EAGER: Engineering Optical Nanobiosensors for Detection of Coronavirus Proteases

冠状病毒已成为呼吸道疾病暴发的主要病原体，包括最近的COVID-19疾病。目前用于检测COVID-19疾病病原体SARS-CoV-2冠状病毒的分析方法是基于逆转录酶偶联聚合酶链反应（RT-PCR）或使用抗体检测病毒抗原的免疫分析。然而，这些检测方法区分活跃感染和过去感染的能力有限。该项目的目标是设计和验证能够报告活性冠状病毒感染的纳米生物传感器。将开发光学纳米生物传感器，检测SARS-CoV-2的两种特征蛋白酶。这些光学纳米生物传感器有望提供活性病毒载量的定量评估，从而可以测量疾病轨迹。基于聚合酶链反应的方法和免疫学方法在区分活动性冠状病毒感染患者和存活患者方面的能力有限。此外，目前可用抗体的不完全特异性阻碍了免疫测定。目前迫切需要一种能够在血浆、血清、痰液或呼出液等“液体活检”中检测冠状病毒活性的技术。本研究的假设是，SARS-CoV-2特征蛋白酶PLpro和3CLpro的蛋白水解活性将提供实时、定量的活性病毒感染标志物。将开发和验证针对这些蛋白酶的光学纳米生物传感器，利用与荧光团和fret猝灭剂相连的磁性Fe/Fe3O4核/壳纳米颗粒的光学特性。随后的体外验证将利用来自感染细胞培养物和临床前模型的生物样品。这项研究建立在堪萨斯州立大学团队先前的工作基础上，该团队开发了生物纳米传感器专利技术，用于超灵敏检测蛋白酶和翻译后修饰，该技术已证明能够检测亚飞摩尔范围内的蛋白酶（例如精氨酸酶）。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。

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.