Nanobody-Induced Aggregation of Gold Nanoparticles:\nA Mix-and-Read Strategy for the Rapid Detection of <i>Cronobacter\nsakazakii</i>

Protein-nanoparticle interactions\nplay a crucial role in both biomedical\napplications and the biosafety assessment of nanomaterials. Here,\nwe found that nanobodies can induce citrate-capped gold nanoparticles\n(AuNPs) to aggregate into large clusters. Subsequently, we explored\nthe mechanism behind this aggregation and proposed the “gold\nnucleation mechanism” to explain this phenomenon. Building\non this observation, we developed a one-step label-free colorimetric\nmethod based on nanobody-induced AuNP aggregation. When nanobodies\nbind to target bacteria, spatial hindrance occurs, preventing further\nAuNPs aggregation. This alteration in surface plasmon resonance properties\nresults in visible color changes. As an example, we present a simple\nand sensitive “mix-and-read” chromogenic immunosensor\nfor <i>Cronobacter sakazakii</i> (<i>C. sakazakii</i>). The experiment can be completed within 20 min, with a visual detection\nlimit of 10<sup>3</sup> CFU/mL and a quantitative detection limit\nof 136 CFU/mL. Importantly, our method exhibits no cross-reactivity\nwith other bacterial species. This strategy harnesses the excellent\nproperties of nanobodies and the optical characteristics of AuNPs\nfor direct and rapid detection of foodborne pathogen.