Detection of salivary pepsin has been given attention as a new diagnostic tool for laryngopharyngeal reflux (LPR) disease, because saliva collection is non-invasive and relatively comfortable. In this study, we prepared polypyrrole nanocorals (PPNCs) on a screen-printed carbon electrode (SPCE) by a soft template synthesis method, using β-naphthalenesulfonic acid (NSA) (for short, PPNCs/SPCE). Gold nanoparticles (GNPs) were then decorated on PPNCs/SPCE by electrodeposition (for short, GNP/PPNCs/SPCE). To construct the immunosensor, pepsin antibody was immobilized on GNP/PPNCs/SPCE. Next, citric acid was applied to prevent non-specific binding and change the electrode surface charge before pepsin incubation. Electrochemical stepwise characterization was performed using cyclic voltammetry, and immunosensor response toward different pepsin concentrations was measured by differential pulsed voltammetry. As a result, our electrochemical immunosensor showed a sensitive detection performance toward pepsin with a linear range from 6.25 to 100 ng/mL and high specificity toward pepsin, as well as a low limit of detection of 2.2 ng/mL. Finally, we quantified the pepsin levels in saliva samples of LPR patients (n = 2), showing that the results were concordant with those of a conventional ELISA method. Therefore, we expect that this electrochemical immunosensor could be helpful for preliminarily diagnosing LPR through the detection of pepsin in saliva.
Keywords: electrochemical immunosensor; laryngopharyngeal reflux; pepsin; polypyrrole nanocorals; saliva.