Terminal deoxynucleotidyl transferase (TdT) is a template-independent DNA polymerase that plays a critical role in immune system development and serves as an important biomarker for acute lymphoblastic leukemia. However, current methods for TdT activity analysis often rely on sophisticated instrumentation and lack simple and portable detection formats. Herein, we report a TdT-enabled multivalent CRISPR/Cas12a lateral flow assay for sensitive and instrument-free detection of TdT activity. In this strategy, TdT-catalyzed poly-adenine (poly-A) extension converts enzymatic activity into adenine-rich DNA scaffolds, which recruit multiple crRNA molecules to trigger multivalent activation of Cas12a. This design effectively bridges TdT activity with CRISPR/Cas12a signal amplification. The activated Cas12a subsequently induces trans-cleavage of a reporter probe, and the cleavage event is translated into a visual signal on a lateral flow strip. The proposed assay enables sensitive detection of TdT with a limit of detection of 0.016 U/mL and a visual detection limit of 0.05 U/mL. In addition, the assay exhibits high specificity toward TdT over other polymerases and demonstrates satisfactory performance in human serum samples with recoveries ranging from 98.8% to 103.7%. This work expands the applicability of CRISPR/Cas12a systems to enzyme activity sensing and provides a simple and practical platform for point-of-care detection of TdT.
