Anhui Key Laboratory of Spin Electron and Nanomaterials, School of Chemistry and Chemical Engineering, Suzhou University, Suzhou, 234000, People's Republic of China.
Mikrochim Acta. 2019 Feb 15;186(3):179. doi: 10.1007/s00604-019-3280-5.
An 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 and the dynamic range extends from 0.02 to 22 U·mL. 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 abstract Schematic 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.
一种基于电致化学发光（ECL）的方法被用于测定尿嘧啶-DNA 糖基化酶（UDG）的活性。该方法基于缺口酶辅助信号放大和催化发夹组装的应用。UDG 可以识别并水解发夹 DNA1（HP1）茎部的尿嘧啶碱基。这导致 HP1 的打开，形成一条直链 DNA。直链 HP1 可以与发夹 DNA2（HP2）杂交，形成 DNA 双链。在缺口酶存在的情况下，它可以识别并切割 DNA 双链中 HP2 的特定序列，随后释放 HP1。它与其他 HP2 杂交，触发 HP2 的连续切割，同时产生大量中间序列（S1）。发夹 DNA3（HP3）通过 Au-S 化学固定在金电极上。在 S1 的存在下，HP3 与 S1 杂交，其发夹结构被打开。这种杂交导致发夹 DNA4（HP4）的置换，并释放 S1 以启动下一个杂交过程。因此，在循环过程后，产生了大量的 HP3-HP4 双链体。随后，通过与打开的 HP4 的冗余部分杂交，将修饰在生物条码 AuNP-CdSe 量子点上的 cDNA 固定在电极上。这导致 ECL 信号的显著放大。该生物传感器对 UDG 具有灵敏性和选择性。检测限为 6 mU·mL，动态范围从 0.02 扩展到 22 U·mL。该方法应用于实际样品并获得了良好的性能，从而为 DNA 修复酶相关的生物医学研究和诊断提供了理想的方法。
