Evaporation-Enhanced Redox Cycling for Rapid Detection of Attomolar SARS-CoV-2 Virions Using Nanolithography-free Electrochemical Devices

In the fight to control the spread of infectious diseases such as COVID-19, simple-to-use, sensitive, scalable, and rapid diagnostics are critical for early infection diagnosis. In this regard, electrochemical biosensors are gaining popularity, especially due to miniaturization which lowers background noise and sample volume. However, miniaturization (using nano- and microfabrication techniques) also reduces the signal level, posing challenges for detecting low virus counts. In this work, we developed microfabricated electrochemical sensors with dual-amplification based on evaporation-enhanced redox cycling (E2RC) in a generator–collector configuration. We propose a novel, scalable, nanolithography-free fabrication method to achieve a controllable sub-micrometer gap between 3D interdigitated microelectrodes (IDEs) by combining photolithography with template-driven electrodeposition. Using the optimum IDE design, we first benchmarked the sensor performance with antigen-coated polymeric virus-mimics with similar size and charge as SARS-CoV-2 virions. Specifically, using continuous measurement in evaporating droplets, we demonstrate rapid and ultrasensitive detection and down to 103 particles/mL. Further investigation into particle charge and size explains the role of electrophoretic enrichment on the overall response. We also characterized the sensors using heat-inactivated SARS-CoV-2 and demonstrate the selectivity for detecting SARS-CoV-2 against HCoV-299E, SARS-CoV S1, and MERS-CoV S1 using magnetic capture with antibody-functionalized magnetic nanoparticles. The developed method provides an affordable, quick, and scalable alternative for detecting charged particles using established magnetic capture techniques. Furthermore, the proposed sensing scheme may find broad application in developing diagnostics ranging from detecting bacteria, extracellular vesicles, and viruses to molecular tagging assays.