Enhanced electrochemiluminescence of luminol at neutral medium using nanochannel-confined Co3O4 nanozyme for highly sensitive detection of tumor biomarker

Sensitive detection of alpha-fetoprotein (AFP) is crucial for early liver cancer screening. In this work, an immunosensor is constructed by combining the immunorecognition interface and enhanced electrochemiluminescence (ECL) of luminol under neutral condition utilizing cobalt oxide (Co 3 O 4 ) nanozymes confined in nanochannels, which realizes highly sensitive detection of AFP. Indium tin oxide (ITO) conductive glass is used as the supporting electrode, and silica nanochannel film (SNF) is simply grown on the ITO surface using the Stöber method. Co 3 O 4 nanozymes are in situ grown within the SNF nanochannels by a one-step electrodeposition method. The rigid 2D nanochannel array of SNF effectively confines the Co 3 O 4 nanozyme, significantly enhancing its stability. Epoxy modification on the external surface of SNF enables covalent attachment of AFP antibodies, leading to recognition interface. Confined Co 3 O 4 exhibits peroxidase-like activity, catalyzing the generation of reactive oxygen species (ROS) from hydrogen peroxide (H 2 O 2 ), enhancing luminol oxidation and boosting the ECL signal under neutral conditions. Upon AFP binding, antibodies capture AFP to form immunocomplexes, causing reduced diffusion of luminol and H 2 O 2 to the electrode surface, decreasing the ECL signal. The constructed immunosensor exhibits a linear range for AFP detection from 100 fg/mL to 10 ng/mL, with a limit of detection (LOD) of 18.5 fg/mL. AFP detection in serum samples from liver cancer patients or healthy individuals is successfully achieved.