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快速部署的诊断工具，能够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，可以以一种廉价、超灵敏、特异性和多路复用的方式检测病毒抗原。我们将把我们的新方法开发成具有原子摩尔灵敏度（与核酸扩增试验相当）检测能力的独立工具，以最小的用户干扰诊断虫媒病毒感染。综合诊断平台将利用一种新的替代方法、微流体集成和一种具有区分虫媒病毒感染能力的多路检测方案。该系统将被设计用于从血清/血浆/血液中启动诊断，并在不到35分钟内使用不到100 μ L的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。这项NIH/NIAID R01拨款的合作工作包括纳米光子工程（Yanik集团）、分子病毒学（Pinsky集团）和传染病流行病学（LaBeaud集团）的整合，与迎风群岛研究和教育基金会（WINDREF）和圣乔治大学团队（Macpherson， Waechter和Noel集团）一起建立和现场测试我们新的即时护理病毒诊断平台。患者样本的初步验证测试将首先在斯坦福医疗机构与LaBeaud和Pinsky团队合作进行。随后，三个原型将转移到格林纳达，最初在中央实验室进行实地测试，然后到资源贫乏的小城镇进行测试。Yanik集团将提供必要的专业知识来整合分子和纳米工程组件，演示一个实用的原型，以及评估根据该提案开发的原型与患者样本的应用（LaBeaud和Pinsky集团）。系统将进行迭代优化，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.