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“新兴病原体”，因为它们的地理传播和对脆弱人群的影响越来越大。迫切需要易于操作和可快速部署的诊断工具，这些工具可以以封闭的样本到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，可以检测病毒抗原在一个廉价的，超灵敏的，具体的，和多重的方式。我们将把我们的新方法发展成独立的工具，具有阿托摩尔灵敏度的检测能力（与核酸扩增测试相当），以最小的用户干扰诊断虫媒病毒感染。集成诊断平台将利用一种新的替代方法，微流体集成和一种具有区分虫媒病毒感染能力的多重检测方案。该系统将被设计为从血清/血浆/血液开始诊断，并在不到35分钟内使用不到100微升的血液样本提供样本到答案的诊断，每次测试的费用为2美元。这项NIH/NIAID R 01资助的合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）的整合，以与向风群岛研究和教育基金会（WINDREF）和圣乔治大学团队（Macpherson，Waechter和诺埃尔组）建立和现场测试我们的新型即时病毒诊断平台。患者样本的初步验证试验最初将在斯坦福大学医疗机构与LaBeaud和Pinsky小组合作进行。随后，三个原型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，然后在小城镇的资源贫乏环境中进行。Yanik集团将为分子和纳米工程组件的整合和实际原型的演示提供必要的专业知识，并评估根据本提案开发的原型与患者样本（LaBeaud和Pinsky Group）的应用。系统将反复优化，并将开发适合现场设置的坚固平台。

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.