四环素双价适配体非酶免标记传感器

doi:10.13560/j.cnki.biotech.bull.1985.2021-1138

生物技术通报 ›› 2022, Vol. 38 ›› Issue (3): 276-284.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1138

四环素双价适配体非酶免标记传感器

兰欣悦¹^,²(), 刘宁宁²^,³, 朱龙佼², 陈旭¹^,², 褚华硕², 李相阳⁴, 段诺⁵, 许文涛¹^,²()

1.中国农业大学食品科学与营养工程学院转基因生物安全性评价（食品安全）重点实验室食品质量与安全北京实验室,北京 100083
2.中国农业大学营养与健康系食品精准营养与质量控制教育部重点实验室,北京 100083
3.河北工程大学生命科学与食品工程学院,邯郸 056000
4.北京农学院食品科学与工程学院,北京 102206
5.江南大学食品学院,无锡 214122

收稿日期:2021-09-04 出版日期:2022-03-26 发布日期:2022-04-06
作者简介:兰欣悦,女,硕士研究生,研究方向：营养与食品安全;E-mail： lanxinyue1210@163.com
基金资助:
山东省重大科技创新工程项目(2019JZZY011014);北京市农业科技项目(PXM2021_014207_000037)

Tetracycline Bivalent Aptamer Non-enzyme Label-free Sensor

LAN Xin-yue¹^,²(), LIU Ning-ning²^,³, ZHU Long-jiao², CHEN Xu¹^,², CHU Hua-shuo², LI Xiang-yang⁴, DUAN Nuo⁵, XU Wen-tao¹^,²()

1. Beijing Advanced Innovation Center for Food Nutrition and Human Health,Key Laboratory of Safety Assessment of Genetically Modified Organism（Food Safety）（MOA）,Beijing Laboratory for Food Quality and Safety,College of Food Science & Nutritional Engineering,China Agricultural University,Beijing 100083
2. Key Laboratory of Precision Nutrition and Food Quality,Department of Nutrition and Health,China Agricultural University,Beijing 100083
3. School of Life Sciences and Food Engineering,Hebei University of Engineering,Handan 056000
4. College of Food Science and Engineering,Beijing University of Agriculture,Beijing 102206
5. School of Food Science and Technology,Jiangnan University,Wuxi 214122

Received:2021-09-04 Published:2022-03-26 Online:2022-04-06

摘要/Abstract

摘要：

四环素（tetracycline,TC）作为广普性抗生素,在畜产养殖业中常因使用不当或滥用致使奶乳制品中出现抗生素残留,对人类健康造成严重威胁。传统检测方法如色谱,酶联免疫分析等,对仪器依赖性较强,且检测体系复杂、时间较长,不能很好地满足现场快速检测需求。因此,本研究设计了四环素双价适配体非酶标记传感器,以硫黄素T（ThT）荧光信号变化的形式响应检测结果,在10 nmol/L-1 μmol/L的四环素浓度范围内的对数值呈现良好的线性关系,R ²达0.99以上,检测限低至96 pmol/L,同时,还实现了在20 min内对牛奶样本中四环素的快速检测,且特异性良好。

关键词: 双价适配体, G-四链体, 非酶免标记传感, 四环素, 定量检测

Abstract:

As a general antibiotic,tetracycline（TC）residue in milk and dairy products is often caused by improper use or abuse during the livestock breeding and processing,which poses a serious threat to human health. Chromatography,enzyme-linked immunosorbent assay and other traditional detection methods are highly dependent on the instrument,as well as the procedure is complicated,the analysis time is prolonged,cannot well meet the requirements of rapid detection on site. Therefore,a non-enzyme-labeled sensor based on bivalent aptamer was established for TC detection,which responds the detection results with the change of thioflavin T（ThT）fluorescence signal. The logarithmic value in the TC concentration range of 10 nmol/L-1 μmol/L showed a good linear relationship,R ² was over 0.99,and the detection limit was as low as 96 pmol/L. At the same time,it also allowed rapid and specificity detection of TC in a milk samples within 20 minutes.

Key words: bivalent aptamer, G-quadruplex, non-enzyme label-free sensing, tetracycline, quantitative detection

兰欣悦, 刘宁宁, 朱龙佼, 陈旭, 褚华硕, 李相阳, 段诺, 许文涛. 四环素双价适配体非酶免标记传感器[J]. 生物技术通报, 2022, 38(3): 276-284.

LAN Xin-yue, LIU Ning-ning, ZHU Long-jiao, CHEN Xu, CHU Hua-shuo, LI Xiang-yang, DUAN Nuo, XU Wen-tao. Tetracycline Bivalent Aptamer Non-enzyme Label-free Sensor[J]. Biotechnology Bulletin, 2022, 38(3): 276-284.

图/表 11

表1 实验所用序列

Table 1 Sequences used in experiment

名称Name	序列Sequence（5'-3'）	碱基数Number of bases/nt
四环素适配体 Tetracycline aptamer（TCA）	CGGTGGTG	8
双价裁剪适配体 Bivalent shortened aptamer（TCSA）	CGGTGGTGCGGTGGTG	16
间隔插入2T Intervening 2T（2T）	CGGTGGTGTTCGGTGGTG	18
间隔插入2A Intervening 2A（2A）	CGGTGGTGAACGGTGGTG	18
间隔插入2C Intervening 2C（2C）	5CGGTGGTGCCCGGTGGTG	18
间隔插入2G Intervening 2G（2G）	5CGGTGGTGGGCGGTGGTG	18
间隔插入1G Intervening 1G（1G）	CGGTGGTGGCGGTGGTG	17
间隔插入3G Intervening 3G（3G）	CGGTGGTGGGGCGGTGGTG	19
间隔插入4G Intervening 4G（4G）	CGGTGGTGGGGGCGGTGGTG	20
间隔插入5G Intervening 5G（5G）	CGGTGGTGGGGGGCGGTGGTG	21

图1 四环素双价适配体非酶免标记传感器原理图

Fig. 1 Schematic diagram of tetracycline bivalent aptamer non-enzyme label-free sensor

图2 双价适配体间隔插入序列激发ThT能力评估 A：双价适配体间隔插入不同类型碱基的荧光强度;B：双价适配体间隔插入不同数量G碱基的荧光强度

Fig. 2 Ability of bivalent aptamer spacer insert sequences to trigger ThT fluorescence A：Fluorescence intensity of different types of bases inserted at bivalent aptamer spacer. B：Fluorescence intensity of different numbers of G bases inserted at bivalent aptamer spacer

图3 可行性验证

Fig. 3 Feasibility verification

图4 荧光强度随反应时间变化曲线

Fig. 4 Fluorescence intensity changing curve with reaction time

图5 不同Captamer:CThT 的荧光强度

Fig. 5 Different fluorescence intensity of Captamer:CThT

图6 TC与序列孵育不同时长的荧光变化

Fig. 6 Fluorescence intensity changes of TC and sequence incubation for different lengths

图7 反应体系及离子浓度优化 A：不同缓冲液体系的荧光强度;Tris：20 mmol/L;NaCl：20 mmol/L;KCl：20 mmol/L;MgCl2：20 mmol/L;B：加入靶标前后不同K+浓度条件下荧光强度的变化

Fig. 7 Optimization of reaction system and ion concen-tration A：Fluorescence intensity of different buffer systems;Tris：20 mmol/L;NaCl：20 mmol/L;KCl：20 mmol/L;MgCl2：20 mmol/L. B：Changes of fluorescence intensity under different K+ concentration conditions before and after adding the target

图8 TC传感器的建立 A：不同TC浓度下传感器荧光光谱;B：标准曲线

Fig. 8 Establishment of TC sensor A：Fluorescence spectrum of sensor under different TC concentration. B：Standard curve

图9 特异性检测

Fig. 9 Specific detection

表2 牛奶样品加标回收

Table 2 Standard addition and recovery of milk samples

添加浓度 Added concentration/（nmol·L^-1）	△荧光强度 △ Fluorescence intensity/（I₀-I_F）			回收浓度 Recycling concentration/（nmol·L^-1）			平均浓度 Average concentration/（nmol·L^-1）+相对标准偏差 RSD	回收率 Recovery rate/%
添加浓度 Added concentration/（nmol·L^-1）	1	2	3	1	2	3	平均浓度 Average concentration/（nmol·L^-1）+相对标准偏差 RSD	回收率 Recovery rate/%
20	109.7	110.8	108.4	21.5	22.3	20.5	21.4±0.7	107.1666667
100	155.9	156.0	155.2	103.9	104.1	101.4	103.1±1.2	103.1333334
500	204.1	202.7	203.6	537.7	512.6	510.8	520.4±12.3	104.0733334

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四环素双价适配体非酶免标记传感器

Tetracycline Bivalent Aptamer Non-enzyme Label-free Sensor

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