Nanozyme-amplified lateral flow nanobody-immunoassay via PEI-mediated coupling strategy for sensitive detection of 3-phenoxybenzoic acid

The integration of nanobody and nanozyme has emerged as a transformative strategy in immunoassays, offering enhanced sensitivity, stability, and versatility compared to conventional antibody-enzyme conjugates. Herein, we successfully developed a ternary nanostructure by integrating silica nanoparticles (SNPs), polydopamine (PDA), and platinum nanoparticles (Pt NPs), yielding SNPs@PDA@Pt (SPP) with robust nanozyme catalytic activity. Notably, the SPP was surface-modified with polyethyleneimine (PEI) to generate positively charged PEI-modified SNPs@PDA@Pt (PSPP). This modification substantially improved the coupling efficiency with anti-3-phenoxybenzoic acid nanobodies (3-PBA Nbs) via electrostatic interactions. The PSPP-based coupling achieved a remarkable efficiency of 92.28 %, representing about a 4.5-fold increase over the SPP method (20.55 %). The PSPP@3-PBA Nbs nanocomposite was successfully integrated into both ELISA and LFIA platforms, achieving remarkable detection limits (0.001 ng/mL for ELISA and 0.003 ng/mL for LFIA). The nanocomposite also showed excellent stability, with no significant loss of immunobinding capacity or catalytic activity over 30 days. This work presents a novel design strategy for constructing high-performance nanozyme-nanobody nanocomposites, which shows great promise for ultra-sensitive 3-PBA detection in resource-limited settings.