A competitive sensitive bio-barcode immunoassay based on bimetallic nanozyme (Au@Pt: gold@platinum) catalysis has been designed for the detection of the pesticide parathion. Gold nanoparticles (AuNPs) were modified with single-stranded thiol oligonucleotides (ssDNAs) and monoclonal antibodies (mAbs) to form AuNP probes; magnetic nanoparticles (MNPs) were coated with ovalbumin (OVA)-parathion haptens as MNP probes, and bimetallic nanozyme (Au@Pt) nanoparticles functionalized with the complementary thiolated ssDNA were used as Au@Pt probes. The Au@Pt probes reacted with the AuNP probes through complementary base pairing. Further, parathion competed with MNP probes to bind the mAbs on the AuNP probes. Finally, the complex system was separated by a magnetic field. The released Au@Pt probes catalyzed a chromogenic system consisting of teramethylbenzidine (TMB). The bimetallic nanozyme-based bio-barcode immunoassay was performed on rice, pear, apple, and cabbage samples to verify the feasibility of the method. The immunoassay exhibited a linear response from 0.01 to 40 μg·kg-1, and the limit of detection (LOD) was 2.13 × 10-3 μg·kg-1. The recoveries and relative standard deviations (RSDs) ranged from 73.12 to 116.29% and 5.59 to 10.87%, respectively. The method was found to correlate well with data obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In conclusion, this method exhibits potential as a sensitive alternative method for the detection of a variety of pesticides, ensuring the safety of fruits and vegetables in agriculture.
Keywords: Au@Pt; AuNPs; bimetallic nanozyme; bio-barcode; immunoassay; parathion.