RESUMO
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.
RESUMO
An electrochemical bioplatform involving screen-printed carbon electrodes modified with rGO/MoS2/AgNPs nanocomposites, the covalent immobilization of the specific capture antibody, and label-free detection has been developed for the determination of Glial Fibrillary Acidic Protein (GFAP). The resulting immunosensor profits the benefits of the rGO high conductivity, the pseudo-peroxidase activity of MoS2 and the electrocatalytic effect provided by AgNPs for improving the reduction current responses of hydrogen peroxide at the electrode surface. GFAP is a biomarker of central nervous system injuries has been proposed for the detection and monitoring of neurological diseases as epilepsy, encephalitis, or multiple sclerosis. For the first time, amperometric detection of the immunosensing event was performed by measuring the electrocatalytic response of hydrogen peroxide reduction at the modified electrode. Several techniques including scanning (SEM) and transmission (TEM) electron microscopies were used for the characterization of the synthesized composite whilst electrochemical impedance spectroscopy (EIS) using the redox probe Fe(CN)63-/4- was employed to evaluate the success of the steps implied in the fabrication of the immunosensor. After optimization of the involved experimental variables, a linear calibration plot for GFAP was constructed over the 0.6-100 ng mL-1 range, and a detection limit of 0.16 ng mL-1 was achieved. The developed immunosensor was successfully applied to the determination of GFAP in human cerebrospinal fluid (CSF) of patients diagnosed with encephalitis.