AbstractAn electrochemiluminescence (ECL) based method is described for the determination of the activity of the enzyme uracil-DNA glycosylase (UDG). It is based on the use of nicking enzyme-assisted signal amplification and catalytic hairpin assembly. UDG can recognize and hydrolyze the uracil bases from the stem of hairpin DNA1 (HP1). This causes the opening of HP1 to form a straight strand DNA. The straight HP1 can hybridize with hairpin DNA2 (HP2) to form a DNA duplex. In the presence of nicking enzyme, it can recognize and cut the specific sequences in the HP2 of the DNA duplex, and a subsequent release of HP1. It hybridizes with other HP2 to trigger the continuous cleavage of HP2, concomitantly generating abundant intermediate sequences (S1). The hairpin DNA3 (HP3) is immobilized on a gold electrode via Au-S chemistry. In the presence of S1, HP3 hybridizes with S1 and its hairpin structure is opened. This hybridization causes displacement from hairpin DNA4 (HP4), and S1 is released to initiate the next hybridization process. Thus, a massive number of HP3-HP4 duplexes is generated after the cyclic process. Subsequently, the cDNA modified on bio-bar-coded AuNP-CdSe quantum dots are immobilized on the electrode by hybridization with the redundant part of the opened HP4. This results in a significant amplification of the ECL signal. This biosensor is sensitive and selective for UDG. The detection limit is 6 mU·mL−1 and the dynamic range extends from 0.02 to 22 U·mL−1. The method was applied to real samples and gained good performance, thereby providing an ideal way for DNA repair enzyme-related biomedical research and diagnosis. Graphical abstractSchematic presentation of the electrochemiluminescence (ECL) detection of uracil-DNA glycosylase (UDG) based on nicking enzyme assisted signal amplification and catalyzed hairpin assembly. The bio-barcoded Au NP-CdSe QDs serve as the ECL signal probes to achieve a significantly signal amplification.

中文翻译：

---

尿嘧啶-DNA糖基化酶活性的电化学发光测定：结合切口酶辅助信号放大和催化发夹组装

摘要描述了一种基于电化学发光 (ECL) 的方法，用于测定酶尿嘧啶-DNA 糖基化酶 (UDG) 的活性。它基于使用切口酶辅助信号放大和催化发夹组装。UDG 可以识别并水解发夹 DNA1 (HP1) 茎中的尿嘧啶碱基。这会导致 HP1 打开以形成直链 DNA。直的HP1可以与发夹DNA2（HP2）杂交形成DNA双链体。在切口酶的存在下，它可以识别并切割DNA双链体HP2中的特定序列，随后释放HP1。它与其他 HP2 杂交以触发 HP2 的连续裂解，同时产生丰富的中间序列 (S1)。发夹 DNA3 (HP3) 通过 Au-S 化学固定在金电极上。在 S1 存在的情况下，HP3 与 S1 杂交并打开其发夹结构。这种杂交导致发夹 DNA4 (HP4) 发生置换，S1 被释放以启动下一个杂交过程。因此，在循环过程之后产生了大量的 HP3-HP4 双链体。随后，在生物条形码的 AuNP-CdSe 量子点上修饰的 cDNA 通过与打开的 HP4 的冗余部分杂交而固定在电极上。这导致 ECL 信号的显着放大。该生物传感器对 UDG 敏感且具有选择性。检测限为 6 mU·mL-1，动态范围从 0.02 扩展到 22 U·mL-1。该方法应用于实际样品并获得了良好的性能，为DNA修复酶相关的生物医学研究和诊断提供了理想的途径。基于切口酶辅助信号放大和催化发夹组装的尿嘧啶-DNA 糖基化酶 (UDG) 的电化学发光 (ECL) 检测示意图。生物条形码 Au NP-CdSe QD 作为 ECL 信号探针，实现显着的信号放大。