Sensitive detection of alkaline phosphatase based on terminal deoxynucleotidyl transferase and endonuclease Ⅳ-assisted exponential signal amplification
- Author:
Ye WEICONG
1
;
Li LONGJIE
;
Feng ZISHAN
;
Tu BOCHENG
;
Hu ZHE
;
Xiao XIANJIN
;
Wu TONGBO
Author Information
1. Department of Pharmaceutical Analysis,School of Pharmacy,Tongji Medical College,Huazhong University of Science and Technology,Wuhan,430030,China
- Keywords:
Alkaline phosphatase;
Terminal deoxynucleotidyl transferase;
Endonuclease Ⅳ;
Exponential amplification
- From:
Journal of Pharmaceutical Analysis
2022;12(4):692-697
- CountryChina
- Language:Chinese
-
Abstract:
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.
