基于荧光分子释放的DNA水凝胶的真菌毒素适配体超灵敏传感器的研究

Since food safety problems attract more and more attention, it is highly desired to develop more sensitive,selective，rapid and convenient detection methods in food safety field.Ochratoxin A and aflatoxin are mycotoxins and naturally occurring toxins produced by certain fungi that can grow on foods such as cereals, nuts, dried fruits, spices, legumes and others under certain environmental conditions.In order to develop an ultrasensitive aptasensor for the mycotoxins,this project is to design the fluorescent signal molecule carrier by pure DNA hydrogel responsing to target toxins.Unlike the conventional hydrogels, the pure DNA hydrogel including the aptamer sequences for target toxins is an ideal carrier for the design of an "on-command" delivery system because of its distinctive characteristics, such as large load capacity, biocompatibility and controllable conformation. In addition ,the DNA hydrogel is totally assembled from oligonucleotide domains with controllable conformation and size.To achieve the signal amplification,the fluorescent molecules are encapsulated by the pure DNA hydrogel and can be released by the stimulus of the target toxins. This stimulated-release process makes it possible for the detection of very low concentration of target by transfering to the aggregated signal by a large number of fluorescent molecules released. The capacity to encapsulate fluorescent molecules and the sensitivity of target recongnization for the pure DNA hydrogel will be discussed as an evaluation indicator for quality of the aptasensor. The size effect for the background signal of loading fluorescent molecules will be also investigated by changing the diameter of the dye molecule from 1nm to 7nm.The general mechnism of successful design of such a signal amplication strategy will also been discussed. Finally, the construction strategy for such aptasensors we developed for ultrasensitive detection of toxins can be a universal method for analyte detections in food safety and other analytical applications.

近年来世界范围内屡屡发生的食品安全事件，使食品的安全检测倍受关注，发展快速超灵敏的检测方法成为亟待解决的问题。本项目设计一种智能响应的全DNA水凝胶释放荧光信号分子的适配体传感器，实现检测信号的放大，用于食品中的赭菌霉素OTA和黄曲霉素AFT的超灵敏检测。将靶分子适配体序列与骨架DNA序列组装成全DNA水凝胶，同时荧光信号分子封装在其中，再由靶分子识别来调控释放封装的大量荧光信号分子，达到少量靶分子释放大量信号分子的放大过程，从而实现对靶分子的超灵敏检测。重点研究全DNA水凝胶的设计和组装，考察其包埋荧光信号分子的能力以及对靶分子的响应敏感度，以及荧光信号分子的尺寸对检测背景及其释放过程的影响，以揭示由全DNA水凝胶中释放荧光信号分子的信号放大方法的构建规律和内在机理。本项目发展的超灵敏、简单、快速的信号放大方法具有非常重要的科学意义和应用价值，可成为食品安全检测中超灵敏检测的普适方法。

1. 根据基金的内容设计了Y1-S和Y2-S的DNA杂交体系，由OTA的适配体作为连接DNA，形成了DNA水凝胶的控制释放体系，该生物传感器在形成水凝胶的过程中可以将HRP包埋在水凝胶中，作为二次放大的酶分子。当靶分子OTA存在时，与其适配体相结合，使凝胶向溶胶转化，同时释放HRP分子，催化溶液中存在的双氧水氧化ABTS2-的反应，使溶液的颜色发生改变，通过测量体系的吸光度，得到吸光度与OTA浓度的关系曲线，检测溶液中OTA的浓度。利用该传感器，可在0.05-0.5μM范围内实现对OTA的定量检测，检出限为10.8nM。同时完成了实际样品中添加标准液回收率的检测，各个浓度的平均回收率为97.6%-102.2%。本章基于全DNA水凝胶的控制释放体系，构建了非标记、高灵敏的生物传感器，通过靶分子与适配体的结合实现了对赭曲霉素（OTA）定量检测，完成了基金的相关设计及内容。.2. 利用 G -四链体脱氧核糖核酸酶在K+存在下可以与hemin形成具有过氧化物酶性质的复合物，可以催化H2O2氧化ABTS反应，增加溶液的吸光度，进行了T4 PNK酶活性检测的传感器设计。该传感器设计方法简便，不需要对使用的寡核苷酸链进行任何标记，也不需要依赖任何昂贵的仪器。通过采用“分段”向“完整”G - 四链体结构转化的设计模式 ,利用该T4 PNK传感器 , 可在0.02 -0.3U/mL浓度范围内实现 T4 PNK的定量检测，检出限为0.014 U/mL，并完成了对两种细胞系的实际样本检测及标准液添加的回收率检测，各个浓度的平均回收率为95.6%-105.7%.。基于“分段”-“完整”转化的 G - 四链体结构构建了非标记、高灵敏的生物传感器，实现了对T4 PNK酶的定量检测，可望在一定程度上提高传感器对实际样本检测的选择性和灵敏度。通过基金的支持成功地完成了这一类基于G -四链体脱氧核糖核酸酶及工具酶的生物传感器的设计。