基于DNAzyme的自锁免洗乳腺癌来源小细胞外囊泡超敏定量检测研究  

Ultrasensitive wash-free quantification of breast cancer-derived small extracellular vesicles via a self-locked DNAzyme nanoprobe

作　　者：陈晓辉 刘海霞 马宁宇 吴倩倩 陈恒屹 陈艺 涂伟[2] 曹静晗 罗阳 Chen Xiaohui;Liu Haixia;Ma Ningyu;Wu Qianqian;Chen Hengyi;Chen Yi;Tu Wei;Cao Jinghan;Luo Yang(Center of Smart Laboratory and Molecular Medicine,School of Medicine,Chongqing University,Chongqing 400044,China;Department of Clinical Laboratory,Fuling hospital,Chongqing University,Chongqing 408000,China;Chongqing Depu Foreign Language School,Chongqing 401320,China)

机构地区：[1]重庆大学医学院智慧检验与分子医学中心,重庆400044 [2]重庆大学附属涪陵医院临床检验科,重庆408000 [3]重庆德普外国语学校,重庆401320

出　　处：《中华检验医学杂志》2025年第3期396-401,共6页Chinese Journal of Laboratory Medicine

基　　金：国家自然科学基金(82241059,82125022);重庆市医学科研项目(2024QNXM028)。

摘　　要：目的构建一种基于DNAzyme自锁式探针的荧光信号放大技术,用于乳腺癌来源小细胞外囊泡(sEV)的免洗超敏检测。方法设计DNAzyme自锁探针,当样本中存在乳腺癌来源sEV时,脱氧核酶DNAzyme-锁链识别其特有的上皮黏附分子EpCAM,打开DNAzyme-锁链的封闭结构,恢复DNAzyme剪切活性,并将底物链上的RNA位点进行循环识别剪切,采用荧光标记剪切链的方式识别检测不同浓度的sEV,并计算检测限和检测范围。结果基于DNAzyme自锁式探针体系能够对乳腺癌来源sEV进行识别并检测到启动循环剪切后的荧光信号。该方法能够对sEV进行免洗识别检测,所检测的sEV浓度与荧光强度有良好的线性趋势(R^(2)=0.98),线性检测范围为1.0×10^(2)~1.0×1.0^(7)颗粒/μl,检测限为59颗粒/μl。结论成功构建了一种乳腺癌来源sEV免洗定量分析检测策略,可实现特定细胞来源sEV的灵敏检测,为肿瘤的诊断提供技术支撑。Objective To develop a self-locked DNAzyme nanoprobe-based fluorescence amplification strategy for wash-free and ultrasensitive detection of breast cancer-derived small extracellular vesicles(sEV).Method A DNAzyme self-locked probe was designed to recognize the epithelial cell adhesion molecule(EpCAM)specifically expressed on breast cancer-derived sEVs.Upon binding to EpCAM,the DNAzyme-lock structure was opened,restoring the DNAzyme cleavage activity.The activated DNAzyme then cyclically cleaved the RNA site on the substrate strand.Fluorescently labeled substrate strands were used to detect sEVs at varying concentrations,and the detection limit and linear range were determined.Results The DNAzyme self-locked probe successfully identified breast cancer-derived sEVs and generated a fluorescent signal through cyclic cleavage.The proposed method achieved wash-free detection of sEVs,with the fluorescence intensity showing a strong linear correlation with sEV concentration(R^(2)=0.98).The linear detection range was 1.0×10^(2)-1.0×1.0^(7)particles/µl,with a detection limit of 59 particles/µl.Conclusion This study established a wash-free and highly sensitive strategy for quantifying breast cancer-derived sEVs,which provides a promising technical approach for the early diagnosis of cancer.

关 键 词：细胞外囊泡 液体活检 脱氧核酶 肿瘤检测 

分 类 号：R737.9[医药卫生—肿瘤]