Novel signal-enhancing immunoassay for ultrasensitive biomarker detection based on laser-induced fluorescence

An innovative signal-enhancing immunoassay for ultrasensitive biomarker detection based on laser-induced fluorescence (LIF) has been developed. A novel LIF optical system with high collection efficiency was constructed by using a parabolic mirror. Carboxyl-functionalized magnetic beads were used to immobilize antibody for achieving a conventional sandwich assay. Fluorescence from Rhodamine 6G (R6G)-labeled antibody was collected by the newly designed optical system. To reduce photobleaching of R6G under laser irradiation, ethanol instead of commonly used aqueous solution was used as assay buffer in the last stage. The newly developed LIF immunoassay displayed excellent analytical performance for α-fetoprotein (AFP) detection in the concentration range from 0.005 to 1.0 ng/mL with a detection limit of 0.0016 ng/mL. The detection limit obtained in this work is about 3 orders of magnitude better than that of conventional enzyme-linked immunosorbent assay (ELISA). In addition, the proposed method exhibited excellent precision, acceptable stability, and good reproducibility. Furthermore, the proposed immunoassay was successfully applied to AFP determination in real serum specimens. Therefore, the present immunoassay was demonstrated to be a powerful tool for ultrasensitive biomarker detection.