Next Generation Plasmon Coupling Nanosensors

核糖核酸(RNA)分子在控制基因表达和控制细胞行为方面具有多种功能。RNA也被一些病毒用来存储遗传信息。这些多样而重要的功能推动了传感器的发展，使其能够对RNA分子进行灵敏的检测和识别。该项目将为两类特定的RNA分子开发RNA传感器：i.MicroRNA(MiRNA)，一种有潜力作为癌症等疾病生物标志物的调控类RNA，以及ii.)病毒核糖核酸能够快速检测核糖核酸病毒，如冠状病毒(新冠肺炎)。所提出的传感器平台包含一条DNA链，在其末端连接到两个金属纳米颗粒，它们的光信号取决于它们的分离。DNA传感器链被设计成当它与特定的RNA靶标结合时改变其结构。这种结构修改改变了纳米颗粒探测器之间的间隔并改变了它们的信号，即使对于小的纳米颗粒，通过散射信号与参考光束的干涉也能灵敏地检测到它们。该传感器将被设计为直接检测miRNA，但对于病毒RNA，耦合的纳米颗粒传感器将与酶信号放大步骤相结合，以获得一个灵敏且快速可重构的检测平台。这项研究将被整合到教育和外联活动中，包括为支持研究的课程开发工作，为波士顿地区市中心的高中生和他们的老师组织一个关于等离子激进学和纳米科学基础的夏季研讨会，旨在激发他们对科学和技术职业的兴趣，以及开发一个Instagram频道，它将为非专业观众提供一个很好的平台来教育他们关于等离子纳米传感器的知识。该项目将结合分子信标，如DNA结构，其末端带有贵金属纳米颗粒(NP)探针与双色干涉散射显微镜(ISCAT)检测，以产生基于等离子激元耦合的传感器，用于两类重要的RNA：i)调控的miRNA和II。)严重急性呼吸综合征冠状病毒2型(SARS-CoV-2)RNA。靶RNA与传感器的结合导致构象从封闭的构象转变为开放的构象，并改变与DNA链结合的NP标记之间的等离子耦合。双色iSCAT显微镜将用于提高灵敏度，以检测与传感器与其目标结合时打开相关的干涉散射信号的变化。传感器的DNA识别元件将被设计为直接与miRNA结合，而对于病毒RNA，等离子体传感器将与CRISPR-CAS13检测和随后的样品扩增相结合，以获得一个灵敏的、可重构的病毒RNA检测平台。分子信标将识别通过CAS13反式裂解底物产生的RNA片段，而不是直接与病毒RNA结合。这一奖项反映了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。

Ribonucleic acid (RNA) molecules have diverse functions in controlling gene expression and controlling cellular behavior. RNA is also used by some viruses to store genetic information. These diverse and important functions motivate the development of sensors that allow for a sensitive detection and identification of RNA molecules. This project will develop RNA sensors for two specific classes of RNA molecules: i.) microRNA (miRNA), a regulatory class of RNA that has potential as biomarkers for diseases such as cancer, and ii.) viral RNA to enable a rapid detection of RNA viruses, such as the corona virus (Covid-19). The proposed sensor platform contains a DNA strand connected at its ends to two metal nanoparticles whose optical signal depend on their separation. The DNA sensor strand is designed to change its structure when it binds to a specific RNA target. This structural modification changes the separation between the nanoparticle probes and alters their signal, which is sensitively detected even for small nanoparticles via interference of the scattered signal with a reference light beam. The sensor will be designed to detect miRNA directly, but for viral RNA the coupled nanoparticle sensor will be combined with an enzymatic signal amplification step to obtain a sensitive and rapidly reconfigurable detection platform. The research will be integrated into education and outreach activities that include development of course work to support the research, organization of a summer workshop on the fundamentals of plasmonics and nanoscience for inner city high school students in the Boston area and their teachers that is designed to excite them about a career in science and technology, and development of an Instagram channel that will provide an excellent platform for educating a lay audience about plasmonic nanosensors.The project will combine molecular beacon like DNA structures whose ends are functionalized with noble metal nanoparticle (NP) probes with two-color interferometric scattering microscopy (iSCAT) detection to generate a plasmon coupling based sensor for two important classes of RNA: i.) regulatory miRNA and ii.) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. Target RNA binding to the sensor induces a conformational change from a “closed” to an “open” conformation and alters the plasmon coupling between the NP labels bound to the DNA strand. Two-color iSCAT microscopy will be applied to enhance the sensitivity for detecting changes in the interferometric scattering signal associated with the opening of the sensor upon binding to its target. The DNA recognition element of the sensor will be designed to bind to miRNA directly, while for the viral RNA the plasmonic transducer will be combined with CRISPR-CAS13 detection and subsequent sample amplification to obtain a sensitive and reconfigurable viral RNA detection platform. Instead of directly binding to viral RNA, the molecular beacon will recognize RNA fragments generated through CAS13 trans-cleavage of a substrate.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.