A label-free ThT-assisted fluorescence detection strategy of alkaline phosphatase activity based on MnO2 nanosheets

Alkaline phosphatase (ALP) is a metalloenzyme, the level of which is clinically significant as an abnormality of ALP activity results in several diseases. In the present study, we introduced a MnO2 nanosheet-based assay for ALP detection employing the adsorption and reduction characteristics of G-rich DNA probes and ascorbic acid (AA), respectively. Ascorbic acid 2-phosphate (AAP) was utilized to act as a substrate for ALP which hydrolyzes AAP generating AA. In the absence of ALP, MnO2 nanosheets adsorb the DNA probe destructing the G-quadruplex formation and showing no fluorescence emission. On the contrary, being present in the reaction mixture ALP hydrolyzes AAP yielding AA, then the AA reduce the MnO2 nanosheets into Mn2+, hence, the probe is free to react with a dye, thioflavin T (ThT), and synthesizes ThT/G-quadruplex to spark high fluorescence intensity. Therefore, under optimized conditions (250 nM DNA probe, 8 mu M ThT, 96 mu g/mL MnO2 nanosheets, and 1 mM AAP) the sensitive and selective measurement of ALP activity can be achieved through the change of fluorescence intensity, with a linear range and a limit of detection of 0.1-5 U/L and 0.045 U/L. Our assay exhibited its potential to assess the ALP inhibitor when in an inhibition assay Na3VO4 inhibited ALP with an IC50 value of 0.137 mM and also was validated in clinical