An Endo IV-driven signal amplifier for sensitive detection of cancer biomarker TdT

Abstract

Terminal deoxynucleotidyl transferase (TdT) is a template-independent DNA polymerase and its function is mainly involved in the development of the human immune system. The activity of TdT is normally suppressed in healthy human cells but is overexpressed in certain diseases, such as leukemia and other malignant blood disorders. Therefore, the detection of TdT activity is crucial for clinical diagnosis and disease progression monitoring. In this study, we developed an Endo IV-driven signal amplifier to assess TdT activity. TdT can recognize primers and add dATP to the 3′ hydroxyl end of the primers, forming a polyA tail. Fluorescent probes (polyT) containing AP sites can bind to the polyA tail and the AP site will be hydrolyzed by Endo IV. Then the fluorescent probe is cut and emits fluorescence. Subsequently, the intact fluorescent probe rebinds to the polyA tail, creating a cyclic process for signal amplification. The detection limit for TdT was 1 × 10⁻⁴ U μL⁻¹, with a linear range of 1 × 10⁻⁴ to 1 × 10⁻¹ U μL⁻¹. This biosensor expands the current TdT detection toolkit. Its excellent specificity and sensitivity in various kinds of biological environments provide a powerful detection tool for clinical TdT testing.