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: 2041345
• 负责人: Hsin-Chih Yeh
• 金额: $ 29.96万
• 依托单位: University of Texas at Austin
• 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=2041345&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 the 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，该病毒从 2019 年 11 月中旬至 2021 年 1 月中旬导致全球超过 200 万人死亡，并继续对其造成影响。 2020 年诺贝尔奖获得者 CRISPR/Cas 技术可用于快速检测任何生物体中的 DNA 序列，这种方法已被许多公司采用，但迄今为止还没有一家公司采用。已经能够匹配“金标准”测试（实时聚合酶链式反应 (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。开发一种比目前用于 SARS-CoV-2 检测的方法更快、更便宜、更灵敏且更方便的 SARS-CoV-2（导致 COVID-19 的病毒）检测方法。通过整合来实现采用尖端技术的 CRISPR/Cas 测定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，以设计用于点-点的集成纳米孔微流体装置。有保证的护理 (POC) 设置（经济实惠、灵敏、具体、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应用。该项目结合了生物化学、纳米工程、光子学和医学外展项目，因此其背后的原理可能适用于与 SARS-CoV-2 无关的其他应用。与这一激动人心的项目相关的技术将提供给 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。该项目的目标是开发高度灵敏且可靠的核酸传感技术。工具基于用于 SARS-CoV-2 检测的 CRISPR/Cas 检测该研究将揭示 Cas 酶对各种复合纳米材料报告基因设计的裂解活性，将优化固态纳米孔，以读取 Cas 检测中纳米材料生产者的裂解模式。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了单分子定量以及易位分子内的分子特征，这比标准读数方法具有显着优势。当今的技术（荧光、试纸条、比色和电化学读数）一旦了解了裂解活性，就可以设计出与特定目标序列相对应的裂解模式的各种生产者，从而能够开发出裂解产物。用于 POC 设置的集成纳米孔-微流体装置，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。

项目成果

Computational study on the binding of Mango-II RNA aptamer and fluorogen using the polarizable force field AMOEBA.

• DOI: 10.3389/fmolb.2022.946708
• 发表时间: 2022
• 期刊: Frontiers in molecular biosciences
• 影响因子: 5

Molecular weight of hyaluronic acid crosslinked into biomaterial scaffolds affects angiogenic potential.

• DOI: 10.1016/j.actbio.2023.08.001
• 发表时间: 2023-08
• 期刊: Acta biomaterialia
• 影响因子: 9.7
• 作者: Joshua Karam;B. Singer;H. Miwa;Limin H Chen;Kajal Maran;Mahdi Hasani;Sarahi Garza;Bianca Onyekwere-B