College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan 250014, China.
School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, China.
J Mater Chem B. 2022 May 4;10(17):3277-3284. doi: 10.1039/d2tb00491g.
DNA glycosylases are engaged in the base excision repair process and play a vital role in maintaining genomic integrity. It remains a challenge for multiplexed detection of DNA glycosylases in cancer cells. Herein, we demonstrate the construction of a dephosphorylation-mediated chemiluminescent biosensor for multiplexed detection of human alkyladenine DNA glycosylase (hAAG) and uracil DNA glycosylase (UDG) in cancer cells. In this biosensor, the generation of chemiluminescence signals relies on the dephosphorylation of 3-(2'-spiroadamantyl)-4-methoxy-4-(3''-phosphoryloxyphenyl)-1,2-dioxetane (AMPPD) catalyzed by alkaline phosphatase (ALP). We design a bifunctional double-stranded DNA (dsDNA) substrate, a biotin-labelled poly-(T) probe, and two capture probes for the hAAG and UDG assay. This assay involves four steps including (1) the cleavage of the bifunctional dsDNA substrate induced by DNA glycosylases, (2) the recognition of the 3'-OH terminus of the primer by TdT and the subsequent TdT-mediated polymerization reaction, (3) the construction of the AuNPs-dsDNA-ALP nanostructures, and (4) the streptavidin-alkaline phosphatase (SA-ALP)-initiated dephosphorylation of AMPPD for the generation of an enhanced chemiluminescence signal. By taking advantage of the unique features of TdT-mediated polymerization and the intrinsic superiority of the ALP-AMPPD-based chemiluminescence system, this biosensor exhibits good specificity and high sensitivity with a detection limit of 1.53 × 10 U mL for hAAG and 1.77 × 10 U mL for UDG, and it can even quantify multiple DNA glycosylases at the single-cell level. Moreover, this biosensor can be applied for the measurement of kinetic parameters and the screening of DNA glycosylase inhibitors, holding great potential in DNA damage-related biomedical research and disease diagnostics.
DNA 糖苷酶参与碱基切除修复过程，在维持基因组完整性方面发挥着至关重要的作用。然而，在癌细胞中对多种 DNA 糖苷酶进行多重检测仍然是一项挑战。在此，我们展示了一种去磷酸化介导的化学发光生物传感器的构建，用于在癌细胞中对人烷基腺嘌呤 DNA 糖苷酶（hAAG）和尿嘧啶 DNA 糖苷酶（UDG）进行多重检测。在该生物传感器中，化学发光信号的产生依赖于碱性磷酸酶（ALP）催化 3-(2'-螺环戊二烯基)-4-甲氧基-4-(3''-磷酸氧代苯基)-1,2-二氧杂环乙烷（AMPPD）的去磷酸化。我们设计了一种双功能双链 DNA（dsDNA）底物、一个生物素标记的聚（T）探针以及两个用于 hAAG 和 UDG 分析的捕获探针。该分析涉及四个步骤：（1）DNA 糖苷酶诱导双功能 dsDNA 底物的切割；（2）TdT 识别引物的 3'-OH 末端，并随后进行 TdT 介导的聚合反应；（3）AuNPs-dsDNA-ALP 纳米结构的构建；（4）链霉亲和素-碱性磷酸酶（SA-ALP）引发 AMPPD 的去磷酸化以产生增强的化学发光信号。利用 TdT 介导的聚合的独特特征和基于 ALP-AMPPD 的化学发光系统的固有优势，该生物传感器具有良好的特异性和高灵敏度，hAAG 的检测限为 1.53×10 U mL，UDG 的检测限为 1.77×10 U mL，甚至可以在单细胞水平上定量多种 DNA 糖苷酶。此外，该生物传感器可用于测量动力学参数和筛选 DNA 糖苷酶抑制剂，在与 DNA 损伤相关的生物医学研究和疾病诊断中具有巨大潜力。
