Engineering a cascaded isothermal signal amplification network using dimeric-palindromic primers for label-free...

Terminal deoxynucleotidyl transferase (TdT), a unique DNA polymerase essential for the human immune system, is highly active in leukemic cells, making it a key biomarker for leukemia. However, existing TdT detection methods are often complex, costly, and radioactive. This study presents a novel, label-free approach for detecting TdT activity using a cascaded isothermal signal amplification network with high efficiency. The method employs a single palindromic primer that self-dimerizes into a dimeric palindromic structure, subsequently extended by TdT to form poly-A chains. These chains undergo multisite polymerization and strand displacement, producing polymerization products converted into double-stranded DNA duplexes via palindrome-based reverse reading. Incorporating SYBR Green I dye, which binds specifically to double-stranded DNA, enables sensitive detection of TdT activity. This approach achieves high sensitivity and specificity, with a detection limit of 0.008 U/mL and a wide linear range of 0.008-50 U/mL. Practical applications were demonstrated with high recovery rates and low variability using mimic real biological samples, revealing the system's potential for biomedical use. The simplicity, robust signal amplification, and reliable assay performance of this method not only advance molecular diagnostic technologies but also offer a valuable new tool for TdT detection in both research and clinical settings.