Sensitive detection of alkaline phosphatase based on terminal deoxynucleotidyl transferase and endonuclease IV-assisted exponential signal amplification.

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.

SUBMITTER: Ye W 

PROVIDER: S-EPMC9463482 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature