RAPID: COVID-19 Detection Through Amplification of Protease-Based Signals

严重急性呼吸综合征冠状病毒2 （SARS-CoV-2）是2019年新型冠状病毒传染病COVID-19的病因。在病毒生命周期的一个关键步骤中，受感染的人类细胞不仅释放病毒颗粒，还释放切割病毒和宿主蛋白质的酶，称为蛋白酶。检测与SARS-CoV-2相关的蛋白酶将为检测COVID-19提供另一种途径。有了这个奖项，化学部门的化学测量和成像项目正在支持博士的研究。麻省大学阿默斯特分校的Sankaran Thayumanavan， Jeanne Hardy和Trisha L. Andrew开发了“智能”取样拭子，当SARS-CoV-2的主要蛋白酶存在时，其颜色会发生变化。当SARS-CoV-2的主要蛋白酶将无色的蛋白质衍生分子分解成单个片段时，拭子的颜色变化就会产生，其中一个片段在分解过程中化学性质发生了变化，因此它吸收或产生了人眼可以看到的光。研究人员设计并制造了蛋白质衍生的探针分子，然后将它们结合到目前使用的临床棉签的纤维中。它们的化学设计使探针分子能够被SARS-CoV-2的主要蛋白酶选择性地分解，即使在存在极低浓度的主要蛋白酶的情况下，也能在智能棉签中产生强烈的颜色。创造必要的化学反应，并将其应用于开发蛋白酶反应智能拭子，可能会导致廉价的测试方法，可供个人在家中使用。由于蛋白酶在病毒感染的生命周期中释放的普遍性，预计新的化学反应和由此产生的技术将对其他病毒的视觉检测有用，这在未来潜在的爆发中可能特别有意义。教育、培训和推广活动的重点是与博士一起工作的研究生。Thayumanavan、Hardy和Andrew在一个跨领域、以团队为导向的研究环境中，讨论了一线测量科学方法的发展，并与公众和K-12学生分享了他们在研究资金如何用于直接影响重大社会危机方面的经验和教训。该项目的目标是开发快速、准确、直接的基于探针的传感器，用于检测病毒蛋白酶的固有催化特性，从而产生清晰的光学读数。目前的工作重点是创造新的传感化学物质，利用专门设计的染料报告剂，并利用它们开发廉价的一次性智能拭子，以检测催化活性的严重急性呼吸综合征冠状病毒2主蛋白酶（CoV2-MP）的存在。一般的研究方法。Thayumanavan， Hardy和Andrew首先通过设计，生产和验证肽来产生蛋白酶识别元件，这些肽可以提供针对目标病毒蛋白酶的最佳特异性。然后将所得的蛋白酶识别元件连接到供体-受体发色团，以创建“前发色团”探针，该探针保持在黑暗（无色，变色或淬灭）状态。暴露于主要的病毒蛋白酶后，游离报告蛋白被释放并产生高强度的比色或荧光信号，其估计检测限可检测亚皮摩尔浓度的CoV2-MP。复杂的报告配方与集成的化学扩增路线，预计提供访问这些检测极限。重要的是，基于蛋白酶的检测策略和拭子测试平台很容易并且通常可用于检测基于其特定蛋白酶结构的潜在未来病毒病原体。这笔拨款是使用《冠状病毒援助、救济和经济安全（关怀）法案》分配给MPS的补充资金提供的资金。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 severe acute respiratory syndrome coronavirus 2, SARS-CoV-2, is the cause of the novel coronavirus infectious disease 2019, COVID-19. During a key step in the viral lifecycle, infected human cells release not only virus particles but also enzymes that cut viral and host proteins, called proteases. Detection of the proteases linked to SARS-CoV-2 would provide an alternative path for testing for COVID-19. With this award, the Chemical Measurement and Imaging Program in the Division of Chemistry is supporting the research of Drs. Sankaran Thayumanavan, Jeanne Hardy, and Trisha L. Andrew at the University of Massachusetts, Amherst to develop "smart" sampling swabs whose color changes when the main protease of SARS-CoV-2 is present. The color change of the swab results when the main protease of SARS-CoV-2 breaks down a colorless, protein-derived molecule into individual pieces, with one of those pieces having its chemical properties changed during the break-down process, so that it absorbs or produces light that can be seen by the human eye. The investigators design and make the protein-derived probe molecules and then incorporate them into the fibers of currently-used clinical swabs. Their chemical design allows the probe molecules to be selectively broken down by the main protease of SARS-CoV-2 and to produce strong color in the smart swabs even when extremely low concentrations of the main protease are present. Creation of the necessary chemical reactions and their application to develop the protease-responsive smart swabs may lead to inexpensive testing methods that are practical for use by individuals at home. Due to the generality of the release of proteases in the lifecycle of viral infections, the new chemical reactions and resulting technologies are anticipated to be useful for visual detection of other viruses, which may be especially meaningful during future potential outbreaks. Educational, training, and outreach activities focus on graduate students working with Drs. Thayumanavan, Hardy, and Andrew in a cross-cutting, team-oriented research environment to address the development of front-line measurement science approaches, with anticipated highlights shared with the public and K-12 students about their experiences and lessons learned about how research funding can be used to directly impact a major societal crisis.The goal of this project is to develop rapid, accurate, straightforward probe-based sensors that detect the inherent catalytic properties of viral protease enzymes, which produce a clear optical readout. Efforts are focused on creating new sensing chemistries that utilize specifically designed dye-based reporters and using them to develop inexpensive, disposable smart swabs to detect the presence of catalytically-active severe acute respiratory syndrome coronavirus 2 main protease (CoV2-MP). The general research approach Drs. Thayumanavan, Hardy, and Andrew take is to first generate protease recognition elements by designing, producing, and validating peptides that provide the optimal specificity to the targeted viral protease. The resulting protease recognition elements are then linked to donor-acceptor chromophores to create "pro-chromophoric" probes that remain in a dark (uncolored, discolored, or quenched) state. Upon exposure to the main viral protease, the free reporter is liberated and produces a highly intense colorimetric or fluorescence signal, with the estimated limits of detection offering sensing of sub-picomolar concentrations of CoV2-MP. Sophisticated reporter formulations with integrated chemical amplification routes are predicted to provide access to these limits of detection. Importantly, the protease-based detection strategy and swab test platform are easily and generally transferable for detecting potential future viral pathogens based on their specific protease structures.This grant is being awarded using funds made available by the Coronavirus Aid, Relief, and Economic Security (CARES) Act supplemental funds allocated to MPS.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.