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荧光探针置换型核酸适配体传感器检测生物小分子

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  • 国家科技期刊平台
  • NETL
  • NSTL
  • 万方数据
利用硫磺素T(ThT)作为置换型荧光探针,分别构建了 3种核酸适配体生物传感器,用于赭曲霉毒素A(OTA)、黄曲霉毒素B1(AFB1)和腺苷(Adenosine)3种生物小分子的快速检测.当靶标分子不存在时,ThT与核酸适配体结合形成复合物,具有较强的荧光响应;加入靶标分子后,ThT的荧光强度减弱,通过监测荧光信号的变化实现对靶标分子的定量检测.详细探究了检测机理,圆二色光谱分析结果表明,加入ThT和靶标分子后,核酸适配体的构象没有明显变化,只是此构象比例增加,因此系统的荧光强度的降低并非由于靶标诱导核酸适配体结构变化,而是因为分子置换作用所致;通过等摩尔连续变化法测定ThT与OTA、AFB1、腺苷的核酸适配体的化学计量比分别为1∶1、1∶1和2∶1,并且解离常数均大于靶标分子与核酸适配体的解离常数.因此,本方法的检测原理是靶标分子置换与核酸适配体结合的荧光探针(ThT),通过测定被靶标分子置换出的ThT荧光强度的变化实现对靶标分子的定量检测.在最优实验条件下,3种小分子的检出限分别为0.8 nmol/L(OTA)、1.3 nmol/L(AFB1)和0.1 μmol/L(腺苷).本方法无需标记、操作简单、检测成本低,具有良好的选择性和较低的检出限.进一步开发了基于本方法的检测试剂盒,可用于实际样品中3种生物小分子的检测.
Aptasensor for Detection of Small Molecules Based on Displacement Fluorescent Probe
By using thioflavin T(ThT)as displacement-based fluorescent probes,three kinds of aptasensors were constructed for rapid detection of three kinds of small molecules such as ochratoxin A(OTA),aflatoxin B1(AFB1)and adenosine.In the absence of target molecule,ThT bound with the aptamer to form an aptamer-ThT complex and exhibited a significant fluorescence response.Upon the addition of target molecule,because of the higher affinity between target and aptamer than that between ThT and the aptamer,ThT was displaced by the target molecule from the aptamer-ThT complex,resulting in weakened fluorescence signal.Based on this principle,the target molecule could be detected quantitatively.Further study through circular dichroism spectra showed that there was no significant change in the conformation of the aptamer after addition of ThT or target molecules.The stoichiometric ratios of ThT to OTAapt,AFB1apt and Adeapt measured through the method of equimolar continuous variation was 1∶1,1∶1 and 2∶1,respectively,and their dissociation constants were all larger than those between the target molecule and its aptamer.Therefore,the principle of this detection method was the displacement of fluorescent probe(ThT)in aptamer-ThT complex by target molecule,resulting in decrease of fluorescence intensity.Under optimal experimental conditions,the limits of detection(LODs)were 0.8 nmol/L for OTA,1.3 nmol/L for AFB1,and 0.10 μmol/L for adenosine,respectively.This method was label-free,simple to operate,with low cost,good selectivity and high sensitivity.The developed assay kit based on this method could be used for actual sample detection.

AptasensorDisplacement fluorescent probeThioflavin TOchratoxin AAflatoxin B1Adenosine

杨成、崔胜楠、王越、王国峰、李程铭、谷双超、薛长颖

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大连理工大学化学学院 大连 116024

大连理工大学生物工程学院 大连 116024

核酸适配体传感器 置换型荧光探针 硫磺素T 赭曲霉毒素A 黄曲霉毒素B1 腺苷

国家自然科学基金中央高校基本科研业务费专项中央高校基本科研业务费专项中央高校基本科研业务费专项电分析化学国家重点实验室开放基金农业部生物毒素检测重点实验室开放基金大连理工大学大学生创新创业训练计划

21876017DUT22YG120DUT21LAB130DUT17LAB18SKLEAC20210520170220221014110489

2024

10.19756/j.issn.0253-3820.231159

分析化学
中国化学会 中国科学院长春应用化学研究所

分析化学

CSTPCD北大核心
影响因子：1.423
ISSN：0253-3820
年,卷(期)：2024.52(5)
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