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

从2019年11月中旬至2021年1月中旬，新冠肺炎导致全球200多万人死亡，并继续对人类生命造成影响，这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求。2020年诺贝尔奖获得者CRISPR/CAS技术可以用于快速检测任何活着的有机体的DNA序列，这提供了一种很有前途的方法。许多公司都在寻求这种方法，但到目前为止，还没有一家公司能够达到“黄金标准”检测(实时聚合酶链式反应)的灵敏度，后者需要4-6个小时才能完成，每次检测的费用约为100美元。因此，本项目的目标是开发一种比目前用于检测SARS-CoV-2(新冠肺炎病毒)的方法更快、更便宜、更敏感、更方便的方法。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。将使用许多先进的分析工具、先进的设备、人工智能和新型纳米材料探测器来解决现有系统的局限性，以设计一种集成的纳米孔-微流控设备，用于确保(负担得起、灵敏、特定、用户友好、快速和坚固、无设备和可交付给最终用户)的护理点(POC)环境中使用。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能适用于与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。与这一激动人心的项目相关的外展项目将提供给K-12学校，吸引年轻人的头脑，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检测的高灵敏和可靠的核酸传感工具，用于SARS-CoV-2的检测。这项研究将揭示CaS酶在各种复合纳米材料报告设计上的切割活性。固态纳米孔将被优化，用于读取CAS分析中纳米材料报告的切割模式，使用深度神经网络对切割特征进行分类。固态纳米孔读出提供单分子量化，并识别转移分子中的分子特征，这比当今的标准读出方法(荧光法、纸条读出法、比色法和电化学读出法)具有显著优势。一旦了解了切割活动，就会设计出各种切割模式与特定靶序列相对应的记者。卵裂产品的鉴定将使用于POC环境的集成纳米孔-微流控设备的开发成为可能，该设备将在多路CRISPR/CAS分析中展示裂解产品的纳米孔和荧光读数。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 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.