A versatile assay for alkaline phosphatase detection based on thymine-HgII-thymine structure generation mediated by TdT.

Alkaline phosphatase (ALP) is a vital hydrolysis enzyme in phosphate metabolism, which catalyzes the hydrolysis of phosphate ester groups in proteins, nucleic acids, and other small molecules. Meanwhile, abnormal ALP expression is associated with occurrence and development of many diseases. Terminal deoxynucleotidyl transferase (TdT) is a widely used tool enzyme in many fields, which randomly adds deoxyribonucleoside triphosphates (dNTPs) at the 3'-OH termini of ssDNA in a template-free manner. In this work, we designed a versatile, convenient, label-free, and highly sensitive fluorescence enhancing assay for ALP activity detection based on the characteristics of ALP, TdT, and thymine-Hg^II-thymine (T-Hg²⁺-T) structure. In the presence of ALP, the 3'-phosphoryl end of the ssDNA-p was hydrolyzed to hydroxyl group, followed by addition of a poly-T tail on its 3' terminal hydroxyl in the mixing solution containing both TdT and dTTPs. Then, the DNA with poly-T tail could interact with Hg²⁺ to form the stable T-Hg²⁺-T mediated metallo DNA duplex, which enhanced the fluorescence intensity of the SG. Under optimal conditions, the proposed system was employed for quantitatively monitoring ALP activity with a dynamic range of 0-2500 mU mL^-1, and the actual detection limit could be down to 0.025 mU mL^-1. And the determination of ALP activity in human serum samples and MCF-7 cells lysates exhibited a good sensing performance, demonstrating its potential applications in biochemical research and clinical diagnosis. Meanwhile, this system could also be applied to both TdT and Hg²⁺ detection.