An Ultrasensitive Electrochemical Biosensor for Nucleic Acid Detection Based on Silver Nanoflower-Stem-Loop Probes

Nucleic acids are critical biomarkers that provide essential information throughout disease progression, making their detection critical to early diagnosis of both infectious and non-infectious diseases. However, existing detection methods, including classical analytical techniques and even most reported biosensors, are often constrained by complex procedures, high costs, and limited sensitivity, with the majority operating at the femtomolar level and failing to achieve single-molecule detection needed for early-stage diagnosis. Here, we report an electrochemical biosensor based on silver nanoflowers (AgNFs) integrated with stem-loop probes (SPs) for universal nucleic acid detection, using Norovirus RNA as a model target to validate the platform. The SPs serve as critical elements in a signal amplification system, converting target binding into a biotin-streptavidin recognition event, which leads to the accumulation of AgNFs-SP complexes on laser-induced graphene (LIG) electrodes and generates a strong electrochemical signal. Under optimized conditions with a 50 min hybridization incubation (total assay time ~60 min), the sensor exhibits a linear response to Norovirus RNA concentrations from 1 aM to 10 fM, with a measured detection limit of 1 aM, achieving single-molecule-level detection capability. For applications requiring faster turnaround, a 20 min hybridization incubation (~30 min total assay time) shifts the linear range to 0.1 fM-1 pM with a measured detection limit of 0.1 fM, offering more rapid quantification when the maximum sensitivity is not required. The proposed biosensor is cost-effective, amenable to miniaturization, and designed as a versatile platform adaptable to other nucleic acid targets by simply modifying the probe sequence, showing broad potential for early diagnosis of various diseases.