Sirivibulkovit K.Voelkle J.Windisch D.J.Muttenthaler M.Lieberzeit P.A.Saetear P.Mahidol University2025-08-302025-08-302025-12-01Biosensors and Bioelectronics Vol.289 (2025)09565663https://repository.li.mahidol.ac.th/handle/123456789/111883Dengue is a significant public health concern, particularly in tropical and subtropical regions, due to its potential severity and widespread impact. Nonstructural protein 1 (NS1) serves as a crucial biomarker for the early and rapid detection of dengue virus infection. This study introduces an electrochemical sensor utilizing an epitope imprinting approach with molecularly imprinted polymer (MIP) nanoparticles to detect dengue NS1. The NS1 epitope, Ac-VHTWTEQYKFQ-CONH<inf>2</inf>, was employed as a template for the solid-phase synthesis of MIP nanoparticles. This epitope is highly conserved among dengue virus serotypes and is specifically recognized by monoclonal antibody 4F6, which does not cross-react with closely related viruses such as Zika virus and Japanese encephalitis virus (JEV), supporting the sensor's high specificity. Characterization of the MIP nanoparticles revealed specific binding sites capable of recognizing the epitope. Two detection strategies were developed: a competitive assay, where the epitope was immobilized on a screen-printed gold electrode (AuSPE) and exposed to MIP nanoparticle suspensions premixed with NS1, and a direct assay, where the MIP nanoparticles coated on AuSPE surfaces detected the epitope in a solution. The competitive assay demonstrated a detection range of 1–100 ng mL⁻¹ with a limit of detection (LOD) of 0.74 ng mL⁻¹, while the direct assay showed a detection range of 5–100 ng mL⁻¹with a LOD of 3.82 ng mL⁻¹. Both assays were extended to detect NS1 in PBS (pH 7.4), achieving a linear range of 5–300 ng mL⁻¹ with a LOD of 4.30 ng mL⁻¹ for the competitive assay and 10–100 ng mL⁻¹ with a LOD of 8.23 ng mL⁻¹ for the direct assay. These LODs cover NS1 levels in blood serum for primary and secondary infections. The electrochemical sensors demonstrated selectivity for NS1 in spiked human serum, offering a cost-effective alternative to natural antibody-based detection methods.ChemistryBiochemistry, Genetics and Molecular BiologyEngineeringArticleSCOPUS10.1016/j.bios.2025.1179152-s2.0-10501397984718734235