EAGER SENTINELS: The PCR-free Biosensor for a Fast, Simple, and Sensitive Detection of RNA.

最近的大流行突出表明，迫切需要快速和准确地检测病毒，以便能够及早诊断和监测疾病，预防今后的大流行，并通过及时的保健减少并发症和死亡的风险。目前，基于聚合酶链式反应(PCR)的诊断试验被广泛应用于病毒检测。尽管分析参数突出，但在最近的新冠肺炎大流行期间，这项技术的重大缺陷已变得明显。更具体地说，基于聚合酶链式反应的检测不仅需要昂贵的实验室设备和训练有素的人员，而且它们也很耗时，不适合护理设备。例如，基于聚合酶链式反应的新冠肺炎检测通常需要两到三天的时间。因此，病毒的传播变得不那么可控。该项目的目的是开发一种快速、简单、经济可行的不需要聚合酶链式反应的生物传感平台。该项目还旨在为本科生提供生物传感器开发的跨学科培训，并为当地高中生提供探索诊断领域的机会。该项目旨在设计并实现一个快速、超灵敏、适应性强的无聚合酶链式反应的病毒RNA检测生物传感平台。具体地说，建议的生物传感器将基于新发现的CRISPR-Cas(簇状规则间隔短回文重复-CRISPR相关)核酸酶，该核酸酶具有高选择性，并可以重新编程以检测各种病毒的RNA。一种新的信号放大方案的开发和整合将允许绕过PCR扩增步骤。这项研究不仅将导致新的RNA传感方法，而且将为开发基于另一种但密切相关的CaS核酸酶的DNA生物传感器提供蓝图。由于易于配置和操作，建议的生物传感器更经济、更快，不需要复杂的设备和人员培训，从而满足当前公共卫生方面的需求。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 recent pandemic has highlighted the urgent need for rapid and accurate detection of viruses to allow for early disease diagnosis and monitoring to prevent future pandemics and to reduce the risk of complications and mortality through timely health care. Currently, diagnostic tests based on polymerase chain reaction (PCR) are widely applied for the detection of viruses. Despite outstanding analytical parameters, significant drawbacks of this technology have become evident during the recent COVID pandemic. More specifically, PCR-based tests require not only expensive laboratory equipment and highly trained personnel, but they are also time-consuming and not well adapted for point-of-care devices. For example, it usually takes two to three days to get the result of a PCR-based COVID-19 test. Consequently, the spread of the virus becomes less containable. The purpose of this project is to develop a fast, easy, and economically feasible biosensing platform that does not require PCR. This project also aims to provide undergraduate students with interdisciplinary training in the development of biosensors, and local high school students with an opportunity to explore the field of diagnostics.This project is to design and realize a rapid, ultrasensitive, and adaptable PCR-free biosensing platform for viral RNA detection. Specifically, the proposed biosensor will be based on the newly discovered CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeat – CRISPR associated) nuclease that shows high selectivity, and can be reprogrammed to detect various viruses’ RNAs. The development and integrations of a novel signal amplification scheme will allow circumvention of the PCR amplification step. Not only will this research lead to the novel RNA sensing approach, but it will also provide a blueprint for developing a DNA biosensor based on an alternative but closely related Cas nuclease. Due to the ease of configuration and operation, the proposed biosensor is more economical, faster, and will not require sophisticated equipment and personnel training, thereby addressing current needs in public health.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.