Liu Birong, Yang Zizhong, Huang Ting, Li Min-Min, Duan Wenjun, Xie Baoping, Chen Jin-Xiang, Dai Zong, Chen Jun
NMPA Key Laboratory for Research and Evaluation of Drug Metabolism, Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, 510515, China.
Center of Clinical Laboratory, The First Affiliated Hospital of Jinan University, Guangzhou, 510630, PR China.
Talanta. 2022 Jul 1;244:123404. doi: 10.1016/j.talanta.2022.123404. Epub 2022 Mar 24.
The highly sensitive detection of low-abundant apurinic/apyrimidinic endonuclease 1 (APE1) activity is of great significance for early diagnosis of disease and pathological research. Many methods for detecting APE1 based on isothermal nucleic acids amplification have been developed for improving its sensitivity. However, some of these methods have certain limitations, such as multiple reaction steps, narrow linear range, and complicated processes for fluorescent labeling. Herein, we develop a highly sensitive and label-free APE1 fluorescence detection method based on rolling circle amplification combined with G-quadruplex (RCA-G4). A hairpin probe (HP) labeled with the AP site can be recognized and cleaved by APE1, leading to the release of the primer sequence, which triggered RCA to produce long chain amplification products with a great amount of repeated sequences. The formed amplicon contains a series G-quadruplex structure, which can be combined with Thioflavin T (ThT) to produce fluorescence and achieve high sensitivity label-free detection of APE1. Benefit from the high amplification efficiency of RCA and the high fluorescence quantum yield of G-quadruplex/ThT, a detection limit as low as 1.52 × 10 U/mL and the linear range from 2 × 10 to 10 U/mL were obtained. The developed RCA-G4 method can be successfully used to detect APE1 in serum samples with a recovery from 96.3% to 105.7%. We believe that this approach is expected to play an important role in APE1-related disease research and drug development.
高灵敏检测低丰度脱嘌呤/脱嘧啶内切酶1（APE1）活性对疾病的早期诊断和病理研究具有重要意义。为提高其灵敏度，已开发出许多基于等温核酸扩增检测APE1的方法。然而，其中一些方法存在一定局限性，如反应步骤多、线性范围窄以及荧光标记过程复杂等。在此，我们开发了一种基于滚环扩增结合G-四链体（RCA-G4）的高灵敏、无标记的APE1荧光检测方法。带有脱嘌呤位点的发夹探针（HP）可被APE1识别并切割，导致引物序列释放，从而引发滚环扩增产生大量重复序列的长链扩增产物。形成的扩增子包含一系列G-四链体结构，其可与硫黄素T（ThT）结合产生荧光，实现对APE1的高灵敏无标记检测。受益于滚环扩增的高扩增效率和G-四链体/ThT的高荧光量子产率，获得了低至1.52×10 U/mL的检测限和2×10至10 U/mL的线性范围。所开发的RCA-G4方法可成功用于检测血清样本中的APE1，回收率为96.3%至105.7%。我们相信，这种方法有望在APE1相关疾病研究和药物开发中发挥重要作用。
