Easy methodology for linker-free SERS detection of vitamin D₃ at trace levels

Vitamin D₃ is a crucial biomarker for several human diseases. Its deficiency is overcome through pharmaceutical and food supplement interventions. Therefore, its detection at biologically relevant low concentrations or trace levels is crucial. SERS is a powerful analytical technique that combines the molecular specificity of Raman spectroscopy with electromagnetic field enhancement at plasmonic nanostructures, enabling single-molecule detection. However, SERS-based detection of vitamin D₃ and its metabolites without any linker molecule is challenging and underexplored. The primary obstacle stems from its molecular structure and physicochemical properties. It is a nonpolar, lipophilic secosteroid with weak affinity for metal surfaces, leading to poor adsorption on conventional SERS substrates. This work elucidates a linker-free SERS methodology for trace-level detection of vitamin D₃ through direct adsorption onto plasmonic silver nanoclusters. Vitamin D₃-like analytes possess a conjugated triene system capable of interacting with Ag nanocluster surfaces through π-metal interactions, facilitated by van der Waals forces and weak charge-transfer contributions. Upon excitation with a laser wavelength resonant with the plasmonic response of the substrate, these π-Ag interactions may enable synergistic electromagnetic and chemical enhancement of Raman-active vibrational modes associated with the conjugated triene framework. If so, the methodology can explicitly exploit the CC stretching Raman vibrational mode at ∼1599 cm^-1 (the most polarizable mode in the conjugated triene system) for quantitative analysis. In this work, using novel walnut kernel-like Ag-nanocluster-based SERS substrates, vitamin D₃ was detected down to 1 nmol/L without the use of linker molecules, aptamers, or immunorecognition elements. The ∼1599 cm^-1 band not only enabled the detection of vitamin D₃ at trace levels but also verified linearity in detection. This approach contrasts with conventional SERS strategies for vitamin D₃ and 25-hydroxyvitamin D₃ detection that rely on sophisticated aptamer- or immunosensor-based architectures, offering a simplified and cost-effective alternative while maintaining high sensitivity.