Amplification Free Detection of SARS-CoV‐2 Using Multi-Valent Binding

We present the development of electrochemical
impedance spectroscopy (EIS)-based biosensors for sensitive
detection of SARS-CoV-2 RNA using multi-valent binding. By
increasing the number of probe−target binding events per target
molecule, multi-valent binding is a viable strategy for improving
the biosensor performance. As EIS can provide sensitive and label-
free measurements of nucleic acid targets during probe−target
hybridization, we used multi-valent binding to build EIS biosensors
for targeting SARS-CoV-2 RNA. For developing the biosensor, we
explored two different approaches including probe combinations
that individually bind in a single-valent fashion and the probes that bind in a multi-valent manner on their own. While we found excellent biosensor performance using probe combinations, we also discovered unexpected signal suppression. We explained the signal suppression theoretically using inter- and intra-probe hybridizations which confirmed our experimental findings. With our best probe combination, we achieved a LOD of 182 copies/μL (303 aM) of SARS-CoV-2 RNA and used these for successful evaluation of patient samples for COVID-19 diagnostics. We were also able to show the concept of multi-valent binding with shorter probes in the second approach. Here, a 13-nt-long probe has shown the best performance during SARS-CoV-2 RNA binding. Therefore, multi- valent binding approaches using EIS have high utility for direct detection of nucleic acid targets and for point-of-care diagnostics.