Sample-to-Answer, Rapid, Multiplexed and PCR-Free Diagnostics of Arboviral Diseases in Resource Limited Settings

节肢动物传播的病毒(虫媒病毒)包括许多最重要的“新出现的病原体”，因为它们在地理上传播，对脆弱的人类种群的影响越来越大。迫切需要易于操作和可快速部署的诊断工具，能够以封闭的样本对答案的方式处理血液样本。在这里，我们建议开发一种新的诊断技术，可以以廉价、超敏感、特异和多重的方式检测病毒抗原。我们将把我们的新方法开发成独立的工具，具有全分子灵敏度(与核酸扩增测试相当)的检测能力，以在最小的用户干扰下诊断虫媒病毒感染。综合诊断平台将利用一种新的替代方法、微流控集成和具有区分虫媒病毒感染能力的多重检测方案。该系统将设计为从血清/血浆/血液启动诊断，并在不到35分钟内使用不到100微米的L血液样本提供样本到答案的诊断，每次测试的成本为2美元。为这项NIH/NIAID R01拨款建议的协作工作涉及纳米光子工程(Yanik Group)、分子病毒学(Pinsky Group)和传染病流行病学(LaBeaud Group)的集成，以与迎风群岛研究和教育基金会(WINDREF)以及圣乔治大学团队(Macpherson、Waechter和Noel小组)一起构建和现场测试我们的新型医疗保健点病毒诊断平台。患者样本的初步验证测试最初将在斯坦福医疗设施与拉博德和平斯基集团合作进行。随后，三个样机将被转移到格林纳达进行实地测试，首先在中央实验室进行，然后在资源匮乏的小城镇进行。亚尼克集团将为分子和纳米工程组件的集成提供必要的专业知识，演示实际原型，并以患者样本(拉博德和平斯基集团)评估根据该建议开发的原型(S)的应用。系统将反复优化，并将开发出适合现场环境的坚固平台。

Arthropod-borne viruses (arboviruses) comprise many of the most important ‘emerging pathogens’ due to their geographic spread and their increasing impact on vulnerable human populations. There is urgent need for easy-to- operate and rapidly deployable diagnostic tools that can handle blood samples in a closed sample-to-answer manner. Here, we propose to develop a novel diagnostic technology that can detect viral antigens in an inexpensive, ultrasensitive, specific, and multiplexed manner. We will develop our novel approach into standalone tool with a detection capability at attomolar sensitivities (comparable to nucleic acid amplification tests) to diagnose arboviral infections with minimal user interference. The integrated diagnostic platform will utilize a novel surrogate approach, microfluidic integration, and a multiplexed detection scheme with the capacity to distinguish arboviral infections. The system will be designed to initiate diagnosis from serum/plasma/blood and provide a sample-to-answer diagnostic within less than 35 minutes using less than 100 µL blood samples at a cost of $2 per test. Collaborative work proposed for this NIH/NIAID R01 Grant involves integration of nanophotonic engineering (Yanik Group), molecular virology (Pinsky Group), and infectious diseases epidemiology (LaBeaud Group) to build and field-test our novel point-of-care viral diagnostic platform with Windward Islands Research and Education Foundation (WINDREF) and St. George’s University teams (Macpherson, Waechter and Noel Groups). Preliminary validation tests with patient samples will be initially performed at Stanford Medical Facility in collaboration with LaBeaud and Pinsky groups. Subsequently, three prototypes will be transferred to Grenada for field-testing initially at central laboratories then to resource-poor settings in small towns. Yanik group will provide the necessary expertise for integration of molecular and nanoengineering components and demonstration of a practical prototype as well as evaluating the application of prototype(s) developed under this proposal with patient samples (LaBeaud and Pinsky Groups). System will be iteratively optimized and a rugged platform suitable for field settings will be developed.