Amperometric Immunosensor for Clenbuterol Based on Enzyme-Antibody Coimmobilized ZrO_2 Nano Probes as Signal Tag
A novel amperometic immunosensor for clenbuterol(CLB) detection based on enzyme-antibody co-immobilized zirconium dioxide(ZrO2) nanoprobes for signal amplification was developed.First,ZrO2 nanoparticles(ZNPs) were used to adsorb glucose oxidase(GOD) enzyme and CLB antibody(anti-CLB) to prepare the signal tag(ZNPs/GOD-anti CLB).Poly(diallyldimethylammonium chloride)(PDDA),hemin and Au nanoparticles(AuNPs) were assembled layer by layer on the surface of multi-walled carbon nanotubes(MWCNTs),which was modified by screen-printed electrodes(SPCE).By this means,the sensor(SPCE │ WCNTs/PDDA/hemin/AuNPs),which could catalyze H2O2 into generate reduction current,was obtained.When CLB was detected,samples and ZNPG were embedded to the bottom of the 96-well plate in advance.Based on the reaction mechanism of competitive enzyme-linked immunoassay,CLB in samples would compete with slab CLB for the combination with the nanoprobe ZNPG.After reaction,glucose(Glu) was added to the 96-well plate,then the GOD catalyzed Glu to generate H2O2,which could be detected by the immunosensor for quantification.Current decline in value(ΔI) was inversely proportional to a certain concentration range of CLB and under the optimal conditions(pH 7.0,incubation at 37 ℃ for 30 min),the immunosensor provided an excellent response for CLB ranging from 0.003 μg/L to 100 μg/L with a detection limit of 1 ng/L(3σ).The sensitivity of this method was 2 orders of magnitude higher than that of the ELISA method due to the high density of GOD on the signal tag,which greatly amplify the detection signal.The average recovery of the standard addition was 93.6% and the relative standard deviation was less than 2.5%,which confirmed that prepared had a high precision.The results showed that the amperometic immunosensor was reusable,sensitive and rapid for on-the site determination of ultra trace amounts of CLB in food samples.