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

2019冠状病毒病的爆发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，从2019年11月中旬到2021年1月中旬，该病毒在全球造成200多万人死亡，并继续对人类生命造成损失。 2020年获得诺贝尔奖的CRISPR/Cas技术可用于快速检测任何生物体中的DNA序列，提供了一种有前途的方法。 许多公司都采用了这种方法，但迄今为止，没有一家公司能够达到“金标准”测试（实时聚合酶链反应（RT-PCR））的灵敏度，该测试需要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SARS-CoV-2（导致COVID-19的病毒）检测方法，该方法比目前用于SARS-CoV-2检测的方法更快，更便宜，更灵敏，更方便。该项目的目标将通过将CRISPR/Cas检测与尖端技术相结合来实现。 现有系统的局限性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针来解决，以设计一种集成的纳米孔微流体设备，用于护理点（POC）设置，这是有保证的（负担得起的，敏感的，具体的，用户友好的，快速和强大的，无设备的，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能适用于与SARS-CoV-2无关的其他应用。 该项目为跨学科研究创造了极好的机会，因为它结合了生物化学，纳米工程，光子学和医学。与这个令人兴奋的项目相关的推广计划将提供给K-12学校，吸引年轻人的头脑，并激励他们追求科学，技术，工程和数学（STEM）学位。该项目的目标是开发一种基于CRISPR/Cas检测的高灵敏度和可靠的核酸传感工具，用于SARS-CoV-2检测。该研究将揭示Cas酶对各种复合纳米材料报告分子设计的切割活性。 将使用深度神经网络对固态纳米孔进行优化，以阅读Cas测定中纳米材料报告分子的切割模式，从而对切割特征进行分类。固态纳米孔读出提供单分子定量，并且还识别易位分子内的分子特征，这与当今的标准读出方法（荧光、纸条、比色和电化学读出）相比具有显著优势。一旦了解了切割活性，将设计其切割模式对应于特定靶序列的各种报告物。裂解产物的鉴定将使一个集成的纳米孔-微流体设备的开发，用于POC设置，将展示在多重CRISPR/Cas检测裂解产物的同时纳米孔和荧光读数。该奖项反映了NSF的法定使命，并已被认为值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

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.