The optical properties of PE allow the use of an inexpensive illumination device; the minimal non-specific binding of PE results in sensitive detection with a wide dynamic range. Additional gains in sensitivity will be achieved through an integrated test feature designed to dissociate immune complexed protein for improved access by the test reagents. In Phase I we will: 1) Optimize the PE reporter (cluster) on anti-NS1 antibody; 2) Develop a smartphone application to allow automated analysis; 3) Develop an on-LFIA strip "zone of dissociation" to aid in analysis of complexed NS1; 4) Develop the use of the control line on the lateral flow strip to allow absolute quantitation of NS1; 5) Design, fabricate and test a prototype of a low cost fluorescence system designed with a materials cost less than $35. 6) Validate the NS1 LFIAs by comparing results with commercially available ELISA and LFIA tests using patient samples. These investigations will demonstrate the performance advantage of our system for detection and analysis of NS1. For phase II we hope to include multiple analytes, such as DENV IgG and IgM, or a multiplex febrile disease test strip (e.g. malaria and DENV).