杂交链式反应介导的发光银纳米簇系统用于miRNA-21的信号放大检测  

Hybridization Chain Reaction-Mediated Luminescent SilverNanocluster System for Amplified Detection of miRNA-21

作　　者：张丽 潘敏 邹芷乔 樊蕾 刘晓庆 ZHANG Li;PAN Min;ZOU Zhi-Qiao;FAN Lei;LIU Xiao-Qing(College of Chemistry and Molecular Sciences,Wuhan University,Wuhan 430072,China)

机构地区：[1]武汉大学化学与分子科学学院,武汉430072

出　　处：《分析化学》2020年第9期1193-1201,共9页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金项目(No.81602610);中央高校基本科研基金项目(No.2042018kf1006)资助。

摘　　要：MicroRNAs(miRNAs)是多种疾病的生物标志物,同时也能为疾病治疗提供潜在靶点,因此,发展简单、快速、灵敏的miRNAs分析方法具有十分重要的意义。本研究结合杂交链式反应(HCR)高的信号放大能力和以DNA为模板合成的银纳米簇(DNA-AgNCs)优异的发光性能,构建了一种免标记的通用型荧光传感器,实现了miRNA-21的快速灵敏检测。将合成银纳米簇(AgNCs)的DNA模板封闭在HCR的反应物(发夹DNA)中,当存在靶标DNA时,发夹DNA的杂交链式组装反应被引发,释放出大量自由的AgNCs模板序列,进而引发近红外荧光DNA-AgNCs的合成,AgNCs的近红外荧光信号强度与引发链DNA的浓度成正相关。进一步通过在检测系统中引入一个封闭有HCR引发链序列的辅助发卡序列,建立通用型HCR-AgNCs传感分析系统,用于靶核酸分子检测。以miRNA-21为模型分析物,只在miRNA-21存在时,此辅助发卡才能被打开,并生成自由的HCR引发链,进而引发HCR反应和AgNCs的合成。本方法检测miRNA-21的线性范围为250 pmol/L^8 nmol/L,线性方程为(F-F 0)/F 0=0.500+0.235 lg(C(pmol/L))(R^2=0.9792),检出限(3σ/S)为19.9 pmol/L。这种基于HCR-AgNCs的检测方法具有免标记、简单、灵敏的优点,可作为通用的核酸传感平台,有望为疾病的临床诊断和治疗提供重要参考。MicroRNAs(miRNAs)are biomarkers and potential therapeutic targets for various diseases.It is important to develop convenient and sensitive methods for miRNAs analysis.Herein,by integrating the high signal amplification capability of hybridization chain reaction(HCR)and the excellent luminescence performance of DNA-templated silver nanoclusters(DNA-AgNCs),a versatile and label-free fluorescent sensor was constructed for highly sensitive miRNA-21 detection.First of all,the DNA template for synthesizing AgNCs was blocked in the hairpin DNA for HCR assembly.Addition of the target DNA could trigger self-assembly of hairpin DNAs,and release numerous free DNA template for the synthesis of near-infrared fluorescent AgNCs.The fluorescence intensity of AgNCs was positively correlated with concentration of trigger DNA.Furthermore,by introducing an auxiliary hairpin containing a blocked HCR initiator sequence,the HCR-AgNCs system could be adapted as a universal sensing platform for target detection.Taking miRNA-21 analysis as an example,in the presence of miRNA-21,the auxiliary hairpin could be opened,releasing the free trigger DNA sequence for HCR,which in turn initiated the HCR reaction and the following synthesis of AgNCs.The results showed that the linear range of this method for miRNA-21 detection was from 250 pmol/L to 8 nmol/L,and the detection limit was as low as 19.9 pmol/L(3σ/S).The HCR-AgNCs system was label-free,sensitive and extendable,which could be used as a universal sensing platform for analyzing different types of targets.This approach may provide a practical method for clinical diagnosis of biomarkers.

关 键 词：MICRORNAS 杂交链式反应 信号放大 银纳米簇 DNA组装 荧光检测 

分 类 号：R440[医药卫生—诊断学] O657.3[医药卫生—临床医学] TP212[理学—分析化学]