EAGER: Engineering Optical Nanobiosensors for Detection of Coronavirus Proteases

冠状病毒已成为呼吸道疾病爆发的主要病原体，包括最近的新冠肺炎病。目前检测新冠肺炎病的病原体SARS-CoV-2冠状病毒的方法是基于逆转录耦合聚合酶链式反应或使用抗体检测病毒抗原的免疫分析方法。然而，这些检测方法在区分活动性和过往感染方面的能力有限。该项目的目标是设计和验证能够报告活跃的冠状病毒感染的纳米生物传感器。将开发检测SARS-CoV-2两种标志性蛋白酶的光学纳米生物传感器。这些光学纳米生物传感器有望提供对活跃的病毒载量的定量评估，从而可以测量疾病的轨迹。基于聚合酶链式反应和免疫学的方法在区分活跃的冠状病毒感染患者和幸存的患者方面能力有限。此外，目前可用的抗体的不完全特异性阻碍了免疫分析。迫切需要一种技术，能够检测诸如血浆、血清、痰或呼出的呼吸冷凝物等“液体活组织检查”中冠状病毒的活性。这项研究的假设是，SARS-CoV-2标志性蛋白水解酶PLPro和3CLPro的蛋白分解活性将提供一个实时、定量的病毒感染活动性标志。利用磁性Fe/Fe3O4核/壳纳米颗粒与荧光团和FRET猝灭剂相连的光学特性，将开发和验证针对这些蛋白酶的光学纳米生物传感器。随后的体外验证将利用来自感染细胞培养和临床前模型的生物样本。这项研究建立在堪萨斯州立大学团队之前的工作基础上，他们开发了获得专利的生物纳米传感器技术，用于超灵敏地检测蛋白水解酶和翻译后修饰，该技术已经证明了检测股骨下角范围内的蛋白水解酶(例如，精氨酸酶)的能力。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。

Coronaviruses have emerged as major pathogens of respiratory disease outbreaks, including most recently COVID-19 disease. Current assays for detection of the SARS-CoV-2 coronavirus, the causative agent of COVID-19 disease, are based on reverse transcriptase-coupled polymerase chain reaction (RT-PCR) or immunoassays which use antibodies to detect viral antigens. However, these assays have limited capacity to discriminate between active and past infections. The goal of this project is to design and validate nanobiosensors capable of reporting active coronavirus infections. Optical nanobiosensors will be developed that detect two signature proteases of SARS-CoV-2. These optical nanobiosensors are anticipated to provide quantitative assessment of active viral load, such that disease trajectories can be measured.PCR-based and immunological methods are limited in their ability to distinguish between patients with active coronavirus infection and patients that have survived the disease. Moreover, immunoassays are hampered by the incomplete specificity of currently available antibodies. A critical need exists for a technology that is capable of detecting the activity of a coronavirus in a "liquid biopsy" such as plasma, serum, sputum or exhaled breath condensate. The hypothesis of this research is that proteolytic activities of SARS-CoV-2 signature proteases PLpro and 3CLpro will provide a real-time, quantitative marker of active viral infection. Optical nanobiosensors specific for these proteases will be developed and validated, leveraging the optical properties of magnetic Fe/Fe3O4 core/shell nanoparticles linked to fluorophores and FRET-quenchers. Subsequent in vitro validation will utilize biosamples from infected cell cultures and pre-clinical models. This research builds on prior work by the Kansas State University team who developed patented bionanosensor technology for the ultra-sensitive detection of proteases and post-translational modifications, which has demonstrated capacity to detect proteases (e.g., arginase) in the sub-femtomolar range.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.