膀胱癌相关miRNA灵敏检测的框架核酸线性放大平台构建  

Framework nucleic acid-based linear amplification platform for sensitive detection of bladder cancer-related miRNAs

作　　者：毛晨宙 张瑞赟 陈海戈[2] 尹芳菲 左小磊 MAO Chenzhou;ZHANG Ruiyun;CHEN Haige;YIN Fangfei;ZUO Xiaolei(Institute of Molecular Medicine,Renji Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200127,China;Department of Urology,Renji Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200127,China)

机构地区：[1]上海交通大学医学院附属仁济医院分子医学研究院,上海200127 [2]上海交通大学医学院附属仁济医院泌尿科,上海200127

出　　处：《上海交通大学学报(医学版)》2025年第3期253-260,共8页Journal of Shanghai Jiao tong University:Medical Science

基　　金：国家重点研发计划(2021YFF1200300);国家自然科学基金(22025404,U24A20497);上海市自然科学基金(19JC1410300)。

摘　　要：目的·构建框架核酸线性放大平台,用于膀胱癌相关微RNA(microRNA,miRNA)灵敏定量检测,实现膀胱癌的早期筛查和精准分型诊断。方法·结合等离子荧光增强芯片与四面体框架核酸高性能探针,以miRNA为靶标,构建框架核酸线性信号放大平台,实现多靶标精准、高通量定量分析。首先利用原子力显微镜(atomic force microscope,AFM)验证四面体结构的有效合成。通过聚丙烯酰胺凝胶电泳(polyacrylamide gel electrophoresis,PAGE)和全内反射荧光显微镜(total internal reflection fluorescent microscope,TIRFM)验证报告单元的信号线性放大能力。比较传感界面基底性能,选取具有信号放大的金岛芯片。通过界面特异度实验,验证检测体系的特异度。选取5种膀胱癌相关miRNA,构建靶标标准曲线,用于定量检测。结果·AFM验证了四面体单体及二聚体结构的有效合成。PAGE和TIRFM表征验证了1~6价荧光报告单元的荧光信号线性放大。为进一步实现信号放大,比较了等离子金岛芯片和传统的玻璃芯片,结果表明金岛芯片具有等离子效应,可显著增强近红外荧光,相较于玻璃芯片信号放大最高可达13.6倍。特异度验证实验显示,该体系信噪比为7~10,特异度良好。基于体系的高特异度,结合框架核酸界面良好的界面调控能力与线性放大,最终实现了靶标双色并行检测,各靶标工作范围为100 fmol/L~10 nmol/L(R²≥0.991),检出限低至100 fmol/L。结论·该平台的构建为生物标志物的高灵敏度定量分析开辟了新的途径。此外,所开发的框架核酸检测平台在膀胱癌及其他重大疾病的临床诊断和预后方面具有很大的潜力。通过早期检测和精准分型,医师可以为患者制定更加个性化的治疗方案,提高治疗效果,减少不必要的治疗和随之而来的不良反应。液体活检技术不仅为膀胱癌的早期筛查提供了新的可能性,也为其他类型癌症的研究和临床应用Objective·To construct a framework nucleic acid-based linear amplification platform for the sensitive and quantitative detection of bladder cancer-related microRNAs(miRNAs),facilitating early screening and accurate diagnosis of bladder cancer.Methods·This study combined a plasma fluorescence-enhanced chip with high-performance tetrahedral framework nucleic acid(tFNA)probes,targeting miRNAs as biomarkers,to construct a framework nucleic acid-based linear signal amplification platform for precise and high-throughput quantitative analysis of multiple targets.First,atomic force microscope(AFM)was used to verify the efficient synthesis of tFNA.The signal linear amplification capability of the reporter unit was verified by polyacrylamide gel electrophoresis(PAGE)and total internal reflection fluorescent microscope(TIRFM).The performance of the sensing interface substrates was compared,and the golden island chip with signal amplification was selected.The specificity of the detection system was verified by an interface specificity experiment.Five bladder cancer-related miRNAs were selected to construct standard curves for quantitative detection.Results·The efficient synthesis of tetrahedral monomer and dimer structures was verified by AFM.PAGE and TIRFM characterization verified the linear amplification of fluorescence signals from 1 to 6 valence fluorescence reporter units.In order to achieve further signal amplification,the plasma island chip and the traditional glass chip were compared.The results showed that the gold island chip exhibited a plasmonic effect,which significantly enhanced the near-infrared(NIR)fluorescence,with a signal amplification of up to 13.6 times compared to the glass chip.The specificity verification experiment showed that the signal-to-noise ratio of the system ranged from 7 to 10,demonstrating high specificity.Based on the high specificity of the system,along with the good interface regulation ability and linear amplification of the framework nucleic acid-based interface,dual-color parallel

关 键 词：膀胱癌 框架核酸 生物传感 荧光芯片 

分 类 号：O652.7[理学—分析化学] R737.14[理学—化学]