Polydopamine nanodots are viable probes for fluorometric determination of the activity of alkaline phosphatase via the in situ regulation of a redox reaction triggered by the enzyme.

Abstract

The authors describe an environmentally friendly and fast (~14min) method for the synthesis of homogeneously distributed fluorescent polydopamine nanodots (PDA-NDs) using KMnO4 as the oxidant. Alkaline phosphatase (ALP) catalyzes the hydrolysis of ascorbic acid 2-phosphate to release free ascorbic acid which undergoes an in-situ redox reaction with KMnO4. Depending on the activity of ALP, more or less KMnO4 is consumed, and this affects the formation of the PDA-NDs. Based on this finding, a sensitive method was worked out to quantify the activity of ALP via real-time formation of fluorescent PDA-NDs. The fluorometric signal (best measured at excitation/emission peaks of 390/500nm) is linear in the 1 to 50mU·mL-1 ALP activity range, and the limit of the detection is as low as 0.94mU·mL-1 (based on 3σ/m). The method was successfully applied to the determination of ALP activity in spiked human serum and in MCF-7 cell lysates. It was also applied in a method to screen for inhibitors of ALP. Graphical abstract Schematic of a fluorometric method for the determination of alkaline phosphatase (ALP) activity. The method is based on the in-situ regulation ofthe formation of fluorescent polydopamine nanodots (PDA-NDs) through the competition between the KMnO4-induced polymerization of dopamine and ALP-directed ascorbic acid 2-phosphate (Asc-2P) hydrolysis. AA: Ascorbic acid.