A nanohybrid composed of Prussian Blue and graphitic CN nanosheets as the signal-generating tag in an enzyme-free electrochemical immunoassay for the neutrophil gelatinase-associated lipocalin.

An enzyme-free electrochemical immunoassay is described for the neutrophil gelatinase-associated lipocalin (NGAL; a biomarker of kidney disease). Prussian Blue (PB) nanoparticles with redox activity were deposited on graphitic CN nanosheets (g-CN) by in-situ reduction. A screen printed electrode (SPCE) was modified with antibody against NGAL, and the PB-g-CN nanohybrid was used as the signal-generating tag for the secondary antibody against NGAL. Upon addition of target NGAL and of secondary antibody, a sandwich is formed on the SPCE. At an applied potential of typically 0.13 V (vs. Ag/AgCl), a well-defined voltammetric peak is observed that results from the presence of PB on the secondary antibody. Under optimal conditions, the peak current increases linearly in the 0.01 to 10 ng·mL NGAL concentration range, and the detection limit is 2.8 pg·mL. An average precision of <12% was accomplished in the batch-to-batch mode. Other disease-related biomarkers do not interfere. The accuracy and inter-laboratory validation of this method were evaluated for target NGAL detection in spiked human serum by using a commercial ELISA. The results obtained by the two methods are in good accordance. Graphical abstract An enzyme-free electrochemical immunoassay was used for detection of neutrophil gelatinase-associated lipocalin by Prussian blut/graphitic-CN nanohybrids as the signal-generation tags.