In situ assembly of porous Au-paper electrode and functionalization of magnetic silica nanoparticles with HRP via click chemistry for Microcystin-LR immunoassay

摘要

A simple, low-cost and sensitive origami electrochemical immunoassay-device was developed based on a novel gold nanoparticle modified porous paper working electrode (Au-PWE) for point-of-care testing. Azide-functionalized Au-PWE was prepared by the functionalization of Au-PWE with 1-azidoundecan-11-thiol. Alkyne end-terminated antibody was prepared with 4-pentynoic acid and antibody by the 1-ethyl-3-(3-(dimethylamino) propyl) carbodiimide hydrochloride and N-hydroxysuccinimide activation reaction. Alkyne-antibody was coupled to azido-Au-PWE by click reaction as a recognition element. Nearly monodispersed sphere-like silica-coated ferroferric oxide (Fe_3O_4@SiO_2) nanoparticles were prepared via the reverse microemulsion method. Azide-functionalized Fe_3O_4@SiO_2 was prepared by the functionalization of silica shell with 3-bromopropyltrichlorosilane followed by substitution with sodium azide. Alkyne-functionalized antibody and horse radish peroxidase were coupled to azide-functionalized Fe_3O_4@SiO_2 by click reaction as signal label. Horse radish peroxidase and ferroferric oxide could catalyze the oxidation of thionine in the presence of hydrogen peroxide. After the sandwich immunoreaction, the current was proportional to the logarithm of the Microcystin-LR. The linear regression equation was i(μA)=119.89+46.27logc_(MC-LR) (μg/mL) in the range from 0.01 to 200μg/mL. The limit of detection was 0.004μg/mL. This immunoassay would provide a universal immunoassay method in environmental monitoring and public health.