基于引物交换反应的非标记荧光探针用于脱嘌呤/脱嘧啶核酸内切酶1的高灵敏检测  

Label-free Fluorescence Probe Based on Primer Exchange Reaction for High Sensitivity Detection of Apurinic/Apyrimidinic Endonuclease 1

作　　者：王芸花 王乐如 杨力改 陈佳政 杜雨润 侯嘉慧 翟翔 赵旭华 于保锋 WANG Yun-Hua;WANG Le-Ru;YANG Li-Gai;CHEN Jia-Zheng;DU Yu-Run;HOU Jia-Hui;ZHAI Xiang;ZHAO Xu-Hua;YU Bao-Feng(Department of Biochemistry and Molecular Biology,School of Basic Medicine,Shanxi Medical University,Taiyuan 030001,China)

机构地区：[1]山西医科大学基础医学院,生物化学与分子生物学教研室,太原030001

出　　处：《分析化学》2025年第3期464-471,共8页Chinese Journal of Analytical Chemistry

基　　金：山西省自然科学基金项目(Nos.202303021221139,202203021222259);中国博士后科学基金项目(No.2024M761887);山西医科大学细胞生理学教育部重点实验室开放基金项目(No.CPOF202301)资助。

摘　　要：脱嘌呤/脱嘧啶核酸内切酶1(Apurinic/apyrimidinic endonuclease 1,APE 1)是一种多功能蛋白质,在DNA修复和基因表达调控中发挥重要作用。由于APE 1在多种癌症中过表达,因此可作为癌症生物标志物,用于辅助临床诊断、指导用药及预后监测。本研究采用引物交换反应(Primer exchange reaction,PER)策略,设计了一种非标记型荧光探针,用于APE 1活性的高灵敏检测。当体系中不存在APE 1时,催化发夹(Catalytic hairpin,HP)的结构稳定,无G-四链体(G-quadruplex)生成。此时,游离的硫磺素T(Thioflavin T,ThT)不发射荧光,体系的背景荧光很低。加入APE 1后,催化发夹上的无嘌呤/嘧啶(Apurinic/apyrimidinic,AP)位点被特异性识别并切割,断裂生成的短核酸序列可直接作为引物引发PER扩增,生成大量G-四链体。游离的ThT嵌入G-四链体结构并发出荧光,导致传感体系的荧光信号显著增强。PER扩增反应策略和催化发夹的低背景设计使此探针展现出很高的灵敏度,线性检测范围为0.001~0.07 U/mL,检出限(3σ)为0.0008 U/mL。此外,引物序列可由APE 1切割直接生成而不需要额外添加,这不仅提高了反应的特异性,还简化了操作步骤。非标记型荧光信号的使用大大降低了实验成本,实现了APE 1的快速检测。利用此荧光探针检测人血清样品中APE 1的含量,回收率在91%~104%之间,表明其在生物酶研究方面拥有巨大的应用潜力。Apurinic/apyrimidinic endonuclease 1(APE 1)is a multifunctional protein that plays important roles in DNA repair and regulation of gene expression.Because APE 1 is overexpressed in various cancers,it can serve as a cancer biomarker for aiding clinical diagnosis,guiding therapy,and monitoring prognosis.On this basis,a label-free fluorescent probe was designed based on the primer exchange reaction(PER)strategy for highly sensitive detection of APE1 activity.In the absence ofAPE 1,the structure ofcatalytic hairpin(HP)was stable and could not form G-quadruplex.Therefore,the background fluorescence of this sensing system was very low due to the dissociation of thioflavin T(ThT).In the presence of APE 1,the apurinic/apyrimidinic(AP)site of HP was cleaved by APE 1 and a short nucleic acid fragment that acted as a primer to initiate PER was generated.After PER reaction,a large number of G-quadruplex were produced,which could specifically bind with ThT and resulted in significant increase of fluorescence signal.The combination of low background design of HP and PER amplification made this biosensor had high sensitivity with a detection limit(3σ)of 0.0008 U/mL.Furthermore,the primer sequence was directly generated by the cleavage of APE 1 without additional addition,which not only increased the specificity of the reaction,but also simplified the experiment procedure.Moreover,the use of labelfree fluorescence signal reduced the cost of the experiment,and realized rapid detection of APE 1.Finally,this sensor was used to detect APE 1 in human serum samples with spiked recoveries of 91%–104%,proving great potential in study of biological enzyme.

关 键 词：脱嘌呤/脱嘧啶核酸内切酶1 引物交换反应 G-四链体 硫磺素T 非标记荧光探针 

分 类 号：O657.3[理学—分析化学]