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

Abstract

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 labelfree 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, multivalent binding approaches using EIS have high utility for direct detection of nucleic acid targets and for point-of-care diagnostics.

Circulating microRNA detection using electrochemical biosensor for rapid diagnosis of liver disease in dogs

“…Screen-printed gold electrodes (DRP-C223BT) were purchased from Metrohm DropSens (Oviedo, Spain) and functionalised with PNA probes following the protocol developed in our earlier studies. 23,24,41 In short, electrodes were electrochemically cleaned using cyclic voltammetry (0–1.6 V potential range, 100 mV s −1 scan rate and 10 cycles) in 100 mM sulfuric acid aqueous solution. The cleaned gold working electrode was promptly incubated with a ternary MCH/HDT/PNA probe layer, comprising 6 μM PNA probe, 100 μM MCH, 200 μM HDT and 5 mM TCEP (reducing reagent), and kept in a humid chamber for 16 h for probe immobilisation.…”