核酸外切酶Ⅲ辅助的DNA铽离子配合物时间分辨荧光检测黄曲霉毒素B1

引用本文: 柳苏月, 田晶晶, 朱龙佼, 黄昆仑, 田洪涛, 许文涛. 核酸外切酶Ⅲ辅助的DNA铽离子配合物时间分辨荧光检测黄曲霉毒素B1[J]. 分析化学, 2021, 49(8): 1327-1334. doi: 10.19756/j.issn.0253-3820.211168 shu

Citation: LIU Su-Yue, TIAN Jing-Jing, ZHU Long-Jiao, HUANG Kun-Lun, TIAN Hong-Tao, XU Wen-Tao. Time Resolved Fluorescence Detection of Aflatoxin B1 with DNA/Terbium Ion Complex Assisted by Exonuclease Ⅲ[J]. Chinese Journal of Analytical Chemistry, 2021, 49(8): 1327-1334. doi: 10.19756/j.issn.0253-3820.211168 shu

核酸外切酶Ⅲ辅助的DNA铽离子配合物时间分辨荧光检测黄曲霉毒素B1

柳苏月 ^1, ,
田晶晶 ^2,3, ,
朱龙佼 ^2,3, ,
黄昆仑 ^2,3, ,
田洪涛 ^{1,
,} ,
许文涛 ^{2,3,
,}

1.
河北农业大学食品科技学院, 保定 071001;
2.
中国农业大学, 北京食品营养与人类健康高精尖创新中心, 北京 100083;
3.
中国农业大学, 农业部农业转基因生物安全评价(食用)重点实验室, 北京 100083

通讯作者: 田洪涛,E-mail:tht631022@163.com; 许文涛,E-mail:xuwentao@cau.edu.cn

基金项目:
国家重点研发计划项目（No.2017YFC1600901）和山东省重点研发计划项目（No.2018YYSP019）资助。

摘要: 基于适配体的高特异性识别能力，结合核酸外切酶Ⅲ（Exo Ⅲ）辅助的核酸信号放大策略和时间分辨荧光测量技术，构建了无标记、超灵敏的适配体荧光生物传感器（Aptasensor），用于检测黄曲霉毒素B1（AFB1）。设计了两个无标记的功能核酸发夹，其中，含有AFB1适体序列的发卡H1作为识别元件，茎部含有富G序列的发卡H2作为信号响应元件。AFB1可特异性结合H1中的适配体序列，从而将H1打开，然后与H2互补配对形成AFB1-H1-H2复合物，继而触发Exo Ⅲ的酶切反应，对复合物中的H2的3'平末端进行消化切割，释放出H2中的富G序列，能够显著增敏铽离子（Tb³⁺）的发射。同时，释放出的AFB1-H1可以自由结合下一个H2，以启动Exo Ⅲ辅助的靶标循环扩增，引起体系中大量富G单链的释放，实现信号放大。随着AFB1浓度增加，体系相对荧光强度增大，可以通过时间分辨发光光谱实现对靶标AFB1的检测，线性检测范围为0.001~0.1 ng/mL，检出限（3σ）为0.9 pg/mL。采用本方法检测玉米样品中的AFB1，回收率在98.5%~103.5%之间，在食品和农产品检测方面具有良好的应用潜力。

关键词:

English

Time Resolved Fluorescence Detection of Aflatoxin B1 with DNA/Terbium Ion Complex Assisted by Exonuclease Ⅲ

1.
College of Food Science Technology, Hebei Agriculture University, Baoding 071001, China;
2.
Beijing Advanced Innovation Center for Food Nutrition and Human Health, China Agricultural University, Beijing 100083, China;
3.
Key Laboratory of Safety Evaluation (Food) of Agricultural Genetically Modified Organisms, Ministry of Agriculture, China Agricultural University, Beijing 100083, China

Corresponding author: TIAN Hong-Tao, ; XU Wen-Tao,

Received Date: 09 March 2021
Revised Date: 21 May 2021
Fund Project:
Supported by the National Key Research and Development Program of China (No.2017YFC1600901) and the Shandong Key Research and Development Program (No.2018YYSP019).

Abstract: Based on the highly specific recognition ability of aptamers, and the combination with an exonuclease Ⅲ (Exo Ⅲ)-assisted nucleic acid signal amplification strategy and the time resolved fluorescence measurement technology, a label-free and ultra-sensitive aptamer fluorescent biosensor (aptasensor) was constructed to detect aflatoxin B1 (AFB1). Two label-free functional nucleic acid hairpins were designed. The hairpin H1 containing AFB1 aptamer sequence was used as the identification element, and the G-rich sequence-containing hairpin H2 of the stem acted as the signal response element. AFB1 could specifically bind to the aptamer sequence in H1, leading to the unfolding of H1 and its complementary paring with H2 to form an AFB1-H1-H2 complex, which consequently triggered the exonuclease Ⅲ (Exo Ⅲ) enzyme digestion. The 3' blunt end of H2 in the complex was digested to release the G-rich sequence in H2, which could significantly enhance the emission of terbium ions (Tb³⁺). Meanwhile, the released AFB1-H1 could freely bind to the next H2 to start Exo Ⅲ-assisted target cyclic amplification, causing the release of considerable G-rich single strands into the system and realizing signal amplification. As the concentration of AFB1 was increased, the relative fluorescence intensity of the system increased. AFB1 could be detected by time-resolved luminescence spectroscopy. The linear detection range was from 0.001 to 0.1 ng/mL, and the detection limit (3σ) was as low as 0.9 pg/mL. This method was used to detect AFB1 in corn samples, with the recoveries of 98.5%-103.5%, showing a good application potential in detection of AFB1 in food and agricultural products.

Key words:

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1.
沈阳化工大学材料科学与工程学院沈阳 110142

核酸外切酶Ⅲ辅助的DNA铽离子配合物时间分辨荧光检测黄曲霉毒素B1

通讯作者: 田洪涛,E-mail:tht631022@163.com; 许文涛,E-mail:xuwentao@cau.edu.cn

English

Time Resolved Fluorescence Detection of Aflatoxin B1 with DNA/Terbium Ion Complex Assisted by Exonuclease Ⅲ

Corresponding author: TIAN Hong-Tao, ; XU Wen-Tao,

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通讯作者: 陈斌, bchen63@163.com

中国化学会秘书处

核酸外切酶Ⅲ辅助的DNA铽离子配合物时间分辨荧光检测黄曲霉毒素B1

通讯作者: 田洪涛,E-mail:tht631022@163.com; 许文涛,E-mail:xuwentao@cau.edu.cn

English

Time Resolved Fluorescence Detection of Aflatoxin B1 with DNA/Terbium Ion Complex Assisted by Exonuclease Ⅲ

Corresponding author: TIAN Hong-Tao, ; XU Wen-Tao,

CCS Chemistry：嵌段共聚物自组装成 “三明治”二维有机材料，实现纳米级压力传感功能

CCS Chemistry：颜徐州、鲍哲南： 你的皮肤可能是一片SPMs

CCS Chemistry：工作高效不疲劳，只须让催化剂动起来

CCS Chemistry：静电组装导向聚合- 聚电解质纳米凝胶量化制备新策略

CCS Chemistry：金黄色葡萄球菌醛基脱氢酶是如何识别特异性底物的？

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