Sensitive Fluorescence Assay for the Detection of Alkaline Phosphatase Based on a Cu-Thiamine System.

The authors describe a novel, facile, and sensitive fluorometric strategy based on a Cu-thiamine (Cu-TH) system for the detection of alkaline phosphatase (ALP) activity and inhibition. The principle of the method is as follows. Under a basic conditions, TH, which does not exhibit a fluorescence signal, is oxidized into fluorescent thiochrome (TC) by Cu. Ascorbic acid 2-phosphate (AAP), which is the enzyme substrate, is hydrolyzed to produce ascorbic acid (AA) by ALP. The newly formed AA then reduces Cu to Cu, which prevents the oxidation of TH by Cu; as a result, the fluorescent signal becomes weaker. On the contrary, in the absence of ALP, AAP cannot reduce Cu; additions of Cu and TH result in a dramatic increase of the fluorescent signal. The sensing strategy displays brilliant sensitivity with a detection limit of 0.08 U/L, and the detection is linear in the concentration range of 0.1 to 100 U/L. This approach was successfully applied to ALP activity in human serum samples, indicating that it is reliable and may be applied to the clinical diagnosis of ALP-related diseases.