A CbAgo-enabled fluorescence assay for sensitive detection of terminal deoxynucleotidyl transferase

Terminal deoxynucleotidyl transferase (TdT) has been recognized as an important biomarker for leukemia diagnosis and prognosis, as its abnormal overexpression is closely associated with malignant lymphoid proliferation and genomic instability. Accurate and sensitive detection of TdT activity is therefore of great significance for understanding polymerase-associated biological processes and supporting analytical and therapeutic research. However, conventional TdT detection methods often involve complex multi-step operations, long incubation times, or amplification reactions, which limit their efficiency and practicality. In this work, a one-pot fluorescence assay integrating TdT-mediated elongation with the programmable endonuclease Clostridium butyricum Argonaute (CbAgo) was developed for rapid, sensitive, and specific TdT activity analysis. In this coupled enzymatic cascade, TdT extends the 5'-phosphorylated A10 primer to generate poly(A) sequences, which hybridize with a fluorophore-quencher reporter to trigger CbAgo-catalyzed cleavage and produce a strong fluorescence signal. Benefiting from the DNA-guided, PAM-independent, and highly stable nature of CbAgo, the proposed system enables homogeneous detection with improved robustness compared with CRISPR-based methods. The assay achieves a detection limit of 6.6 × 10⁻⁴ U/µL and a linear range from 0.00002 to 0.01 U/µL, showing excellent selectivity, reproducibility, and satisfactory performance in complex biological matrices and cell lysates. This study demonstrates a simple and versatile fluorescence platform for efficient TdT activity quantification with high stability and operational simplicity.