Ultrasensitive, Rapid, Amplification-Free RNA Virus Detection Using Nanodimer-Based Nucleic Acid Target Sequence Recognition

核糖核酸(RNA)病毒是对公众健康和全球经济的主要威胁。从普通感冒到危及生命的出血热，许多人类疾病都是由RNA病毒引起的。在过去的几十年里，人类免疫缺陷病毒、埃博拉病毒、登革病毒以及最近的冠状病毒(新冠肺炎)等核糖核酸病毒给全球卫生和全球经济带来了巨大的负担。对人类样本和环境中存在的RNA病毒进行快速和高灵敏度的检测，对于跟踪病毒污染率、防止快速传播、制定预防措施和及时实施治疗至关重要。流行的基于聚合酶链式反应(PCR)的病毒检测需要很长的时间，需要专门的实验室仪器和训练有素的人员。本项目旨在创建一种新型的生物传感微系统，该系统可以在不需要PCR扩增的情况下提供超灵敏、快速、可靠的RNA病毒检测。该项目将产生一种便携、快速、经济的RNA病毒检测方法，用于护理点检测、大流行预防、反生物恐怖主义和环境监测。此外，这项跨学科研究将支持阿克伦大学培养多样化的研究生和本科生。该研究成果还将支持阿克伦大学的几门关于微系统和生物材料的研究生课程。该项目的技术范围分为多项任务：1)制造一种表面声预过滤芯片，它将快速去除连续流动中的微尺度杂质，否则会导致传感通道堵塞；2)研究一种基于独特的电扩散凝胶效应的新型纳米颗粒聚焦方法，它可以显著提高病毒粒子的浓度，从而在几秒钟内输出数千倍的信号；3)研究一种基于创新的纳米二聚体解离实验的病毒检测芯片，原理上可以检测到单一病毒分辨率的病毒，4)研究在阻性脉冲传感器阵列上应用独特的信号复用技术，这将使RNA病毒的高通量、数字化检测成为可能。5)将这三个芯片集成到一个微系统中，并展示其在使用灭活模型病毒快速检测超低丰度RNA病毒方面的有效性。这项研究将以超高分辨率和数字化能力推进病毒检测领域，这是目前最先进的方法难以实现的。它还将为下一代高通量现场生物传感系统的开发产生大量新的知识和创新。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。

Ribonucleic acid (RNA) viruses are a major threat to public health and global economy. Numerous human diseases, from the common colds to life-threatening hemorrhagic fevers are caused by RNA viruses. In the past decades, RNA viruses such as human immune deficiency virus, Ebola virus, dengue virus, and most recently coronavirus (COVID-19) impose significant burdens on global health and global economy. Rapid and high-sensitivity detection of the presence of RNA viruses in human specimens and the environment is crucial to track the rate of viral contamination, prevent fast spreading, and establish preventive measures and timely administration of treatments. The popular polymerase chain reaction (PCR)-based virus detection takes lengthy turn-around time and requires specialized laboratory instruments and trained personnel. This project aims to create a novel biosensing microsystem that can provide ultra-sensitive, rapid and reliable detection of RNA viruses with no need of PCR amplification. The project will result in a portable, rapid, economical RNA virus detection method for point-of-care-testing, pandemic prevention, anti-bioterrorism, and environmental monitoring. Furthermore, this cross-disciplinary research will support the development of a diverse cohort of graduate, undergraduate students at the University of Akron. The research results will also support several graduate-level courses on microsystems and biomaterials at the University of Akron.The technical scope of the project are divided into multiple tasks: 1) create a surface acoustic pre-filtering chip that will rapidly remove microscale impurities in continuous flow, which would otherwise cause clogging of the sensing channels, 2) research a novel nanoparticle focusing method based on unique electro-diffusio-phoresis effect, which can significantly increases the virion concentration and thus output signals thousands of times within seconds, 3) research a virus detection chip based on an innovative nanodimer dissociation assay, which in principle, can detect viruses with single virus resolution, 4) research unique signal multiplexing applied on a resistive pulse sensor array, which will enable high throughput, digital detection of RNA viruses, and ultimately 5) integrate the three chips into a microsystem and demonstrate its utility for rapid detection of ultra-low abundance RNA viruses using an inactivated model virus. The research will advance the field of virus detection with ultra-high resolution and digitization capability, which are difficult to achieve using current state-of-the-art methods. It will also generate numerous new knowledges and innovations for the development of the next generation high-throughput, onsite biosensing systems in general.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.