有机污染物的传统检测方法需要使用色谱、质谱、光谱等大型仪器。这些仪器不仅价格昂贵，且操作复杂，对技术人员要求相应较高。这些问题制约了此类方法在日常分析检测中的应用。而公众需要便携的日常检测手段来认知环境样品或食品中污染物的含量，以便更好地保障公共安全。因此，本文研究了基于核酸适配子和血糖仪的污染物便携检测技术，这种方法可用于环境污染物的便携检测，具有用户友好、快速、廉价的特点。 论文首先进行了核酸适配子的筛选工作。结构转换SELEX筛选环孢霉素A（CsA）核酸适配子的筛选结果表明，用该法进行CsA核酸适配子的筛选过程中，随着筛选轮数的推进，随机DNA文库的中间序列发生了碱基缺失和突变，这导致了随机DNA文库的退化，阻碍了筛选的进行。因此，研究了普通SELEX筛选的关键步骤，通过NH2-PEG-SH，CsA被成功地固定到琼脂糖树脂上，用所合成的CsA-琼脂糖树脂可进行CsA核酸适配子的普通SELEX。 然后进行了核酸适配子的截短研究。Rd29C33是我们前期用结构转换SELEX所筛的三聚氰胺核酸适配子，然而其长度较长（88 nt），从技术经济性上考虑，该核酸适配子并不利于实际应用。本研究对Rd29C33进行了截短。等温热滴定（ITC）结果表明，Rd29C33与三聚氰胺、三聚氰酸、三聚氰酸一酰胺以及三聚氰酸二酰胺结合的解离常数分别为0.91±0.13 μM、423±16 μM、8130 μM及1383 μM。而经截短所得的三聚氰胺核酸适配子Rd29C33-T7（34 nt）与三聚氰胺结合的解离常数则为0.95±0.03 μM，同时，该核酸适配子对三聚氰胺的结构类似物无亲和作用。 第三，用圆二色谱、非变性PAGE及荧光光谱对Rd29C33、Rd29C33-T6和Rd29C33-T7的结构进行了表征，结果表明这三种核酸适配子可能形成了正向平行的G-四聚体结构。在此基础上，利用其荧光光谱建立了一种免标记的三聚氰胺生物检测方法。 最后，在上述对核酸适配子研究的基础上，构建了基于核酸适配子和血糖仪的污染物生物便携检测传感器。分别选择三聚氰胺（有害食品添加剂）、赭曲霉素A（生物毒素）及奎宁（PPCPs）为靶标，建立了检测这三种物质的便携检测方法。用核酸适配子和血糖仪检测三聚氰胺、赭曲霉素A和奎宁的检测限分别为0.33 μM、3.31 μg/L和0.14 μM。
Conventional detection methods for environmentally relevant organic pollutants include chromatography, mass spectrometry et al. Although these methods can provide accurate information about environmental samples, they require the use of expensive instruments, careful calibration and a relatively long time to obtain results, making it difficult for on-site and real-time detection. However, to ensure the public safety, it is important to develop easier detection methods which are both cheap and convenient to use by the public. In this thesis, we used DNA aptamers and a commercially available personal glucose meter (PGM) to detect environmentally relevant pollutants. As aptamer is the key element of all aptasensors, we were firstly focused on the study of the aptamer. Structure-switching SELEX was employed in the isolation of aptamer against CsA. The results indicated that mutations and deletions of DNA bases in the immobilization sequence region of the random DNA pool occurred as the selection proceeded. As a result, the selection was hindered by the degenerated DNA pool. To allow the successful isolation of CsA aptamer using the conventional SELEX, we further immobilized CsA onto the agarose resin through the NH2-PEG-SH linker. Rd29C33 was a melamine aptamer which was selected by structure-switching SELEX by us previously. Although this aptamer showed high structure-switching activity, we know nothing about its affinity against melamine. In this thesis, ITC was employed to determine the affinity between aptamers and its targets. ITC results indicated that the dissociation constant of Rd29C33 to melamine was 0.91±0.13 μM while dissociation constants of 423±16 μM，8130 μM and 1383 μM were respectively obtained towards cyanic acid, ammelide and ammeline. Rational truncations of Rd29C33 (88 nt) was conducted according to the structural prediction. After the truncation, a truncation variant Rd29C33-T7 ( 34 nt) was obtained with a dissociation constant of 0.95±0.03 μM against melamine and no binding to the melamine analogues. Afterwards, different methods including CD, native PAGE and fluorescence spectrometry were employed to determine the structure of different melamine aptamers. These results indicated that melamine aptamers may have formed parallel G-quadruplex. A label-free biosensor for melamine detection was then developed using Rd29C33-T7 and TO. Finally, portable detection of different environmentally relevant contaminants based on DNA aptamers and a PGM were realized. Three contaminants including melamine (one kind of hazardous food addictives), OTA (one kind of biotoixins) and quinine (one kind of PPCPs) were chosen as the detection targets. Detection limits of 0.33 μM, 3.31 μg/L and 0.14 μM were obtained for melamine, OTA and quinine, respectively.
