基于黏性末端链置换扩增的电化学发光/电化学比率传感器用于miRNA检测
Ratiometric Electrochemiluminescence/Electrochemistry Sensor for miRNA Based on Toehold-Mediated Strand Displacement Amplification
金小亚 1张莹 2林悦 3林振宇3
作者信息
- 1. 温州医科大学附属第一医院感染内科,院内感染管理处,温州 325000
- 2. 福建医科大学附属第一医院中心实验室,福建省肿瘤精准诊疗重点实验室,福州 350001;福州大学化学学院,食品安全与生物分析教育部重点实验室,福建省食品安全分析与检测技术重点实验室,福州 350116
- 3. 福州大学化学学院,食品安全与生物分析教育部重点实验室,福建省食品安全分析与检测技术重点实验室,福州 350116
- 折叠
摘要
基于黏性末端链置换(TMSD)非酶扩增技术构建了一种简单、高灵敏的电化学发光/电化学比率传感方法用于miRNA的检测.本方法以修饰在电极上的亚甲基蓝(MB)产生的EC信号作为内参比信号,并将Ru(phen)32+嵌入到通过分支杂交链式反应(bHCR)产生的DNA纳米微粒中作为ECL探针.目标物存在时,MB脱离电极表面并使ECL信标修饰在电极上,从而使EC信号减弱,ECL信号增强,采用ECL和EC信号比值(ECL/EC)作为输出信号,降低了潜在干扰,提高了传感器的重现性和灵敏度.体系的ECL/EC比值与miRNA-21的浓度在10 fmol/L～500 pmol/L范围内具有良好的响应,检出限为6.5 fmol/L.与单一使用ECL信号的方法相比,在测定1 pmol/L的miRNA目标物时,响应信号的相对标准偏差从8.2%下降到4.1%.本方法成功应用于复杂细胞样品中miRNA-21的检测.
Abstract
A simple and highly sensitive method for detection of miRNA was constructed by combining the high-efficiency thiolated-mediated strand displacement(TMSD)non-enzymatic amplification and the high-reproducibility electrochemiluminescence/electrochemistry(ECL/EC)ratio sensing technique.The EC signal generated by methylene blue(MB)modified on the electrode was used as the internal reference signal,and Ru(phen)32+was embedded in DNA nanoparticles produced by the branched hybridization chain reaction as the ECL probe.The presence of target miRNA facilitated the detachment of MB from electrode surface and the modification of ECL beacon on the electrode,resulting in a decrease of the EC signal and an enhancement of the ECL signal.The method of using the ratio of ECL signal to EC signal reduced potential interference and improved reproducibility and sensitivity of the biosensor.The ratio of ECL/EC signals exhibited linear relationship with miRNA-21 concentration in the range from 10 fmol/L to 500 pmol/L,with a detection limit of 6.5 fmol/L,and the relative standard deviation of the assay was reduced from 8.2%to 4.1%when determining 1 pmol/L target miRNA-21 compared to that using single ECL signal.This method was preliminarily applied to the detection of miRNA-21 in complex cell samples.
关键词
电化学/电化学发光/比率传感器/黏性末端链置换/miRNAKey words
Electrochemistry/Electrochemiluminescence/Ratiometric biosensor/Toehold-mediated strand displacement/miRNA引用本文复制引用
基金项目
国家自然科学基金(22174018)
浙江省基础公益研究计划(TGD23H030004)
温州市基础性科研项目(Y20220021)
浙江省卫生健康科技计划(2022KY887)
出版年
2024
国家科技期刊平台
NSTL
万方数据