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

• A rapid non-competitive immunoassay was developed for organophosphorus pesticide (OPs) detection. • The biotinylated anti-immunocomplex peptide SIC2 fusion protein replaced the phage borne peptide. • A biotinylated fusion protein-streptavidin-peroxidase signal amplification module was prepared. • Immunomagnetic beads (IMBs) as solid-phase carriers significantly reducing incubation time. • This proposed method demonstrated high sensitivity, efficiency and satisfactory recovery rates. 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 employs 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 receptor domain (BAD) genes. These fusion constructs were expressed in E. 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 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).