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

目前迫切需要准确的测量工具来检测样品中是否存在新型冠状病毒SARS-CoV-2，以便将这些信息用于诊断2019年新型冠状病毒传染病（COVID-19）。以分钟为单位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。使用目前的一些快速检测途径发现了高得惊人的病毒漏检率（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，这些途径更有可能产生假阴性。该项目的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”反应源于与病毒核酸中特定化学物质的目标相互作用。该项目由乔治亚州立大学的王刚利博士及其同事领导。反应迅速加强，以便可能在几分钟内检测到微量病毒。该设计原理提供了几个优点，例如大大简化了样品预处理，快速的循环时间，简化了一步检测，大大减少了假阴性结果。这些基本品质为未来超越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难得的机会，通过前沿研究来解决当前的社会和全球危机。此外，该项目为佐治亚州立大学的学生和博士后提供了丰富多样的教育体验，佐治亚州立大学是一个拥有大量少数民族学生和低收入学生的机构。科学概念和成果通过课堂教学、当地活动、参加科学会议和出版物传播。通过正在进行的美国国家科学基金会本科生研究项目和亚特兰大科学节，以及佐治亚州立大学最近建立的其他几个导师项目，额外的教育和推广活动为亚特兰大都会区和其他地区的许多人提供了机会。在电化学信号传感器中，通过氧化还原循环的原位信号放大机制是其实现的智力优势。这种新颖的设计大大提高了检测下限（LoD），与现有方法竞争，甚至可能超过现有方法，并消除了对容易出错的多步骤样品处理的需要，如富集、标记或扩增。该概念用于单实体电化学，与大多数正在使用的核酸分析工具中采用的样品扩增策略有根本不同。原则上，预计LoD将接近最大水平，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电极固定化探针对其的识别。将关键元件作为传感器的组成部分进行预组装，可以建立近零背景，这是提高LoD和解决单个核酸结合的前提。与目标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随后能够在传感器电极和溶液中的共反应物之间反复介导电子转移。信号传导机制进一步减轻了对非特异性吸附的担忧。单个传感器被组装成阵列，以同时检测多个目标rna。多路信号读出设计代表了增强传感器性能的另一个智能优点。循环伏安法和脉冲伏安法获得的低LoD的机理见解对于制定最佳和可推广的测量方法和协议至关重要。选择合成样品、常见生物基质、病毒RNA提取和病毒感染细胞裂解物来建立校准概况和检测策略。将结果与实验室分析技术（如逆转录聚合酶链反应）的结果进行比较，进一步验证了传感器的有效性。除了为快速测试提供定性的是/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大流行以及生物传感界的其他当前和未来需求具有重要价值。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。

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.