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.