Fluorometric determination of the activity and inhibition of terminal deoxynucleotidyl transferase via in-situ formation of copper nanoclusters using enzymatically generated DNA as template
1.Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry & Chemical Engineering,Shaanxi Normal University,Xi’an,China;2.Department of Chemistry,Qinghai Normal University,Xining,China
Abstract:
The authors report on a simple strategy for sensitive determination of the activity of terminal deoxynucleotidyl transferase (TdT) using copper nanoclusters (CuNCs) as fluorescent probes. TdT-polymerized long chain AT-rich DNA serves as a template for the synthesis of the CuNCs, and TdT activity is detected fluorometrically at excitation/emission wavelengths of 340/570 nm. The protocol relies on the target-triggered formation of dsDNA polymers and in-situ formation of CuNCs. The calibration plot is linear in the 0.7 to 14 U L?1 activity range, with a 60 mU L?1 detection limit (at a signal-to-noise ratio of 3). The protocol was applied to determine TdT activity in acute lymphatic leukemia cells. This approach is selective, simple, convenient and cost-efficient because a complex DNA sequence is not required. In our perception, the method provides a viable new platform for monitoring the activity and inhibition of TdT.
Graphical abstract Based on the target-triggered formation of dsDNA polymers and in-situ formation of CuNCs with strong fluorescence, a turn-on fluorescence assay for TdT activity is presented.
