Fluorescent dye DITO-1 has almost no fluorescence in the absence of nucleic acid. G bases in single strand DNA can induce maximum fluorescent enhancement followed by the A bases when it binds the DITO-1. However, the incorporation efficiency of the dATP was higher than dGTP in terminal transferase (TdT) polymerization. As a consequence, ploy (A)_n, rather than ploy (G)_n via TdT polymerization had the superior photoluminance when it binded DITO-1 fluorescent dye. Here, we developed a high selective and sensitive sensing strategy for assaying TdT and T4 polynucleotide kinase activity (T4 PNK) based on the ploy (A)_n-DITO-1 fluorescent probe. An increasing amounts of TdT enzyme could promote the distinct incorporation of dATP on the DNA primer and form poly (A)_n ssDNA with a difference in length. A good linear relationship between the ΔF and the concentrations of TdT in a range of 0.2–50 U/mL was obtained and the detection limit was 0.05 U/mL. Based on the experimental results for TdT, we further expanded the application of this method for detection of a series of concentrations of T4 PNK. The ΔF and the logarithm concentrations of T4 PNK in the range of 0.1–10 U/mL showed a good linear response and the detection limit of 0.02 U/mL was obtained. In addition, the detection of T4 PNK in Hela cell lysate was achieved, demonstrating that the proposed method had the potential application in complex system. The ploy (A)_n-DITO-1 fluorescent probe had the excellent properties of one-step readout, robustness for target detection in complex system, and easiness operation, and showed the great potential in clinical diagnostics, inhibitor screening, and drug discovery.

荧光染料DITO-1在没有核酸的情况下几乎没有荧光。单链 DNA 中的 G 碱基在与 DITO-1 结合时可以诱导最大的荧光增强，其次是 A 碱基。然而，dATP 在末端转移酶 (TdT) 聚合中的掺入效率高于 dGTP。因此，ploy (A) _n与通过 TdT 聚合的ploy (G) _n相比，在结合 DITO-1 荧光染料时具有优异的光亮度。在这里，我们开发了一种基于 ploy (A) _n -DITO-1 荧光探针的高选择性和高灵敏度传感策略，用于检测 TdT 和 T4 多核苷酸激酶活性 (T4 PNK)。增加量的 TdT 酶可以促进 dATP 在 DNA 引物上的明显掺入并形成聚 (A)_n长度不同的 ssDNA。ΔF与TdT浓度在0.2~50 U/mL范围内具有良好的线性关系，检测限为0.05 U/mL。基于 TdT 的实验结果，我们进一步扩展了该方法在检测一系列浓度的 T4 PNK 中的应用。ΔF和T4 PNK的对数浓度在0.1-10 U/mL范围内表现出良好的线性响应，检测限为0.02 U/mL。此外，还实现了对Hela细胞裂解液中T4 PNK的检测，表明该方法在复杂系统中具有潜在的应用价值。策略 (A) _n-DITO-1荧光探针具有一步读出、复杂系统中靶点检测鲁棒性、操作简便等优良特性，在临床诊断、抑制剂筛选和药物发现等方面显示出巨大潜力。