Magnetic-bead sandwich immunosensing with multienzyme immunogold nanoprobe and nanochannel electrode for sensitive electrochemiluminescence detection of tumor biomarker

Sensitive tumor biomarker detection is vital to early cancer diagnosis, therapy monitoring, and prognosis. Herein, a signal-off electrochemiluminescence (ECL) immunosensing platform was developed by combining enzyme-loaded immunogold nanoprobes, magnetic bead-based sandwich recognition, and nanochannel modified electrode, that enables sensitive detection of carcinoembryonic antigen (CEA) as proof-of-concept demonstration. Functionalization of low-cost ITO electrode with silica nanochannel film (SNF) produced nanochannel electrode, which enabled the electrostatic enrichment of the cationic tris(2,2′-bipyridyl)ruthenium(II) (Ru(bpy) 3 2+ ) emitter and resulted in a 119-fold ECL enhancement over bare ITO. Gold nanoparticles (AuNPs) were used to co-load multiple glucose oxidase (GOD) together with detection antibody (dAb), yielding GOD/dAb@Au nanoprobes. In the presence of CEA, magnetic beads functionalized with capture antibody (cAb) selectively bound the target and subsequently associated with the GOD/dAb@Au probes to form an enzyme-bearing sandwich immunocomplex. Application of an external magnetic field enabled rapid separation of the immunocomplex from sample matrix. Using the SNF/ITO electrode and a Ru(bpy) 3 2+ /tripropylamine (TPrA) solution containing glucose (Glu), the GOD-mediated production of H 2 O 2 acted as a quencher, decreasing the ECL signal in a CEA-dependent manner. The immunosensor demonstrated a linear detection range for CEA from 10 fg/mL to 100 ng/mL, with a limit of detection (LOD) as low as 5.3 fg/mL. CEA detection in fetal bovine serum was realized. Additionally, the SNF/ITO electrode could be regenerated and reused.