A Portable Surface-Free Electrochemical Aptamer-Based Biosensor for Multiplex Detection of Arboviruses

Mosquito-borne arboviruses, including dengue virus (DENV), chikungunya virus (CHIKV), and yellow fever virus (YFV), represent persistent global public health threats, especially in tropical and subtropical regions. Here, we report a portable, surface-free electrochemical aptamer-based biosensor capable of multiplexed detection of DENV, CHIKV, and YFV antigens directly from human serum. The device integrates antibody-functionalized magnetic nanoparticles and aptamer@alkaline phosphatase conjugates within a cartridge-based system for in-solution recognition and magnetic separation, eliminating the need for electrode surface modification. This surface-free, solution-phase configuration overcomes the mass-transport limitations and labor-intensive electrode functionalization steps inherent to conventional electrochemical biosensors, enabling robust antigen detection directly in serum. The hybrid antibody–aptamer architecture achieves detection limits of 0.89, 1.42, and 1.31 ng·mL –1 for DENV, CHIKV, and YFV, respectively, with excellent reproducibility (RSD < 12%). Clinical validation using 80 ELISA-confirmed positive and 50 negative serum samples demonstrated 100% diagnostic sensitivity, specificity, and accuracy, outperforming RT-PCR (98.8%, 84%, and 93.1%, respectively) and surpassing RT-PCR in early stage infection detection. The assay provides a quantitative readout within 30 min, making it a powerful point-of-care platform for rapid field diagnostics.