Colorimetric multiplexed immunoassay for sequential detection of tumor markers

In this paper, a very simple and easily-operated colorimetric multiplexed immunoassay method for sequential detection of tumor markers has been presented. Magnetic microparticles which are conjugated with biotinylated antibodies are firstly added into the test solution. After fast magnetic collection, these complexes are separated from non-specific proteins. Through different enzymatic reactions of 3,3',5,5'-tetramethylbenzidine (TMB) and o-phenylenediamine (OPD) catalyzed by horseradish peroxidase molecules which are loaded on the surfaces of gold nanoparticles, two antigens carcinoembryonic antigen and alpha-fetoprotein can be detected even with naked eyes. The detection limit obtained from the spectrophotometric measurements is as low as 0.02 ng/mL. This proposed method also has high specificity and reproducibility, as well as excellent efficiency of 94 min for the detection of serum samples. So, this new multiplexed immunoassay method might be a promising approach for the diagnosis of cancer and some other diseases in clinical applications.