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

合作研究：基于Cas检测和固态纳米孔的超灵敏核酸传感工具

• 批准号: 2041340
• 负责人: MinJun Kim
• 金额: $ 27.94万
• 依托单位: Southern Methodist University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2041340&HistoricalAwards=false
• 关键词: Collaborative; Research; Ultrasensitive; Nucleic; Acid

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.

Covid-19 的爆发清楚地证明了对快速、廉价且方便的病毒检测方法的迫切需要，从 2019 年 11 月中旬到 2021 年 1 月中旬，Covid-19 导致全球超过 200 万人死亡，并继续对人类造成生命损失。 荣获 2020 年诺贝尔奖的 CRISPR/Cas 技术可用于快速检测任何生物体中的 DNA 序列，提供了一种有前景的方法。 许多公司都在采用这种方法，但迄今为止还没有一家公司能够与“金标准”测试（实时聚合酶链式反应 (RT-PCR)）的灵敏度相媲美，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比目前用于 SARS-CoV-2 检测的方法更快、更便宜、更灵敏、更方便的 SARS-CoV-2（导致 COVID-19 的病毒）检测方法。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。 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现有系统的局限性，设计一种集成的纳米孔微流体装置，用于护理点 (POC) 设置，该装置是 ASSURED（经济实惠、灵敏、特异、用户友好、快速且稳健、无需设备且可交付给最终用户）。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绝佳的机会。与这一令人兴奋的项目相关的外展计划将向 K-12 学校提供，吸引年轻人并激励他们攻读科学、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 检测的高度灵敏且可靠的核酸传感工具，用于 SARS-CoV-2 检测。该研究将揭示 Cas 酶对多种复合纳米材料报告基因设计的裂解活性。 固态纳米孔将被优化，用于在 Cas 测定中读取纳米材料报告基因的裂解模式，使用深度神经网络对裂解特征进行分类。固态纳米孔读数提供单分子定量，还可以识别易位分子内的分子特征，这比当今的标准读数方法（荧光、纸条、比色和电化学读数）具有显着优势。一旦了解了切割活性，就会设计出其切割模式与特定靶序列相对应的各种报告基因。裂解产物的鉴定将有助于开发用于 POC 设置的集成纳米孔-微流体装置，该装置将在多重 CRISPR/Cas 测定中展示裂解产物的同步纳米孔和荧光读数。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。