Thioglucose-stabilized gold nanoparticles as a novel platform for colorimetric bioassay based on nanoparticle aggregation

Gold nanoparticles stabilized with thioglucose (TGlu-AuNPs), which have carboxyl groups on the particle surface as anchoring sites for covalent immobilization of biomolecules, were prepared by the chemical reduction of HAuCl4 using 1-thio-β-d-glucose as a reducing and stabilizing agent, and their application to colorimetric bioassay was demonstrated using the carbohydrate–lectin system. p-Aminophenyl α-d-mannose (Man-NH2) was covalently attached by a conventional method to the activated carboxyl groups on the TGlu-AuNPs. On addition of Con A to the Man-AuNPs, multiple binding events occurred between Con A and the mannoses immobilized on the particle surface. This Con A-induced aggregation resulted in a significant red shift in local surface plasmon resonance. The binding isotherm showed a sigmoidal curve, indicating cooperativity in the binding of Con A and the Man-AuNPs. In addition, Hill plots showed two nonequivalent binding modes, with the Kd values for high- and low-affinity binding of 11.3 and 66.5 pM, respectively, which was significantly lower than that for methyl-α-d-mannose binding to Con A. The enhanced binding affinity between Man-AuNPs and Con A involves the cluster effect of the carbohydrate groups on the AuNPs. A linear correlation curve was obtained in the range 10–100 nM (R2 = 0.983). The limit of detection (LOD) for Con A was 9.0 nM in aqueous buffer, which is comparable to that of other conventional methods such as ELISA.