Label-free fluorescence detection of alkaline phosphatase activity using a G-triplex based dumbbell-shaped probe

Based on the fluorescence enhancement property of the G-triplex (G3)-Thioflavin T (ThT) complex, a fluorescent biosensor was successfully constructed for detection of ALP using a G3-based dumbbell-shaped probe (DP). In this work, calf intestinal ALP (CIP) can act on the 5'-terminal phosphate of DP, thereby regulating the subsequent DNA ligation reaction and enzyme cleavage of the DP nick. When the DP is digested by exonuclease, the released G3 can bind to ThT, resulting in enhanced fluorescence signal. The linear range of the sensor for CIP detection is 0.00002-0.002 U/μL, and the detection limit is 1.8 × 10^-5 U/μL. The proposed method has the advantages of simplicity, no fluorophore labeling, and low cost, which was successfully applied to the screening of enzyme inhibitors and ALP determination in human serum samples. To the best of our knowledge, this is the first report of a biosensor using G3-ThT as the signal indicator for ALP detection, which should promote the further exploitation of applying G3-ThT complex in the field of various targets recognition and analysis.