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

描述（由申请人提供）：Daktari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使用，仅需最少的培训；总动手时间为60秒。最初的Daktari CD4 Assay已经开发出来，可以在不到10分钟的时间内以低成本从10 μL的指棒血液样本中进行CD4细胞计数。在这个项目下，我们将在Daktari平台上开发一种HCV病毒载量测定方法。HCV病毒载量测定将包括血液采集、样品处理、HCV捕获和电检测，在一个集成的一次性塑料盒中。在第一阶段，我们将建立整个HCV病毒的磁捕获和裂解，以及释放的病毒蛋白的电检测的可行性。首先，将在血浆和全血中评估选定的一组HCV捕获剂的结合特异性和敏感性。同时，我们将优化和评估基于三明治免疫分析法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检测释放的HCV核心抗原。电检测方法将利用Daktari的新型电极设计。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裂解以及电检测整合到Daktari微流体盒中，产生一个完整的丙型肝炎病毒载量测定，准备进行商业规模扩大，用于Daktari的手持式仪器。首先，HCV捕获方案，包括磁性混合和使用具有抗HCV捕获剂功能的微颗粒进行浓缩，将被优化并适应Daktari药筒的生产格式。其次，将整合第一阶段确定的电检测模块，并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HCV病毒载量检测试剂盒。最后，将对目前的Daktari仪器进行改进，以纳入HCV病毒粒子磁捕获所需的永磁体，以及用于试剂运输的附加机械驱动器。最终的成果将是一种简单、低成本的仪器，能够用于初级保健水平，量化循环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.