基于聚多巴胺纳米粒子的荧光共振能量转移法检测microRNA

doi:10.7503/cjcu20150016

高等学校化学学报 ›› 2015, Vol. 36 ›› Issue (6): 1061.doi: 10.7503/cjcu20150016

基于聚多巴胺纳米粒子的荧光共振能量转移法检测microRNA

马立娜¹, 柳扶摇^1,², 高嘉雪^1,², 王振新¹()

1. 中国科学院长春应用化学研究所, 电分析化学国家重点实验室, 长春130022
2. 中国科学院大学, 北京 100049

收稿日期:2015-01-07 出版日期:2015-06-10 发布日期:2015-05-05
作者简介:联系人简介: 王振新, 男, 博士, 研究员, 博士生导师, 主要从事高通量分析和生物纳米技术方面的研究. E-mail:wangzx@ciac.ac.cn
基金资助:
国家自然科学基金(批准号: 21127010)资助

Polydopamine Nanoparticle-based Fluorescence Resonance Energy Transfer Assay for microRNA Detection^†

MA Lina¹, LIU Fuyao^1,², GAO Jiaxue^1,², WANG Zhenxin^1,^*()

1. State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry,Chinese Academy of Sciences, Changchun 130022, China
2. University of Chinese Academy of Sciences, Beijing 100049, China

Received:2015-01-07 Online:2015-06-10 Published:2015-05-05
Contact: WANG Zhenxin E-mail:wangzx@ciac.ac.cn
Supported by:
† Supported by the National Natural Science Foundation of China(No.21127010)

摘要/Abstract

摘要：

基于聚多巴胺纳米粒子(PDA NPs)对Cy5标记单链DNA(Cy5-ssDNA)探针的荧光猝灭效应以及脱氧核糖核酸酶Ⅰ(DNaseⅠ)选择性切割DNA/RNA杂合结构中单链DNA的特性, 建立了一种用于微小核糖核酸(miRNA)检测的新型恒温信号放大方法. 在优化的实验条件下, 体系的相对荧光强度(F_R)与miR-21浓度的对数值成正比; 对miR-21检测的线性范围为10 pmol/L~100 nmol/L, 检出限达7 pmol/L. 血清加标实验结果表明, 该方法可用于生理环境下miR-21的检测.

关键词: 聚多巴胺纳米粒子, 微小核糖核酸, 脱氧核糖核酸酶Ⅰ, 荧光共振能量转移

Abstract:

A simple, rapid and low-cost polydopamine nanoparticle(PDA NP)-based fluorescence resonance energy transfer(FRET) assay with deoxyribonucleaseⅠ(DNaseⅠ)-assisted target recycling signal amplification was developed for microRNA(miRNA) detection. Under the optimized experimental conditions, the recovery ratio of Cy5 fluorescence intensity increased linearly with logarithm of miR-21 concentration from 10 pmol/L to 100 nmol/L, with a detection limit of 7 pmol/L. Moreover, satisfactory results were obtained while the proposed method was applied to detect miRNA in 10% human serum.

Key words: Polydopamine nanoparticle, microRNA, DNaseⅠ, Fluorescence resonance energy transfer

中图分类号:

O657

TrendMD:

马立娜, 柳扶摇, 高嘉雪, 王振新. 基于聚多巴胺纳米粒子的荧光共振能量转移法检测microRNA. 高等学校化学学报, 2015, 36(6): 1061.

MA Lina, LIU Fuyao, GAO Jiaxue, WANG Zhenxin. Polydopamine Nanoparticle-based Fluorescence Resonance Energy Transfer Assay for microRNA Detection^†. Chem. J. Chinese Universities, 2015, 36(6): 1061.

图/表 7

Table 1 Oligonucleotide sequences used in experiments*

Name	Sepuence	Number of bases
P-miR-21	5'-Cy5-TCAACATCAGTCTGATAAGCTA-3'	22
miR-21	5'-UAGCUUAUCAGACUGAUGUUGA-3'	22
P-7f	5'-Cy5-AACTATACAATCTACTACCTCA-3'	22
let-7f	5'-UGAGGUAGUAGAUUGUAUAGUU-3'	22
let-7a	5'-UGAGGUAGUAGGUUGUAUAGUU-3'	22

Fig.1 TEM micrographs of as-prepared PDA NPs with different average diameters(A—C) and their UV-Vis spectra(D) Average diameter/nm: (A), a. 94±9; (B), b. 206±15; (C), c. 306±17.

Scheme 1 Schematic representation of PDA NP-based FRET assay with DNase I-assisted target recycling signal amplification for the detection of target miRNA

Fig.2 Effects of quenching time of 94(A), 206(B) and 306 nm(C) PDA NPs on quenching efficiency in the concentration of 20 μg/mL

Fig.3 Effects of concentration of PDA NPs(A), cleavage/reaction time(B), NaCl concentration(C) and MgCl2 concentration(D) on the recovery ratio of Cy5 fluorescence intensity(FR) in the presence of 66 nmol/L Cy5-ssDNA

Fig.4 Fluorescence spectra of Cy5 in the presence of various concentrations of miR-21 in buffer(A) and 10% human serum(B), respectively and calibration curves of recovery ratio of Cy5 fluorescence intensity(FR) vs. logarithmic concentration of miR-21(C) in buffer(a) and 10% human serum(b), respectivelyThe error bars mean standard deviations(n=6).

Fig.5 Specificity evaluations for miRNA detection in buffer and 10% human serum, respectivelyThe concentration of the each miRNA targets is 100 nmol/L.

参考文献 27

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基于聚多巴胺纳米粒子的荧光共振能量转移法检测microRNA

Polydopamine Nanoparticle-based Fluorescence Resonance Energy Transfer Assay for microRNA Detection^†

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基于聚多巴胺纳米粒子的荧光共振能量转移法检测microRNA

Polydopamine Nanoparticle-based Fluorescence Resonance Energy Transfer Assay for microRNA Detection†

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Polydopamine Nanoparticle-based Fluorescence Resonance Energy Transfer Assay for microRNA Detection^†