Targeting the Viral Signature Through gFET: Ultrasensitive Monitoring of dsRNA, a Universal Viral Biomarker

Sabine SZUNERITS, List, Danube Private University, Wiener Neustadt, Austria (sabine.szunerits@dp-uni.ac.at)
Manova Santhosh, List, Danube Private University, Wiener Neustadt, Austria
Christophe RITZENTHALER, Cnrs, Institut De Biologie Moléculaire Des Plantes, Strasbourg, France
Subhankar SAHU, Cnrs, Institut De Biologie Moléculaire Des Plantes, Strasbourg, France

Double-stranded RNA (dsRNA) is a generic viral infection biomarker and represents a prototypal pathogen-associated molecular pattern (PAMP) recognized during host immune response. Monitoring dsRNA can be crucial for the detection of emerging infectious diseases in particular those cause by unknown viruses. However, dsRNA-centered approaches for viral sensing remain underrepresented in the current literature. In this research, the diagnostic ability of a graphene-based field-effect transistor (gFET) for the sensitive and specific detection of viral dsRNA (V-dsRNA) is outlined by leveraging the B2 protein’s remarkable dsRNA binding properties. The dsRNA-binding domain of B2 (from Flock house virus) is a 73 amino acid biorecognition scaffold for dsRNA with a nanomolar-order binding affinity for dsRNA. Herein, the B2 is structurally engineered to contain a 6× histidine-tag and is site-specifically immobilized on a NTA/Ni²⁺ functionalized graphene channel in an oriented fashion. This resulted in a in improved sensor responses to synthetics as well as viral-dsRNA probes, outperforming gFET devices with covalent integrated B2. Spiking 0.01× PBS samples with bacteriophage MS2 dsRNA isolated from Escherichia coli cells, revealed a limit of detection (LoD) of 30 pg mL⁻¹ (~60 fM). The sensitivity of this technology was benchmarked against gFETs using the gold standard J2 antibody (for dsRNA) as recognition element rather than B2, which resulted in a LoD of 0.97 ± 0.16 nM. The developed B2: gFET sensor showed excellent selectivity towards V-dsRNA with no response to ssRNA as well as ds/ssDNA probes. The first results on the sensing of dsRNA in mosquito samples will be highlighted in addition. This strategy can be considered as a first step towards implementing a dsRNA-centered transistor-based viral surveillance strategy.