The simultaneous determination of multiple analytes has been an urgent demand in screening of antibiotic residues in food products of animal origin due to its higher analysis efficiency. Five aminoglycoside antibiotics (AGAs) have been monitored in milk, including gentamicin (GEN), kanamycin (KAN), neomycin (NEO), and streptomycin/dihydrostreptomycin (STR/ diSTR). A chemiluminescence microarray immunoassay (CLMIA) based on nitrocellulose membrane had been developed for the detection of multiple AGAs, which the LODs for STR, KAN, NEO, and GEN were 4.74 ng/mL, 4.97 ng/mL, 2.99 ng/ mL, and 4.42 ng/mL respectively. To improve the sensitivity of immunoassay, single-well carbon tubes (SWCNTs) were utilized as solid support for loading horseradish peroxidase-labelled goat anti-mouse antibody to obtain the multi-enzyme particles. After the optimization of usage of multi-enzyme particles and antibodies, the enhanced CLMIA was established and evaluated. The LODs were 1.25 ng/mL for STR, 0.64 ng/mL for KAN, 0.38 ng/mL for GEN, and 0.39 ng/mL for NEO, which was improved by threefold, sevenfold, 11-fold, and sevenfold compared with the conventional CLMIA developed. These methods presented higher specificity and repeatability. Finally, the enhanced CLMIA based on CNT-assisted multienzyme particles was utilized to analyze twenty-five milk samples from local market and dairy farm, which all the results were below the LOD. The enhanced CLMIA showed the great application potential for the detection of multiple targets simultaneously and provided efficient tool for the screening of pollutants in food.
Summary
Owing to the increasing incidences of antibiotic residues being found in foods of animal‐origin, rapid and reliable screening methods for detecting these residues are urgently required. This study aimed to develop a robust and ultrasensitive colorimetric immunoassay based on nanohybrids for the rapid screening of kanamycin (KAN) residues in milk. The HAuCl4 solution was directly reduced on the multiwalled carbon nanotubes (MWCNTs) using a one‐step synthesis method to obtain the Au/MWCNTs nanohybrids. The Au/MWCNTs exhibited mimetic peroxidase activity and higher affinities for the TMB and H2O2 compared with horseradish peroxidase and other nanohybrids. A direct competitive enzyme‐linked immunosorbent assay (dcELISA) based on Au/MWCNTs was developed and employed for KAN detection in milk samples under optimised conditions. The dcELISA showed high sensitivity toward KAN residues, with a detection limit of 11.2 pg/mL. Moreover, this novel immunoassay displayed high precision and accuracy in milk samples, with a recovery rate ranging from 94.3 to 124.5% and a coefficient variation from 7.8 to 11.3%. Eight real milk samples were found to contain a KAN concentration in the range of ND–0.26 ng/mL. Therefore, this dcELISA based on the Au/MWCNTs nanohybrid has promising potential for applications in food monitoring and quality control.
SummaryAminoguanidine (AG), a specific inhibitor of diamine oxidase (DAO), can be used to investigate the γ‐aminobutyric acid (GABA) accumulation pathways. The effect of AG on GABA accumulation pathways of tea fresh leaves remains to be explored. Our results indicated AG concentration did not significantly affect GAD activity, but high concentration solution made GABA‐T activity increase. AG (1 mm) maximally, but not entirely, blocked the polyamine degradation pathway, but had no effect on GAD and GABA‐T. The polyamine degradation pathway was responsible for 26.8% and 17.0% of GABA formation under hypoxic conditions involving immersion and immersion–vacuum, respectively, because DAO and PAO activity was not completely inhibited.
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