A new washing-free immunosensor for tumor marker detection based on functionalized Fe3O4 submicron particles

In this submission, a novel washing-free immunosensor was designed for chemiluminescence immunoassay (CLIA) of tumor marker using human Alpha-fetoprotein (AFP) as a representative. Reactive groups were introduced on the surface of the solvothermally prepared Fe 3 O 4 magnetic submicron particles (MSPs) and the dual-functional polyethylene glycol (PEG) linkers were modified to reduce the non-specific adsorption capacity of the particle surface and further to immobilize N -(4-aminohexyl)- N -ethylisoluminol (AHEI) and anti-AFP antibody on the surface of the particles. After incubation with AFP and horseradish peroxidase (HRP) labeled anti-AFP antibody respectively, the formed sandwich-type immunocomplexes on the particle surface could lead to the close proximity between AHEI and HRP, thus eventually leads to an increase in the detected chemiluminescence (CL) signal. As a bound HRP labeled anti-AFP antibody produces much stronger CL signal than an unbound one, this approach eliminates the time- and labor-consuming washing steps usually required of such assays. Under the optimal conditions, this present method shows a wide quantitative range from 1 to 250 ng mL −1 with a detection limit of 1.751 ng mL −1 . The detection limit was sufficient for measuring physiological (0–20 ng mL −1 ) and clinical (>20 ng mL −1 ) AFP levels. This method can also be applied in serum samples, and also avoid the problem of particle aggregation caused by multiple separation and washing steps.