CAREER: Ultrasensitive Multiplex Nanopore Biosensing for Point-of-Care Testing of Infectious Diseases

正在进行的2019冠状病毒病全球大流行提醒了我们诊断检测技术的重要性。目前的即时检测（POCT）技术价格低廉，易于使用、储存和运输。然而，与基于实验室的诊断测试相比，它们的简单性导致敏感性和特异性的显著丧失。这项NSF CAREER研究计划的主要目标是开发一种新的纳米孔检测技术，以合理的低成本实现对传染病的超灵敏检测。一旦该项目成功完成，所提出的纳米孔测试将满足POCT技术的迫切需求，其精度将超过目前的POCT甚至实验室测试技术。该平台技术在改变现有传染病POCT的现有模式方面具有巨大潜力。此外，一旦发现新的疾病和生物标志物，它可以很容易地进行最小优化修改，以便在诊所和护理点快速部署。该项目还包括教育和推广活动，例如为高中学生组织暑期研究研讨会和实习，介绍生物医学工程和纳米技术的基础知识，向当地社区广泛介绍我们的研究，并启动传染病研究研讨会，使当地临床医生、生物医学工程师和科学家之间产生额外的协同作用。我们将特别关注来自弱势群体的学生和第一代大学生，他们构成了我们学生群体的重要组成部分。这项职业计划将为本科生和高中生带来成就奖和研究出版物的作者身份。在个性化医疗时代，在护理点快速准确地定量多种生物标志物是成功控制和管理传染病暴发的基础。经典的即时检测（POCT）技术价格低廉，易于使用、储存和运输。但是，与基于实验室的体外诊断相比，它们的简单性导致敏感性和特异性的显著丧失。纳米孔技术具有单分子分析能力、便携性和低成本等优点，是一种很有前途的替代技术。然而，现有的纳米孔技术不适合检测复杂人类样本中的分析物，只能分析特定尺寸的带电生物标志物，并且不兼容设备。该职业计划的主要目标是开发下一代纳米孔生物传感器，以便在护理点对人体血液中的多种传染病生物标志物进行超灵敏检测和定量。该计划有三个研究目标：(1)开发和优化具有前所未有的灵敏度、鲁棒性和可重复性的纳米孔生物传感器，用于各种用途；(2)通过自动免疫沉淀和新的信号转导机制实现敏感和特异性的生物标志物检测；(3)展示循环蛋白质组学生物标志物的超灵敏多重定量及其在传染病诊断和预后中的应用。该方法有几个创新之处：采用多种扩增方法实现亚飞摩尔水平的检测限；利用特殊的单链DNA结构作为生物标记物的检测替代物，使读出的信号易于与其他生物分子区分，防止误报；集成的微流控自动免疫沉淀模块可以减少样品到答案的时间和分析方案中的人为错误。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

The ongoing global pandemic of COVID-19 has reminded us the importance of diagnostic testing technologies. Current point-of-care testing (POCT) technologies are inexpensive and easy to use, store, and transport. However, their simplicity causes a significant loss of sensitivity and specificity comparing to lab based diagnostic tests. The major goal of this NSF CAREER research program is to develop a new nanopore testing technology to enable ultrasensitive detection of infectious diseases at a reasonably low cost. Upon the successful completion of this project, the proposed nanopore test will meet the urgent need for a POCT technology with accuracy that exceeds current POCTs or even lab-based testing technologies. This platform technology presents great potential for changing the current paradigm of POCT for existing infectious diseases. Also, it can be readily modified with minimal optimization as soon as new diseases and biomarkers are identified for rapid deployment in clinics and at the point of care. This project also includes education and outreach activities, such as organizing a summer research workshop and internship for high school students on the fundamentals of Biomedical Engineering and Nanotechnology, broadly presenting our research to local communities, and starting an infectious disease research symposium that brings an additional synergy among local clinicians, biomedical engineers, and scientists. Special attention will be given to students from underrepresented groups and first-generation college students, who constitute a significant part of our student population. This CAREER program should result in achievement awards and authorships on research publications for undergraduate and high school students.In the era of personalized medicine, rapid and accurate quantification of multiple biomarkers at the point-of-care is fundamental to a successful control and management of infectious disease outbreaks. Classical point-of-care testing (POCT) technologies are inexpensive and easy to use, store, and transport. But their simplicity causes a significant loss of sensitivity and specificity comparing to lab based in vitro diagnostics. The nanopore technology is a promising alternative because of its single-molecule analysis capacity, portability, and low cost. However, existing nanopore technologies are not suitable for detecting analytes in complex human samples, can only analyze charged biomarkers of certain sizes, and are not device compatible. The major goal of this CAREER program is to develop a next generation nanopore biosensor to enable ultrasensitive detection and quantification of multiple infectious disease biomarkers from human blood at the point of care. The program has three research objectives: (1) Develop and optimize a nanopore biosensor with unprecedented sensitivity, robustness, and reproducibility for various purposes; (2) Enable sensitive and specific biomarker detection by automated immunoprecipitation and novel signal transduction mechanisms; (3) Demonstrate ultrasensitive multiplex quantification of circulating proteomics biomarkers and its applications in infectious disease diagnosis and prognosis. Proposed approach has several innovative elements: several amplification methods are employed to achieve the sub-femtomolar level detection limit; special single strand DNA structures are used as detection surrogates for biomarkers, so that the readout signal can be easily differentiated from other biomolecules to prevent false-positives; an integrated microfluidic automatic immunoprecipitation module can reduce sample-to-answer time and human errors in the assay protocol.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.