Glycosylated protein-functionalized gold nanoparticle-based detection of heat-labile enterotoxin from complex samples

Abstract

Enterotoxigenic Escherichia coli O78:H11 is a food-borne pathogen that can cause “traveler’s diarrhea.” The pathogenicity of E. coli O78:H11 is attributed to the expression of enterotoxin, particularly heat-labile enterotoxin (LT). LT is a pentameric protein composed of one A and five B subunits. The B subunit of LT (LT-B) contains the binding moieties toward Galβ(1→4)Glc. Chicken ovalbumin (COA) is a glycoprotein composed of Galβ(1→4)Glc termini and abundant (∼54%) in chicken egg white (CEW) proteins. COA decorated with ∼17 additional galactosides, obtained by reacting CEW proteins with lactose (Galβ[1→4]Glc) through the Maillard reaction, was used as a probe molecule against LT-B. The generated CEW derivatives, mainly dominated by glycated COA, were immobilized on gold nanoparticles (Au@Lac-CEW NPs) by reacting with aqueous tetrachloroaurate through one-pot reactions. Au@Lac-CEW NPs had a good binding affinity toward LT-B with a dissociation constant of ∼1.86 × 10−7 M. A colorimetric method by using Au@Lac-CEW NPs as sensing probes against LT-B (≥31 nM) that can be visualized was demonstrated. The limit of detection against LT-B was reduced to ∼4 nM as revealed by matrix-assisted laser desorption/ionization mass spectrometry as a detection tool.