Collaborative Research: Ultrasensitive Nucleic Acid Sensing Tools Based on Cas Assays and Solid-State Nanopores

Covid-19的发病清楚地证明了对快速、廉价和方便的病毒检测方法的迫切需要。2019年11月中旬至2021年1月中旬，Covid-19在全球造成200多万人死亡，并继续对人类生命造成损害。获得2020年诺贝尔奖的CRISPR/Cas技术，可用于快速检测任何生物体的DNA序列，提供了一种很有前途的方法。这种方法已经被许多公司采用，但迄今为止还没有一种方法能够与“金标准”测试（实时聚合酶链反应（RT-PCR））的灵敏度相媲美，后者需要4-6小时才能完成，每次测试的成本约为100美元。因此，该项目的目标是开发一种检测SARS-CoV-2（导致COVID-19的病毒）的方法，这种方法比目前用于SARS-CoV-2检测的方法更快、更便宜、更敏感、更方便。该项目的目标将通过将CRISPR/Cas分析与尖端技术相结合来实现。现有系统的局限性将通过使用一些先进的分析工具、先进的设备、人工智能和新型纳米材料探针来解决，设计一个集成的纳米孔微流控装置，用于定点护理（POC）环境，确保（经济实惠、敏感、特异性、用户友好、快速和强大、无设备，并可交付给最终用户）。该传感器平台的成功开发将提供广泛的其他用途，因为其背后的原理可以应用于与SARS-CoV-2无关的其他应用。该项目结合了生物化学、纳米工程、光子学和医学，为跨学科研究创造了极好的机会。与这个激动人心的项目相关的外展项目将提供给K-12学校，吸引年轻人并激励他们攻读科学、技术、工程和数学（STEM）学位。本项目的目标是开发一种基于CRISPR/Cas检测的高灵敏度、高可靠性的核酸检测工具，用于检测SARS-CoV-2。本研究将揭示Cas酶在多种复合纳米材料上的裂解活性。固体纳米孔将被优化，用于在Cas分析中使用深度神经网络对解理特征进行分类，以读取纳米材料报告的解理模式。固态纳米孔读出提供单分子定量，还可以识别易位分子中的分子特征，这比目前的标准读出方法（荧光、纸条、比色和电化学读出）具有显著优势。一旦了解了裂解活动，就可以设计出各种裂解模式与特定靶序列相对应的报告子。裂解产物的鉴定将有助于开发用于POC设置的集成纳米孔微流控装置，该装置将在多重CRISPR/Cas分析中演示裂解产物的同时纳米孔和荧光读数。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 urgent need for rapid, inexpensive, and convenient methods to detect viruses has been clearly evidenced by the onset of Covid-19, which caused the death of over 2 million prople worldwide from mid November 2019 to mid January 2021, and continues to take its toll on human life. The 2020 Nobel Prize winning CRISPR/Cas technology, which can be used to rapidly detect DNA sequences in any living organism, offers a promising approach. This approach has been pursued by many companies, but none to date has been able to match the sensitivity of the “gold standard” test (real-time polymerase chain reaction (RT-PCR)), which requires 4-6 hours for completion and costs ~$100 per test. Thus the goal of this project is to develop a method for SARS-CoV-2 (the virus responsible for COVID-19) detection that is faster, cheaper, more sensitive, and more convenient than the methods presently used for SARS-CoV-2 detection. The project’s goals will be achieved by integrating CRISPR/Cas assays with cutting-edge technologies. Limitations of existing systems will be addressed using a number of advanced analysis tools, advanced devices, artifical inteligence, and novel nanomaterial probes to design an integrated nanopore-microfluidic device for use in point-of-care (POC) settings that is ASSURED (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and deliverable to end users). Succesful development of this sensor platform will offer a wide range of other uses, as the principles behind it may be applied to other applications that are not related to SARS-CoV-2. The project creates excellent opportunities for interdisciplinary research, as it combines biochemistry, nanoengineering, photonics, and medicine. Outreach programs related to this exciting project will be offered to K-12 schools, attracting young minds and inspiring them to pursue science, technology, engineering and mathematics (STEM) degrees. The goal of this project is to develop a highly sensitive and reliable nucleic acid sensing tool based on CRISPR/Cas assays for SARS-CoV-2 detection. The research will reveal the cleavage activities of Cas enzymes on a variety of composite nanomaterial reporter designs. Solid-state nanopores will be optimized for reading the cleavage patterns of nanomaterial reporters in the Cas assays using a deep neural network to classify the cleavage signatures. Solid-state nanopore readout provides single-molecule quantification and also identifies molecular signatures within the translocating molecules, which has significant advantages over the standard readout methods of today (fluorescence, paper-strip, colorimetric, and electrochemical readout). Once the cleavage activities are understood, a variety of reporters whose cleavage patterns correspond to specific target sequences will be designed. Identification of the cleavage products will enable the development of an integrated nanopore-microfluidic device for use in POC settings that will demonstrate simultaneous nanopore and fluorescence readings of cleavage products in multiplexed CRISPR/Cas assays.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.