Label-free DNA hairpin probe for real-time monitoring of alkaline phosphatase activity

Abstract

We have developed a new methodology for the label-free detection of alkaline phosphatase (ALP) activity based on a DNA hairpin and thioflavin T (ThT) probes. In the presence of ALP, the 3′-phosphate of the primer DNA is dephosphorylated, triggering a primer extension reaction to open the hairpin probe and leading to release of the G-quartets. These then bind to ThT to form ThT/G-quadruplexes with obvious fluorescence generation. The method is highly sensitive, and the ALP detection limit was estimated to be 0.001 U mL⁻¹, which is superior or comparable to those of reported assays. The assay avoids complicated modifications or chemical labeling, making it simple and cost-effective. Furthermore, the inhibition of ALP activity by the inhibitor theophylline is demonstrated, indicating the potential for the discovery and characterization of ALP-targeted drug candidates in pharmaceutical development.