Unravelling porous silicon (PSi)-based surface-enhanced raman spectroscopy (SERS) substrate fabrication formula sensitive to salivary dengue non-structural protein 1 (NS1)

Dengue fever remains a major public health issue in Malaysia, with annual cases consistently exceeding 100,000 since 2014. Although the mortality rate is relatively low at around 0.2%, timely diagnosis is essential for effective treatment. One of the most promising biomarkers for early detection is the non-structural protein 1 (NS1), which can be detected as early as the first day of infection, well before the production of antibodies such as IgM, IgG and IgA. This makes NS1 an attractive target for diagnostic assays. Blood-based tests are commonly used, but their invasive nature may discourage early medical consultation. Since NS1 is also present in saliva, it offers a less invasive diagnostic alternative; however, reported sensitivity in saliva detection remains limited at only 64.7%. This limitation underscores the need for more sensitive and reliable detection methods. In this study, a surface-enhanced Raman spectroscopy (SERS) substrate coated with silver nanoparticles (AgNPs) was fabricated using an electrochemical etching method combined with drop-casting deposition, with the aim of detecting low concentrations of NS1 in saliva. Etching durations were varied to optimize the porous silicon structure, with the optimum condition determined from the highest Raman peak intensity and narrowest full width at half maximum (FWHM). AgNPs of various sizes (5 nm, 25 nm, 50 nm, 75 nm and 110 nm) were also tested to identify the most effective configuration for SERS enhancement. The resulting substrates were characterized using Raman spectroscopy, field emission scanning electron microscopy (FESEM) and energy-dispersive X-ray spectroscopy (EDX). Performance evaluation using Rhodamine 6G (R6G) as a model analyte showed that the substrate coated with 75 nm AgNPs exhibited the best enhancement, achieving an enhancement factor (EF) of 148.56 and a limit of detection (LOD) of 0.01 mg/mL. For NS1 detection, the 75 nm AgNPs-coated PSi SERS substrate was functionalized with dengue antibodies. This functionalized substrate achieved a detection limit of 0.001 mg/mL (equivalent to 1000 ng/mL), which lies within the clinically relevant detection range. Overall, this work highlights the potential of 75 nm AgNPs-coated PSi SERS substrate as a sensitive, non-invasive platform for dengue detection in saliva. These findings provide a foundation for developing portable and cost-effective diagnostic tools that could facilitate early disease detection and timely intervention.