Label-Free Detection of Salivary Pepsin Using Gold Nanoparticle/Polypyrrole Nanocoral Modified Screen-Printed Electrode

Abstract

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.

Advanced Biomimetic Nanomaterials for Non-invasive Disease Diagnosis

“…Cyclic voltammetry was used for electrochemical characterization, and differential pulse voltammetry was employed to determine the response of the immunosensor to different pepsin concentrations. The sensor showed a linear range of 6.25-100 ng/mL, high specificity, and a detection limit of 2.2 ng/mL (Lee et al, 2018).…”

“…Inflammatory diseases (Bardhan et al, 2014;Lee et al, 2018;Liu T. et al, 2018) (Li et al, 2016;Apilux et al, 2018) + + − − − − Metabolic disease (Zhao et al, 2019) − − + − − − Cardiovascular disease Eom et al, 2020) + − − + − − MRI, magnetic resonance imaging; OI, optical imaging; USI, ultrasound imaging. +, applicable; -, no report.…”

Suppressing Non-Specific Binding of Proteins onto Electrode Surfaces in the Development of Electrochemical Immunosensors

“…Different concentrations of pepsin (0, 6.25, 12.5, 25, 50 and 100 ng/mL) were added to the Ab/CA/AuNPs/PPNCs/SPCE EI platform (Figure 10). LOD of this sensor platform was found to be 2.2 ng/mL pepsin [144].…”

“…Detection of pepsin antigen in saliva using the [Fe (CN) 6 ] 3−/4− redox probe. LOD of this sensor platform was found to be 2.2 ng/mL pepsin [144].…”

Portable and Rapid Fluorescence Turn-On Detection of Total Pepsin in Saliva Based on Strong Electrostatic Interactions

“…To date, the commonly used analytical methods, such as enzyme-linked immunosorbent assays, electrochemical immunosensors, spectrophotometry, and molecularly imprinted analysis, have been reported to detect pepsin. − However, these approaches are limited in their use for rapid on-site analysis because they typically require complex experimental procedures. Fluorescent sensors have been widely used, retaining the advantages of high sensitivity and rapid response. − Recently, strategies based on pepsin digestion resulting in fluorescence quenching of protein-coated fluorescent dyes or nanomaterials have been reported for pepsin detection. − Interestingly, the isoelectric point (pI) of pepsin is quite low, and it has the strongest activity at around pH 2.0 .…”