A terminal extension-actuated isothermal exponential amplification strategy toward the ultrasensitive and versatile detection of enzyme activity in a single cell

Abstract

Terminal deoxynucleotidyl transferase (TdT) plays an important role in regulating a wide range of genomic processes. The sensitive and accurate detection of cellular TdT activity, particularly at the single-cell level, is highly significant for leukemia-associated biomedical and biological studies. Nevertheless, owing to the limited sensitivity of the existing TdT assays, the quantification of TdT activity at the single-cell level remains a big challenge. Herein, a simple but ultrasensitive method for assaying TdT activity is proposed based on terminal extension actuated loop-mediated isothermal amplification (TEA-LAMP). By using the TdT-induced extension product as an actuator, TdT activity is amplified twice by terminal extension and LAMP in an exponential manner and finally converted to a remarkably amplified fluorescent signal. In this study, as low as 2 × 10−8 U/μL TdT can be clearly detectable with the elegant TEA-LAMP strategy. Such an ultrahigh sensitivity enables the direct determination of TdT activity in individual single cells. In the meantime, by employing TdT as a co-factor, this strategy can also be applied to detecting other enzymes that can catalyze the DNA terminal hydroxylation. This work not only reports the up-to-now most sensitive TdT detection strategy at a single-cell level but also opens the new gate for versatile enzyme activity detection.