EAGER: Engineering Optical Nanobiosensors for Detection of Coronavirus Proteases

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.