A one-step functionalized 3D-printed electrochemical biosensor for serotype-selective detection of dengue NS1

Rapid and selective detection of dengue virus serotypes remains challenging because of the co-circulation of antigenically related arboviruses. We developed a fully 3D-printed electrochemical biosensor for dengue virus serotype 2 non-structural protein 1 (DENV-2 NS1). The integrated three-electrode platform incorporated a graphite/polylactic acid working electrode modified in a single drop-casting step with preassembled monoclonal antibody-gold nanoparticle conjugates (mAb-AuNP). DENV-2 NS1 binding was monitored by cyclic voltammetry using [Fe(CN)₆]^3-/4- as a redox probe; immunocomplex formation partially blocked interfacial electron transfer and decreased the cathodic peak current. The response stabilized after 15 min and was linear from 0.15 to 0.75 μg L^-1 (1.75-8.74 pM), with R² = 0.9922 and a limit of detection of 0.061 μg L^-1 (0.71 pM). Three independently prepared biosensors showed a relative standard deviation of 3.4%, indicating good fabrication reproducibility. The immunosensor produced a substantially greater response to DENV-2 NS1 than to DENV-1 NS1, Zika virus NS1, or chikungunya virus E2 protein. In 100-fold-diluted human serum spiked at 0.15 and 0.25 μg L^-1, standard-addition analysis yielded recoveries of 106.0 ± 1.3% and 97.7 ± 1.1%, respectively. These findings demonstrate that one-step mAb-AuNP functionalization of fully 3D-printed electrodes provides a rapid, reproducible, and serotype-selective approach to NS1 detection, warranting further validation with clinical samples.