Facile and sensitive fluorescence sensing of alkaline phosphatase activity using NMM/G-quadruplex

Herein, we are presenting an approach that utilizes the λ exonuclease (λ exo) cleavage reaction in combination with the formation of G-quadruplex, thereby providing a label-free fluorometric tool for simply and accurately determining alkaline phosphatase (ALP) activity and inhibition. A hairpin probe (HP) with 5'-phosphoryl termini and 3'-end containing a G-rich region, is designed. Taking advantage of the efficient enzyme reactions, namely the λ exo cleavage reaction, the G-rich DNA fragment is released from HP and folds into a stable G-quadruplex in the presence of potassium ions, thus greatly enhance the fluorescence of N-methyl mesoporphyrin IX (NMM) (a specific G-quadruplex binder). However, in the presence of ALP, the 5'-phosphoryl of the HP is dephosphorylated. The yielding 5'-hydroxyl end product hampers the λ exo cleavage reaction. HP maintains its stem-loop structure. Thus the formation of the G-quadruplex is prohibited, and this results in weak fluorescence of NMM. The fluorescence intensity exhibits a linear correlation to ALP concentration in the range of 1-50 U/L with a detection limit of 0.75 U/L. Additionally, inhibition effect of sodium orthovanadate has also been investigated. This study offers a simple yet sensitive method for ALP activity assay.