Determination of the activity of alkaline phosphatase by using nanoclusters composed of flower-like cobalt oxyhydroxide and copper nanoclusters as fluorescent probes.

The authors describe a sensitive fluorometric method for the determination of the activity of alkaline phosphatase (ALP). It is based on the use of a composite prepared consisting of flower-like cobalt oxyhydroxide (CoOOH) and copper nanoclusters (CuNCs). On formation of the CuNC-CoOOH aggregates, the fluorescence of the CuNCs is quenched by the CoOOH sheets. If, however, the CoOOH sheets are reduced to Co(II) ions in the presence of ascorbic acid (AA), fluorescence recovers. AA is formed in-situ by hydrolysis of the substrate ascorbic acid 2-phosphate (AA2P) as catalyzed by ALP. Thus, the ALP activity can be detected indirectly by kinetic monitoring of the increase in fluorescence, best at excitation/emission wavelengths of 335/410 nm. The assay allows ALP to be determined in 0.5 to 150 mU·mL-1 activity range and with a 0.1 mU·mL-1 detection limit. The method was successfully applied to the determination of ALP activity in (spiked) human serum samples. The assay has attractive features in being of the off-on type and immune against false positive results. Graphical Abstract A fluorescent bioassay is reported for the determination of the activity of alkaline phosphatase (ALP). It is exploiting the ascorbic acid (AA)-induced decomposition of nanoclusters composed of flower-like cobalt oxyhydroxide and copper nanoclusters. ALP catalyzes hydrolysis of ascorbic acid 2-phosphate (AA2P) and dephosphorylation to form AA.