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

描述（由申请人提供）：Daktari诊断平台-基于微流体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而设计，只需接受最少的培训;总动手时间为60秒。最初的Daktari CD 4 Assay开发用于在不到10分钟的时间内以低成本对10 μL手指针刺血样进行CD 4细胞计数。在该项目下，我们将在Daktari平台上开发HCV病毒载量测定。HCV Viral Load Assay将血液采集、样本处理、HCV捕获和电检测集成在一次性塑料检测盒中。在第一阶段，我们将建立完整的HCV病毒的磁性捕获和裂解，并释放病毒蛋白的电检测的可行性。首先，将在血浆和全血中评价选定的一组HCV捕获剂的结合特异性和灵敏度。同时，我们将优化和评估基于夹心免疫测定的电检测方法的检测阈值，以使用金纳米颗粒/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、HCV捕获和裂解以及电检测集成到Daktari微流控盒中，产生一个完整的HCV病毒载量检测试剂盒，可用于Daktari的手持式仪器中，供商业规模扩大使用。首先，将优化HCV捕获方案（包括使用第I阶段确定的抗HCV捕获剂功能化的微粒进行磁性混合和浓缩），并使其适应Daktari检测盒生产形式。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（包括试剂储存和处理），最终形成完整的HCV Viral Load Assay检测盒。最后，将修改当前Daktari仪器，以纳入磁性捕获HCV病毒体所需的永磁体，以及用于试剂运输的额外机械致动器。最终的交付成果将是一个简单、低成本的工具，能够在初级保健水平上使用，以量化循环中的HCV，并确定需要治疗的活动性HCV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.