Amperometric Immunosensor for Determination of Clenbuterol Based on Enzyme-Antibody Coimmobilized ZrO2 Nano Probes as Signal Tag

Abstract

Abstract 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). While 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 can catalyze H2O2 into generate reduction current, was obtained. When CLB was detected, the samples and ZNPG were embedded to the bottom of the 96-well plate in advance. On the basis of 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, and then the GOD catalyze 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. Under the optimal conditions (pH 7.0, incubation time 30 min at 37 °C), the immunosensor provided an excellent response for CLB ranging from 0.003 to 100 μg L−1 with a detection limit of 1 ng L−1 (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 could greatly amplify the detection signal. The average recovery of the samples was 93.6%, the coefficient of intragroup variation was less than 2.5%, which confirmed that the amperometic immunosensor prepared had a high precision. The immunosensor was reusable, sensitive and rapid for the analysis of ultra trace amounts of CLB in food samples in real time.