A sensitive fluorescent sensor for quantification of alpha-fetoprotein based on immunosorbent assay and click chemistry

A novel fluoresencent immunosensor for determination of cancer biomarkers such as alpha-fetoprotein (AFP) was designed by utilizing both the high specificity of antigen-antibody sandwich structure and the high sensitivity of the click chemistry based fluorescence detection. Instead of an enzyme or fluorophore, the CuO nanoparticles are labeled on the detection antibody, which was not susceptible to the change of the external environments. The CuO nanoparticles which were modified on the sandwich structure can be dissolved to produce Cu2+ ions with the help of HCl and then the Cu2+ ions were reduced by sodium ascorbate to produce Cu+ ions which triggered the Cu+ catalyzed alkyne-azide cycloaddition (CuAAC) reaction between the weak fluorescent compound (3-azido-7-hydroxycoumarin) and propargyl alcohol to form a strong fluorescent compound. A good linear relationship was observed between the fluorescence increase factor of the system and the concentration of AFP in the range of 0.025-5.0 ng/mL with a detection limit of 12 pg/mL (S/N=3). The proposed fluorescent sensor had been applied to detect AFP in the human serum samples and gave satisfactory results.