Fluorometric determination of ascorbic acid by exploiting its deactivating effect on the oxidase–mimetic properties of cobalt oxyhydroxide nanosheets

Nanosheets of cobalt oxyhydroxide (CoOOH) are found to exhibit oxidase–like activity and can catalyze the oxidation of the substrate 3,3′,5,5′–tetramethylbenzidine (TMB) by oxygen in weakly acidic solution even in the absence of hydrogen peroxide. On the basis of this property, a fluorometric assay was designed in which the blue oxidation product of TMB (oxTMB) reduces the intensity of the fluorescence of albumin–stabilized gold nanoclusters (BSA–AuNCs). The fluorescence of the AuNCs under 502 nm photoexcitation has a peak at 620 nm which overlaps the absorption band of oxTMB. As a result, fluorescence is reduced. The findings were used to develop a method for the fluorometric quantitation of ascorbic acid. Ascorbic acid is capable of destroying some of the CoOOH nanosheets, and hence catalytic oxidation is retarded and fluorescence will be less strongly reduced (i.e., reabsorbed). Fluorescence intensity increases in the 0.75 to 20 μM ascorbic acid concentration range, and the detection limit is 0.2 μM. The method is well reproducible (the relative standard deviation of 1.6% for 11 replicates at a 10 μM ascorbic acid level). The practicability of the method was demonstrated by the analysis of ascorbic acid in tablets and juices. Graphical abstract Schematic of a fluorometric assay for ascorbic acid (AA). It is based on the finding that AA retards the cobalt oxyhydroxide nanosheet-catalyzed oxidation of tetramethylbenzidine to form a colored product. This reduces the inner filter effect caused by the blue oxidation product on the red fluorescence of bovine serum albumin–stabilized gold nanoclusters.