Selective generation of gold nanostructures mediated by flavo-enzymes to develop optical biosensors

This paper explores, for the first time, the use of flavo-enzymes for the enzymatic generation of gold nanomaterials. It has been demonstrated that when the oxidation of glucose by GOx is carried out in the presence of Au(III), the in-situ formation of gold nanomaterials is observed. Moreover, depending on the experimental conditions, either nanoparticles (AuNPs) or nanoclusters (AuNCs) are better observed, whose spectroscopical properties can be related to the concentration of glucose. Working at pH 6, only AuNCs with fluorescence at 420 nm (λ_ex=335 nm) are obtained (linear relationship from 6.0·10⁻⁵ M to 1.5·10⁻³ M glucose). However, when the enzymatic reaction is performed at pH 8, AuNPs (λ_max=580 nm) are also obtained (linear relationship from 5.5·10⁻⁴ M to 2.0·10⁻³ M glucose). Mathematical equations describing the variation of fluorescence and absorbance values during the reaction have been proposed. The results obtained suggest that AuNCs are formed using GOx as nucleation seeds. Since AuNPs belong to the branched-type, it is suggested that they are obtained by AuNC coalescence. From these models the AuNP molar absorptivity per atom was obtained (2.0(±0.3)·10³ M⁻¹cm⁻¹). Finally, the method has been applied to the determination of glucose in orange juice and human plasma samples. Comparing the results with the GOx/HRP/TMB method and a commercial glucometer, no significant differences (P=0.05) are obtained.