Determination of the activity of alkaline phosphatase based on the use of ssDNA-templated fluorescent silver nanoclusters and on enzyme-triggered silver reduction

Abstract

The authors describe a fluorometric method for the determination of the activity of the enzyme alkaline phosphatase (ALP). It is based on the finding that the formation of ssDNA-templated fluorescent silver nanoclusters (AgNCs) is inhibited by enzyme-assisted silver reduction. On addition of ALP, ascorbic acid 2-phosphate (AAP) is hydrolyzed to form ascorbic acid (AA). The generated AA reduces silver ion to form metallic silver which hampers the formation of fluorescent silver nanoclusters, this leading to low fluorescence. Conversely, in the absence of ALP, AAP cannot reduce Ag(I). As a result, fluorescent AgNCs are being formed. Fluorescence intensity correlates linearly in the 1–100 U·L−1 ALP concentration range, with a detection limit of 0.63 U·L−1. The method was applied to the determination of ALP in spiked fetal calf serum samples. It is cost-effective and convenient, does not require labels, or probes, or complicated operations.