Multilayers enzyme-coated carbon nanotubes as biolabel for ultrasensitive chemiluminescence immunoassay of cancer biomarker

A novel and ultrasensitive chemiluminescence immunoassay (CLIA) method based on multiple enzyme layers assembled multiwall carbon nanotubes (MWCNTs) as signal amplification labels was developed by employing luminol–H2O2-HRP-bromophenol blue (BPB) enhanced chemiluminescence (CL) system for the detection of a cancer biomarker in human serum samples, as exemplified by the measurement of alpha-fetoprotein (AFP) as a model protein. In this study, horseradish peroxidase (HRP) was assembled onto MWCNTs templates layer-by-layer (LBL) through electrostatic interactions with polyion PDDA, and further conjugated with AFP secondary antibodies (Ab2) as the enzyme label. The resulting LBL assembly could maximize the ratio of HRP/Ab2 which could amplify the sensitivity greatly. To the best of our knowledge, it was the first time for this strategy applied in CLIA to date. Under the optimum conditions of luminol–H2O2-HRP-BPB CL system and the sandwich immunoreactions, a linear range from 0.02 to 2.0 ng/mL (R = 0.9980) was obtained with the detection limit of 8.0 pg/mL (3σ) which was two orders of magnitude lower than standard ELISA method. Furthermore, accurate detection of AFP in human serum samples was also demonstrated by comparison to ELISA assays. From the above results, such signal amplification strategy proposed by the novel CNT-LBL enzyme label showed an excellent promise for ultrasensitive detection of cancer biomarkers in clinical laboratory.