Disposable label-free electrochemical immunosensor based on gold nanoparticles-Prussian blue for neutrophil gelatinase-associated lipocalin detection in urine samples

Neutrophil gelatinase-associated lipocalin (NGAL) is a remarkable biomarker for assessing acute kidney injury. In this study, we developed a novel label-free NGAL electrochemical immunosensor based on gold nanoparticles (AuNPs) and Prussian blue (PB) without an external mediator. The AuNPs-PB based immunosensor was fabricated on a custom gold-electrode (AuE)-based polypropylene (PP) substrate. We systematically assessed and optimized key experimental parameters, including the process of AuNPs-PB electrodeposition, antibody concentration, and incubation time. The immunosensor response toward NGAL was determined using differential pulse voltammetry, where the decrease in the oxidation current response of the PB redox probe correlating with the increase in NGAL concentration. Our results demonstrated that the synergistic benefits of both AuNPs and PB significantly improved electrochemical activity for NGAL detection and provided a highly stable sensor across a range of pH values. The label-free immunosensor exhibited two linear ranges: 0.10−1.40 ng mL−1 and 1.40−25.0 ng mL−1, with a low detection limit of 0.094 ng mL−1. The developed NGAL immunosensor displayed high selectivity and excellent reproducibility. Furthermore, NGAL detection was completed within 30 min and the immunosensor exhibited storage stability for six weeks. Notably, NGAL levels determined in human urine samples using this developed label-free immunosensor showed good agreement with the results obtained from the enzyme-linked immunosorbent assay. This novel label-free NGAL immunosensor provides great potential in developing NGAL point-of-care testing applications.