Alkaline phosphatase(ALP)is widely expressed in human tissues.ALP plays an important role in the dephosphorylation of proteins and nucleic acids.Therefore,quantitative analysis of ALP plays a vital role in disease diagnosis and the development of biological detection methods.Terminal deoxynucleotidyl transferase(TdT)catalyzes continuous polymerization of deoxynucleotide triphosphates at the 3'-OH end of single-stranded DNA in the absence of a template.In this study,we developed a highly sensitive and selective method based on TdT and endonuclease Ⅳ(Endo Ⅳ)to quantify ALP activity.After ALP hydrolyzes the 3'-PO4 end of the substrate and generates 3'-OH,TdT can effectively elongate the 3'-OH end with deoxynucleotide adenine triphosphate(dATP)and produce a poly A tail,which can be detected by the poly T probes.Endo Ⅳ digests the AP site in poly T probes to generate a fluorescent signal and a new 3'-OH end,leading to the generation of exponential fluorescence signal amplification.The substrate for TdT elongation was optimized,and a limit of detection of 4.3×10-3 U/L was achieved for ALP by the optimized substrate structure.This method can also detect ALP in the cell lysate of a single cell.This work has potential applications in disease diagnosis and biomedical detection.

Weicong Ye;Longjie Li;Zishan Feng;Bocheng Tu;Zhe Hu;Xianjin Xiao;Tongbo Wu

Department of Pharmaceutical Analysis,School of Pharmacy,Tongji Medical College,Huazhong University of Science and Technology,Wuhan,430030,China;Department of Biopharmaceutics,School of Life Science and Technology,Wuhan Polytechnic University,Wuhan,430023,China;Department of Translational Medicine,Institute of Reproductive Health,Tongji Medical College,Huazhong University of Science and Technology,Wuhan,430030,China

药物分析学报（英文）

[1]Weicong Ye;Longjie Li;Zishan Feng;Bocheng Tu;Zhe Hu;Xianjin Xiao;Tongbo Wu-.Sensitive detection of alkaline phosphatase based on terminal deoxynucleotidyl transferase and endonuclease Ⅳ-assisted exponential signal amplification)[J].药物分析学报（英文）,2022(04):692-697

deoxynucleotide,triphosphates,hydrolyzes,dATP

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