RESUMEN
In situ enzymatic generation of bimetallic nanoparticles, mainly Au/Pt, overcomes the drawbacks (continuous absorbance drift, modest LOQ, and long-time reaction) observed when AuNP alone are produced. In this study, Au/Pt nanoparticles have been characterized by EDS, XPS, and HRTEM images using the enzymatic determination of tyramine with tyramine oxidase (TAO) as a model. Under experimental conditions, the Au/Pt NPs show an absorption maximum at 580 nm which can be related to the concentration of tyramine in the range 1.0 × 10-6M to 2.5 × 10-4M with a RSD of 3.4% (n = 5, using 5 × 10-6M tyramine). The Au/Pt system enables low LOQ (1.0 × 10-6 M), high reduction of the absorbance drift, and a significant shortening of the reaction time (i.e., from 30 to 2 min for a [tyramine] = 1 × 10-4M); additionally, a better selectivity is also obtained. The method has been applied to tyramine determination in cured cheese and no significant differences were obtained compared to a reference method (HRP:TMB). The effect of Pt(II) seems to involve the previous reduction of Au(III) to Au(I) and NP generation from this oxidation state. Finally, a three-step (nucleation-growth-aggregation) kinetic model for the generation of NPs is proposed; this has enabled us to obtain a mathematical equation which explains the experimentally observed variation of the absorbance with time.