Tetracycline Bivalent Aptamer Non-enzyme Label-free Sensor

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

Abstract

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

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.

Figures/Tables 11

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

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

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

Fig. 3 Feasibility verification

Fig. 4 Fluorescence intensity changing curve with reaction time

Fig. 5 Different fluorescence intensity of Captamer:CThT

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

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

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

Fig. 9 Specific detection

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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