Highly sensitive electrochemiluminescent immunoassay for detecting neuron-specific enolase (NSE) based on polyluminol and glucose oxidase-conjugated glucose-encapsulating liposome

Neuron-specific enolase (NSE) is a cancer biomarker for diagnosing small cell lung carcinoma, carcinoids, islet cell tumors, and neuroblastomas. In this study, we developed an ultrasensitive electrochemiluminescence (ECL)-based immunoassay to determine NSE, consisting of the separation and sensing elements. First, super-paramagnetic iron oxide (Fe3O4) nanoparticles were synthesized and attached to primary NSE monoclonal (Ab1-Fe3O4) to prepare a separating nanosystem. Then D-glucose-containing liposomes (Glu@LP) were prepared and covalently attached to a second monoclonal antibody using Sulfo-SMCC (Glu@LP-Ab2). Afterward, to develop a sensing electrode, gold nanoparticles (AuNPs) were electrochemically formed on a screen-printed electrode (SPE), and multi-walled carbon nanotubes (MW) were deposited on the SPE. Glucose oxidase (GOx) enzymes were conjugated with the carboxylic groups of MW on the sensing electrode. Finally, polyluminol (PLu) was electropolymerized on the electrode. Ab1-F3O4 and Glu@LP-Ab2 were used to capture NSE (Glu@LP-Ab(2)-(NSE)-Ab1-F3O4). ECL signals from polyluminol (PLU-GOx-MW-Au-SPE) and hydrogen peroxide, produced by oxidation of glucose by GOx reaction, were proportional to the concentration of captured NSE. Immunoassay's detection limit (LOD) and linear range (LDR) were found to be 12.8 fg.mL(-1) (at S/N = 3) and 100 fg.mL(-1) to 100 ng.mL(-1), respectively. This immunoassay resulted in acceptable accuracy with recoveries in the 98.2 - 102.6 % range and high reproducibility with RSD of less than 3.4%.