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This work described a simple and feasible colorimetric immunoassay. Using hemin (a horseradish peroxidase (HRP) mimic enzyme) and the mimic enzyme-chromogenic substrate system, it can qualitatively and quantitatively detect α-fetoprotein (AFP) at an ultralow concentration. The glucose oxidase (GOx) catalyzed oxidation of glucose leads to the formation of gluconic acid and hydrogen peroxide (H2O2). The latter can oxidize 4-aminoatipyrine (4-AAP) to chromogenic products, and the reaction was catalyzed by hemin. With the increase of H2O2, the absorbance increased, and the color of the solution changed from colorless to pink. On the basis of the system, monitored by recording the color or absorbance (λ = 505 nm), a new immunoassay protocol with GOx-labeled anti-AFP detection antibody was designed. A wide linear dependence was obtained in the range from 0.075 to 280 ng mL with a low detection limit (LOD) of 0.0247 ng mL (S/N = 3).

A Novel Colorimetric Immunoassay of Ultrasensitive Alpha-Fetoprotein Sensing in Magnetic Bead-Based Mimic Enzyme-Chromogenic Substrate System