Colorimetric determination of ascorbic acid and the activity of alkaline phosphatase based on the inhibition of the peroxidase-like activity of citric acid-capped Prussian Blue nanocubes

Colorimetric methods are described for the determination of ascorbic acid (AA) and alkaline phosphatase (ALP). Both assays are based on the inhibition of the peroxidase (POx)-like activity of Prussian Blue nanocubes (PB NCs) capped with citric acid. They catalyze the oxidation of 3,3,5,5-tetramethylbenzidine (TMB) by H2O2 to produce a blue color with an absorption maximum at 652nm. On addition of AA, the PB NCs are reduced to Prussian White (PW) which does not act as a POx mimic. This results in a decreased rate of the formation of the blue coloration whose intensity decreases with increasing concentration of AA. The assay allows AA to be quantified with a 35nM detection limit (at 3 sigma/m). The hydrolysis of AA phosphate by ALP leads to the formation of AA which can be quantified by the above method. Based on this, the activity of ALP can be determined by measurement of the intensity of the blue coloration thus formed. The method can be used to determine the activity of ALP with a detection limit as low as 0.23UL(-1).Schematic presentation of a method forcolorimetric determination of ALP activity. AA obtained by ALP-catalyzed hydrolysis of ascorbic acid phosphate (AAP) inhibits the intrinsic peroxidase-like activity of PB NCs by reducing Prussian Blue nanocrystals(PB NCs) to form inactive Prussian White (PW).