Electrochemical immunoplatform for the determination of multiple salivary biomarkers of oral diseases related to microbiome dysbiosis

Several diseases of the oral cavity are related to compositional and functional shifts in the oral microbiome. The analysis of saliva is an attractive alternative for the diagnosis and prognosis of these diseases. Samples can be obtained by no invasive procedures and processing is relatively simple. However, sensitive and selective analytical methods are needed to make the diagnosis as specific as possible. In this work, four salivary biomarkers of oral diseases: interleukin-6 (IL-6), receptor activator of NF-kB ligand (RANKL), protein arginine deiminase 4 (PAD4) and the corresponding antibody (anti-PAD-4) were selected as targets for their simultaneous determination using an electrochemical immunosensing platform. Sandwich-type amperometric immunoassays were implemented using horseradish peroxidase (HRP)/H₂O₂/hydroquinone (HQ) for application to the analysis of saliva of six volunteers. The developed method provides excellent sensitivity, selectivity, and wide linear ranges with LOD values of 0.09 pg mL⁻¹ (IL-6), 0.10 pg mL⁻¹ (RANKL); 0.09 ng mL⁻¹(PAD4) and 14.5 ng mL⁻¹ (anti-PAD4) and allows the accurate analysis of saliva without matrix effects, using 25 μL of raw sample. The developed methodology is competitive with commercial ELISA kits available only for a single biomarker determination, while the assay for the four biomarkers can be completed in less than two hours.