An enzyme-free HCR-amplified colorimetric biosensor based on a Cu²⁺/rGO nanozyme for detection of dengue virus

Rapid and cost-effective detection of dengue infection, especially during the early stages of disease and in resource-limited settings, remains a significant public health concern. Recent advances in nanomaterial-based biosensors have provided promising opportunities for the development of simple, sensitive, and affordable nucleic acid detection platforms. In the present study, a Cu²⁺-modified reduced graphene oxide (Cu²⁺/rGO) nanostructure-based colorimetric biosensor integrated with hybridization chain reaction (HCR) was developed and assessed for the detection of a dengue virus target sequence. The nanozyme exhibited peroxidase-like activity and catalyzed the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H₂O₂), generating a visible colorimetric signal. Following HCR-mediated double-stranded DNA formation, the catalytic activity of the nanostructure was suppressed, resulting in reduced color intensity and enabling signal-off detection. The proposed biosensor showed a linear response over 0.5-10 nM, with detection limits of 1.36 nM in buffer and 1.6 nM in human serum samples spiked with synthetic target DNA. The color variation was directly visible to the naked eye. The assay showed good linearity (R² = 0.9868) while avoiding the need for thermocyclers and complex instrumentation. The Cu²⁺/rGO nanozyme preserved nearly 79% of its catalytic performance after 10 days of storage. Overall, the results suggest that the developed platform may provide a practical and cost-effective biosensing strategy for preliminary nucleic acid screening in resource-limited settings and complement conventional polymerase chain reaction (PCR)-based assays.