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

严重急性呼吸综合征冠状病毒2(SARS-CoV-2)已经在全球肆虐，感染了200多万人的新冠肺炎，迄今夺走了数十万人的生命。阻止这一大流行的主要挑战之一是开发诊断工具，以便在护理地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，这种方法往往需要在集中式实验室设置中进行长时间的周转。到目前为止，对SARS-CoV-2感染的有限和延迟检测已成为阻止该病毒继续在社区传播的瓶颈。在这一快速应用中，研究人员将通过开发一种新型的基于纳米材料的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。对检测实际病毒的方法的验证将允许对新冠肺炎患者进行快速筛查和隔离，这对于在当前和未来的大流行期间切断传播链至关重要，包括在当前隔离结束后可能出现的新一波SARS-CoV-2病毒。该研究项目还将为研究生和本科生提供物理、生物和纳米技术方面的培训机会。现有的SARS-CoV-2活动性感染检测依赖于通过逆转录聚合酶链式反应方法检测病毒RNA，这通常需要在中央实验室设置较长的周转时间。为了打破这一瓶颈并阻止大流行，需要快速检测病毒的替代方法。研究人员计划开发适体连接的纳米等离子传感器，用于实时检测病毒感染细胞中高度丰富的病毒蛋白，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条技术进步：1)两个相连的金纳米颗粒之间的局部表面等离子共振耦合，对其颗粒间的距离敏感；2)具有明确的二级和三级结构的单链DNA或RNA适配子，它可以识别特定的蛋白质，与抗体具有很强的结合亲和力；以及3)基因调控中由适配子和调节结构域组成的自然产生的核糖开关，当适配子结构域与目标分子结合时，它可以改变构象。首席研究员将利用文献中已报道的高亲和力适配子序列来特异性识别目标病毒蛋白，并通过模仿核糖开关和引入调控序列来设计构象可切换的序列。使用计算设计的序列，联合首席研究员将合成纳米等离子传感器，并通过实验验证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，成本也更低，这对大量生产检测试剂盒至关重要。当拟议的研究项目成功完成后，所开发的传感器将提供另一种在护理地点实时快速检测SARS-CoV-2的方法，并可用作研究病毒感染机制的科学工具。该项目由化学、生物工程、环境和运输系统(CBET)分部和既定的激励竞争研究计划(EPSCoR)共同资助。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 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.