Biotinylated fusion protein-streptavidin-peroxidase complex based non-competitive magnetic immunoassay for rapid detection of organophosphorus pesticides

Phage-displayed anti-immunocomplex peptide-based non-competitive immunoassays have shown great potential for detecting organophosphorus pesticide (OPs) residues. However, the phage-borne peptides raise safety concerns and require secondary reagents, which limit their commercial application. To address these challenges, a new approach has been developed. This study employed the SIC2 peptide, which recognized the immune complex of single-chain variable fragment 5 (scFv5) and OPs, to reveal its potential for detecting OPs residues. The SIC2 peptide was fused with glutathione S-transferase (GST) and biotin acceptor domain (BAD) genes. These fusion constructs were expressed in Escherichia coli BL21 (DE3) and then biotinylated. The biotinylated fusion protein bound to streptavidin-horseradish peroxidase (SA-HRP) conjugates through the biotin-streptavidin system, enhancing the detection signal via the catalytic oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB). Based on this, and using immunomagnetic beads, a rapid non-competitive magnetic immunoassay (NCMIA) was developed for detecting OPs. The results showed that under optimal conditions, the limits of detection (LODs) for 12 OPs ranging from 1.04 to 13.18 ng mL^-1, and the incubation time of this assay needed 11 min. The intra- and inter-batch recovery rates of 3 OPs in cucumber, cabbage and lettuce ranged from 79.91 % to 103.72 %, with coefficients of variation ranging from 0.26 % to 14.98 %. In addition, the recoveries of parathion determined by NCMIA were consistent with the results of parallel analyses using gas chromatography tandem mass spectrometry (GC–MS/MS).