RAPID: Microelectrode Array Sensors for SARS-CoV-2 and Other RNA Viruses

目前迫切需要精确的测量工具来检测样本中是否存在新型冠状病毒SARS-CoV-2，以便这些信息可用于诊断2019年新型冠状病毒传染病（COVID-19）。在几分钟的时间轴内获得检测结果对于限制病毒传播和帮助个人和社区做出适当决定至关重要。使用当前的一些快速检测途径发现了惊人的高病毒存在率（假阴性）。这些途径通常依赖于耗时且资源密集的样品扩增，并且由于其复杂的多步骤样品处理，这些途径更可能产生假阴性。该项目由格鲁吉亚州立大学的Gangli Wang博士及其同事领导，其近期目标是设计和开发电化学传感器，其对SARS-CoV-2存在的“开启”反应是由病毒核酸中特定化学物质的靶相互作用引起的。 响应迅速加强，以便在几分钟内对病毒进行痕量检测。该设计原理提供了几个优点，例如大大简化的样品预处理，快速的周转时间，以及简化的一步检测，大大减少了假阴性结果。这些基本特性为未来超越SARS-CoV-2的使用点应用铺平了道路。该项目为基础测量科学提供了一个难得的机会，通过尖端研究解决持续的社会和全球危机。此外，该项目为格鲁吉亚州立大学的学生和博士后研究员提供了丰富多样的教育经验，该大学有大量少数民族学生和低收入背景的学生。通过课堂教学、地方活动、参加科学会议和出版物传播科学概念和成果。额外的教育和推广活动提供机会，许多在亚特兰大都会区和超越通过一个正在进行的NSF本科研究计划和亚特兰大科学节，以及其他几个最近建立的导师计划在格鲁吉亚州立大学。使智能的优点是原位信号放大机制，在信号上的电化学传感器通过氧化还原循环。这种新的设计大大提高了检测下限（LoD），使其与现有方法竞争并可能超过现有方法，并消除了对易出错的多步样品处理（如富集、标记或扩增）的需求。在单实体电化学中采用的概念与大多数核酸分析工具中采用的样品扩增策略根本不同。原则上，预计LoD接近最大水平，即，核糖核酸（RNA）的单拷贝，其中响应时间通过统计分析相关。传感器对SARS-CoV-2特异性RNA序列的特异性是基于它们被电极固定探针识别。通过将关键组件预组装为传感器的组成部分，可以建立近零背景，这是提高LoD并解决单个核酸结合的先决条件。与靶序列的结合开启了设计的电子转移途径：识别探针上的氧化还原分子然后能够重复介导传感器电极和溶液中的共反应物之间的电子转移。信号开启机制进一步缓解了非特异性吸附问题。单个传感器被组装成阵列，以同时检测多个靶RNA。多路信号读出设计代表了增强传感器性能的另一个智力优势。从循环伏安法和低LoD实现与脉冲伏安法的机制的见解被认为是制定最佳和可推广的测量方法和协议的关键。选择合成样品、常见生物基质、病毒RNA提取物和病毒感染的细胞裂解物来建立校准曲线和检测策略。比较结果与那些从实验室分析技术，如逆转录聚合酶链反应，提供了进一步验证的传感器的功效。除了为快速检测提供定性的是/否答案外，传感器方法还可以在宽动态范围内提供绝对浓度的定量信息，这对COVID-19大流行具有重要价值，以及其他方面-该奖项反映了NSF的法定使命，并被认为是值得通过使用基金会的知识价值和更广泛的影响审查标准。

There is an urgent need for accurate measurement tools to test for the presence of the novel coronavirus SARS-CoV-2 in samples, so that this information may be used for diagnosing the novel coronavirus infectious disease 2019 (COVID-19). Obtaining test results on the timeline of minutes is essential to limiting spread of the virus and helping individuals and communities make appropriate decisions. Alarmingly high rates of missed virus presence (false negatives) have been found using some of the current fast testing routes. Those routes typically rely on sample amplification that is time-consuming and resource-intensive, and due to their complicated multi-step sample treatments, those routes are more likely to generate false negatives. The immediate goal of this project, led by Dr. Gangli Wang and colleagues at Georgia State University, is to design and develop electrochemical sensors whose "switched-on" response to SARS-CoV-2 presence results from target interactions with specific chemical species in the nucleic acids of the virus. The response is rapidly intensified to allow for trace-level detection of virus, potentially within minutes. The design principle provides several benefits, such as drastically simplified sample pretreatment, fast turn-around time, and simplified one-step detection with greatly decreased false negative outcomes. These essential qualities pave the way for future point-of-use applications that go beyond SARS-CoV-2. The project provides a rare opportunity for fundamental measurement science to address an ongoing societal and global crisis through cutting-edge research. In addition, the project provides a rich and diverse educational experience for students and postdoctoral fellows at Georgia State University, an institution with large numbers of minority students and students from low-income backgrounds. Scientific concepts and results are disseminated through classroom teaching, local events, attendance at scientific meetings, and publications. Additional educational and outreach activities provide opportunities to many in the Atlanta Metro Area and beyond through an on-going NSF undergraduate research program and the Atlanta Science Festival, as well as several other recently established mentor programs at Georgia State University. The enabling intellectual merit is the in-situ signal amplification mechanism in signal-on electrochemical sensors via redox cycling. The novel design drastically improves the lower limit of detection (LoD) to be competitive with and possibly surpass that of existing methods and eliminates the need for error-prone, multi-step sample treatments, such as enrichment, labeling, or amplification. The concept, employed in single-entity electrochemistry, is fundamentally different from the sample amplification strategy adopted in most nucleic acid analysis tools being used. In principle, the LoD is anticipated to approach utmost levels, i.e., single copies of ribonucleic acid (RNA) where the response time is correlated through statistical analysis. The sensor specificity to the SARS-CoV-2 specific RNA sequence(s) is based on their recognition by electrode-immobilized probes. With key components pre-assembled as integral parts of the sensor, near-zero background may be established, which is the prerequisite to improve the LoD and resolve binding of single nucleic acids. Binding with the target sequence turns on the designed electron-transfer pathway: the redox molecule on the recognition probe is then abled to repeatedly mediate electron transfers between the sensor electrode and the co-reactants in solution. The signal-on mechanism further mitigates nonspecific adsorption concerns. Individual sensors are assembled into arrays to simultaneously detect multiple target RNAs. The multiplex signal readout design represents another intellectual merit for enhanced sensor performance. The mechanistic insights from cyclic voltammetry and low LoD achieved with pulse voltammetry are envisioned as critical for the formulation of optimal and generalizable measurement methods and protocols. Synthetic samples, common biomatrices, viral RNA extractions, and virus-infected cell lysates are selected to establish calibration profiles and detection strategies. Comparison of the results with those from laboratory analysis techniques, such as reverse-transcription polymerase chain reaction, provides further validation of the sensor efficacy. In addition to offering qualitative Yes/No answers for fast testing, the sensor approach may provide quantitative information in absolute concentrations over a wide dynamic range, an outcome of great value to the COVID-19 pandemic, as well as to other on-going and future needs in the biological sensing community.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.