Gold nanocluster-based ratiometric fluorescent probes for hydrogen peroxide and enzymatic sensing of uric acid

A method is described for ratiometric fluorometric assays of H₂O₂ by using two probes that have distinct response profiles. Under the catalytic action of ferrous ion, the 615 nm emission of protein-stabilized gold nanoclusters (under 365 nm photoexcitation) is quenched by H₂O₂, while an increased signal is generated with a peak at 450 nm by oxidizing coumarin with the H₂O₂/Fe(II) system to form a blue emitting fluorophore. These decrease/increase responses give a ratiometric signal. The ratio of the fluorescences at the two peaks are linearly related to the concentration of H₂O₂ in the range from 0.05 to 10 μM, with a 7.7 nM limit of detection. The detection scheme was further coupled to the urate oxidase catalyzed oxidation of uric acid which proceeds under the formation of H₂O₂. This method provides an simple and effective means for the construction of ratiometric fluorometric (enzymatic) assays that involve the detection of H₂O₂. Graphical abstract Under catalysis by ferrous ion, hydrogen peroxide quenches the luminescence of gold nanoclusters (AuNCs) and oxidizes coumarin into a fluorescent derivative, which rendered fluorescence ON and OFF at two distinct wavelengths for ratiometric measurements.