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

正在进行的新冠肺炎全球大流行提醒我们诊断检测技术的重要性。目前的护理点检测(POCT)技术价格低廉，易于使用、存储和运输。然而，与基于实验室的诊断测试相比，它们的简单性会导致灵敏度和特异度的显著下降。NSF职业研究计划的主要目标是开发一种新的纳米孔测试技术，以合理的低成本实现对传染病的超灵敏检测。在该项目成功完成后，拟议的纳米孔测试将满足对POCT技术的迫切需求，其精度超过目前的POCT，甚至超过基于实验室的测试技术。这一平台技术为改变现有传染病的POCT模式提供了巨大的潜力。此外，一旦新的疾病和生物标记物被确定用于临床和护理点的快速部署，它可以很容易地进行最小优化修改。该项目还包括教育和外展活动，例如为高中生组织一个关于生物医学工程和纳米技术基础的暑期研究研讨会和实习，向当地社区广泛展示我们的研究，并开始一个传染病研究研讨会，在当地临床医生、生物医学工程师和科学家之间带来额外的协同效应。将特别关注代表不足群体的学生和第一代大学生，他们是我国学生总数的重要组成部分。这一职业计划应该为本科生和高中生带来成就奖和研究出版物上的作者。在个性化医学时代，在护理地点快速准确地量化多个生物标志物是成功控制和管理传染病爆发的基础。传统的护理点检测(POCT)技术价格低廉，易于使用、存储和运输。但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。纳米孔技术由于其单分子分析能力、可移植性和低成本，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体样本中的分析物，只能分析某些大小的带电生物标志物，并且不能与设备兼容。这项职业计划的主要目标是开发下一代纳米孔生物传感器，以便能够在护理点对人类血液中的多种传染病生物标志物进行超灵敏检测和量化。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、稳定性和重复性的纳米孔生物传感器，以满足各种目的；(2)通过自动免疫沉淀和新的信号转导机制实现灵敏和特异的生物标记物检测；(3)展示循环蛋白质组学生物标记物的超灵敏多重定量及其在传染病诊断和预后中的应用。建议的方法有几个创新之处：使用几种扩增方法来达到亚毫微摩尔水平的检测极限；使用特殊的单链DNA结构作为生物标志物的检测替代品，以便读出的信号可以很容易地与其他生物分子区分开来，防止假阳性；集成的微流控自动免疫沉淀模块可以减少分析协议中的样本到答案的时间和人为错误。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.