RAPID: Aptamer-Linked Nano-Plasmon Sensor for Rapid Detection of SARS-CoV-2

严重急性呼吸系统综合征冠状病毒2 （SARS-CoV-2）在全球造成严重破坏，迄今已有200多万人感染了COVID-19，数十万人丧生。阻止大流行的主要挑战之一是开发诊断工具，以便在护理点实时快速检测冠状病毒。现有的活动性感染检测依赖于用扩增方法检测病毒遗传物质，这往往需要在集中的实验室设置中花费较长的周转时间。迄今为止，对SARS-CoV-2感染的有限和延迟检测已成为阻止该病毒持续社区传播的瓶颈。在这一快速应用中，研究人员将通过开发一种新型纳米材料传感器来打破这一瓶颈，该传感器具有实时检测病毒蛋白质的能力。验证检测实际病毒的方法将允许快速筛查和隔离COVID-19患者，这对于在当前和未来的大流行期间打破传播链至关重要，包括在当前隔离结束后可能出现的新一波SARS-CoV-2。该研究项目也将为研究生和本科生提供物理、生物和纳米技术界面的培训机会。对活性SARS-CoV-2感染的现有检测依赖于通过逆转录聚合酶链反应方法检测病毒rna，这通常需要在集中实验室设置中较长的周转时间。为了打破这一瓶颈并阻止大流行，需要采取其他方法来快速检测病毒。研究人员计划开发与适体连接的纳米等离子体传感器，用于实时检测病毒感染细胞中大量存在的病毒蛋白，包括刺突糖蛋白和核衣壳蛋白的受体结合域。这种新颖的方法结合了纳米和生物工程领域的几项技术进步：1)两个相连的金纳米颗粒之间的局部表面等离子体共振耦合对它们的粒子间距离敏感；2)具有明确的二级和三级结构的单链DNA或RNA适体，可以识别具有与抗体相当的强结合亲和力的特定蛋白质；3)天然存在的核开关由适体和基因调控中的调节结构域组成，通过适体结构域结合靶分子可以改变构象。首席研究员将利用文献中报道的高亲和力适体序列特异性识别目标病毒蛋白，并通过模拟核糖开关和引入调节序列设计构象“可切换”序列。利用计算设计的序列，联合首席研究员将合成纳米等离子体传感器，并通过实验验证其识别目标病毒蛋白的功能。与用于传感器设计的抗体相比，核苷酸的合成要容易得多，也便宜得多，这对于大量生产检测试剂盒至关重要。一旦项目顺利完成，研制的传感器将为现场实时快速检测SARS-CoV-2提供一种替代方法，也可作为研究病毒感染机制的科学工具。此外，这项应用的资助将促进学生在物理学、生物学和纳米技术方面的训练。该项目由化学、生物工程、环境和运输系统（CBET）部门和促进竞争研究的既定计划（EPSCoR）共同资助。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 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been wreaking havoc around the globe, infecting more than 2 million people with COVID-19 and taking hundreds of thousands of lives to date. One of the major challenges in stopping the pandemic is the development of diagnostic tools for the rapid detection of the coronavirus at point-of-care in real time. Existing tests for active infections rely on the detection of virus genetic materials with amplification methods that often require long turn-around times in a centralized laboratory setup. The limited and delayed detection of SARS-CoV-2 infections so far has become the bottleneck for stopping the continued community transmission of the virus. In this RAPID application, the investigators will break this bottleneck by developing a novel nanomaterial-based sensor with the capability to detect virus proteins in real time. Validation of the method for detecting actual virus will allow fast screening and isolation of COVID-19 patients, critical for breaking the chain of transmission during current and future pandemics, including potential new waves of the SARS-CoV-2 after the end of the current quarantine. This research project will also provide training opportunities for both graduate and undergraduate students at the interface of physics, biology, and nanotechnology. Existing test for active SARS-CoV-2 infections relies on the detection of virus RNAs by the reverse transcription polymerase chain reaction method, which usually requires long turn-around times in a centralized laboratory setup. In order to break this bottleneck and to stop the pandemic, alternative approaches for fast virus detection are required. The investigators plan to develop aptamer-linked nano-plasmon sensors for real-time detection of virus proteins that are highly abundant in virus-infected cells, including the receptor-binding domain of the spike glycoprotein and the nucleocapsid protein. This novel approach combines several lines of technological advancements in nano- and bio-engineering: 1) the localized surface plasmon resonance coupling between two linked gold nanoparticles that is sensitive to their inter-particle distance; 2) single-stranded DNA or RNA aptamers with well-defined secondary and tertiary structures that can recognize specific proteins with strong binding affinities comparable to antibodies; and 3) naturally-occurring riboswitches comprised of an aptamer and a regulatory domains in gene regulation that can change conformations upon binding the target molecule by the aptamer domain. The principal investigator will utilize high-affinity aptamer sequences that have been reported in the literature to specifically recognize the targeted virus proteins and design conformationally “switchable” sequences by mimicking riboswitches and introducing regulatory sequences. Using the computationally designed sequences, the co-principal investigator will synthesize the nano-plasmon sensors and experimentally validate the functionality in recognizing the targeted virus proteins. Compared to antibodies used for sensor design, nucleotides are much easier and cheaper to synthesize, which is critical for producing testing kits in large quantities. Upon successful completion of the proposed research project, the developed sensors will offer an alternative approach for rapid detection of SARS-CoV-2 at the point-of-care in real time and can also be used as a scientific tool to study the virus infection mechanism. Moreover, funding of this application will foster student training at the interface of physics, biology, and nanotechnology.This project is jointly funded by the Chemical, Bioengineering, Environmental and Transport Systems (CBET) Division and the Established Program to Stimulate Competitive Research (EPSCoR).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.