Copper chromogenic reaction based colorimetric immunoassay for rapid and sensitive detection of a tumor biomarker

A new colorimetric immunoassay method was developed for the rapid and sensitive detection of a tumor biomarker of carcinoembryonic antigen (CEA) by combination of a magnetic bead (MB)-based sandwich immunoassay and a copper chromogenic reaction. The magnetic immunoassay platform was constructed through the covalent immobilization of the capture antibody on the surface of carboxylated magnetic beads. After immuno-recognition of CEA, signal antibody-functionalized copper oxide nanoparticle (CuO NP) probes were applied for sandwich immunoreaction to form an immunocomplex. The CuO NP labels quantitatively captured onto the immunocomplex were then dissolved in acid solution to release high-content copper ions. Based on the coordination of these ions with the newly synthesized chromogenic agent of 1,2-diphenyl-2-(2-(pyridin-2-yl)hydrazono)ethanone, a red complex was produced for the colorimetric signal readout, resulting in the successful construction of a sensitive immunoassay method for CEA detection. Under the optimum conditions, this method showed a wide linear range over three orders of magnitude and a low detection limit of 26 pg/mL. Besides, this method showed excellent performance with low cost, rapid and convenient operation as well as satisfactory reproducibility, stability and accuracy, thus providing great potentials for practical applications.