Digital detection of hepatitis E antigen tailored for multiple genotypes using graphene transistors functionalized...

Hepatitis E virus (HEV), an emerging zoonotic pathogen and a major cause of acute hepatitis, requires advanced diagnostics for multi-genotype detection, quantification, and digital analysis to improve diagnosis and outbreak control. This study presents the development of a digital biosensor using graphene field-effect transistors (GFETs) functionalized with llama-derived nanobodies (Nbs) for detecting HEV ORF2 proteins. Using a Nb library, the features of high affinity and multi-genotype ORF2 recognition were demonstrated by ELISA and surface plasmon resonance (SPR) spectroscopy. These Nbs exhibited exceptional affinity for ORF2, with dissociation equilibrium constants (K_D) reaching a value of 2.64 pM for genotype 3. The GFET sensors were co-functionalized with Nbs and polyethylene glycol (PEG), an antifouling element that enhances specificity. The sensors displayed robust signal responses to HEV-1 and HEV-3 ORF2, achieving amplitudes as high as 22.5 mV and a limit of detection (LOD) in the nanomolar range. Testing in spiked serum samples confirmed the biosensors' capability to distinguish positive and negative samples. A quantitative model was used to correlate antigen concentration with GFET response (ΔV_cnp). We validated graphene interfacial transduction by comparing ΔV_cnp with antigen surface density (Γ_ORF2) estimated by SPR. Furthermore, a theoretical model using Poisson-Boltzmann and Grahame equations explained key FET sensor behaviors related to ionic screening and molecular distance. This Nb-functionalized GFET biosensor represents a significant advancement in multi-genotype HEV antigen detection, with the added advantage of being digital, thereby facilitating the rapid monitoring and containment of HEV cases.