Development of a Microfluidic Point-of-Care HCV Viral Load Assay

描述(由申请人提供)：达克塔里诊断平台基于微流控样品处理和电化学检测，专为护士或初级卫生保健工作者设计，只需最少的培训；总上手时间为60秒。最初的达克塔里CD_4检测方法是为了从10μ的L手指血样中进行CD_4细胞计数，时间不到十分钟，成本很低。在这个项目下，我们将在达克塔里平台上开发一种丙型肝炎病毒载量分析。丙型肝炎病毒载量分析将把血液采集、样本处理、丙型肝炎病毒捕获和电子检测整合在一个一次性塑料试剂盒中。在第一阶段，我们将建立整个丙型肝炎病毒的磁性捕获和裂解，以及释放的病毒蛋白的电子检测的可行性。首先，将评估一组选定的丙型肝炎病毒捕捉剂在血浆和全血中的结合特异性和敏感性。同时，我们将优化和评估一种基于夹心免疫分析的电检测方法的检测阈值，利用金纳米颗粒/银沉积信号放大级联来检测释放的丙型肝炎病毒核心抗原。电检测方法将利用达克塔里的新电极设计。如果这两项研究的数据确定了技术可行性，该项目的第二阶段将把血液采集、丙型肝炎病毒捕获和溶血以及电子检测集成到达克塔里的微流控色谱盒中，生产出完整的丙型肝炎病毒载量分析，准备用于达克塔里的手持式仪器进行商业放大。首先，将对丙型肝炎病毒捕获方案进行优化，并使其适应达克塔里药盒的生产格式。该方案包括利用第一阶段确定的抗丙型肝炎病毒捕获剂功能化的微粒进行磁性混合和浓缩。其次，将整合第一阶段中确定的电气检测模块，并将设计和测试流体路径的布局(包括试剂存储和处理)，最终形成完整的丙型肝炎病毒载量分析试剂盒。最后，将对目前的达克塔里仪器进行改装，使之包括磁力捕获丙型肝炎病毒粒子所需的永久磁铁，以及用于试剂运输的额外机械致动器。最终交付的将是一种简单、低成本的工具，能够用于初级保健水平，以量化循环中的丙型肝炎病毒，并识别需要治疗的活动性丙型肝炎病毒感染患者。

DESCRIPTION (provided by applicant): The Daktari diagnostic platform-based on microfluidic sample processing and electrochemical detection-is designed for use by a nurse or primary health care worker with minimal training; total hands-on time is 60 seconds. The initial Daktari CD4 Assay has been developed for performing CD4 cell counts from a 10 μL fingerstick blood sample in less than ten minutes, at low cost. Under this project, we will develop an HCV Viral Load Assay on the Daktari platform. The HCV Viral Load Assay will incorporate blood collection, sample processing, HCV capture, and electrical detection in an integrated, disposable plastic cartridge. In Phase I, we will establish the feasibility of whole HCV virus magnetic capture and lysis, and electrical detection of released viral protein. First, the binding specificity and sensiivity of a selected panel of HCV capture agents will be evaluated in both plasma and whole blood. In parallel, we will optimize and evaluate the threshold of detection of an electrical detection method based on a sandwich immunoassay, to detect released HCV core antigen using a gold-nanoparticle/silver deposition signal amplification cascade. The electrical detection method will make use of Daktari's novel electrode design. Should the data from these two studies determine technical feasibility, Phase II of the project will integrate blood collection, HCV capture and lyss, and electrical detection into a Daktari microfluidic cartridge, yielding a complete HCV Viral Load Assay ready for commercial scale-up, for use in Daktari's handheld instrument. First, the HCV capture protocol, consisting of magnetic mixing and concentration using microparticles functionalized with the anti-HCV capture agent identified in Phase I, will be optimized and adapted to the Daktari cartridge production format. Second, the electrical detection module identified in Phase I will be integrated, and the layout of the fluidic paths (including reagent storage and handling) will be designed and tested, ultimately leading to a complete HCV Viral Load Assay cartridge. Finally, the current Daktari instrument will be modified to incorporate the permanent magnets required for magnetic capture of HCV virions, and an additional mechanical actuator for reagent transport. The final deliverable will be a simple, low-cost instrument capable of being used at the primary care level, to quantify circulating HCV and identify patients with active HCV infection in need of treatment.