Wu Xiao, Diao Donglin, Lu Zhangwei, Han Yu, Xu Shi, Lou Xinhui
Department of Chemistry, Capital Normal University.
College of Life Sciences, Capital Normal University.
J Vis Exp. 2018 Mar 21(133):56814. doi: 10.3791/56814.
Phthalic acid esters (PAEs) areone of the major groups of persistent organic pollutants. The group-specific detection of PAEs is highly desired due to the rapid growing of congeners. DNA aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward highly hydrophobic small molecule targets, such as PAEs, is rarely reported. This work describes a bead-based method designed to select group-specific DNA aptamers to PAEs. The amino group functionalized dibutyl phthalate (DBP-NH2) as the anchor target was synthesized and immobilized on the epoxy-activated agarose beads, allowing the display of the phthalic ester group at the surface of the immobilization matrix, and therefore the selection of the group-specific binders. We determined the dissociation constants of the aptamer candidates by quantitative polymerization chain reaction coupled with magnetic separation. The relative affinities and selectivity of the aptamers to other PAEs were determined by the competitive assays, where the aptamer candidates were pre-bounded to the DBP-NH2 attached magnetic beads and released to the supernatant upon incubation with the tested PAEs or other potential interfering substances. The competitive assay was applied because it provided a facile affinity comparison among PAEs that had no functional groups for surface immobilization. Finally, we demonstrated the fabrication of an electrochemical aptasensor and used it for ultrasensitive and selective detection of bis(2-ethylhexyl) phthalate. This protocol provides insights for the aptamer discovery of other hydrophobic small molecules.
邻苯二甲酸酯（PAEs）是主要的持久性有机污染物类别之一。由于同系物数量迅速增加，对PAEs进行特定类别检测的需求极为迫切。DNA适配体已越来越多地被用作生物传感器平台上的识别元件，但针对高疏水性小分子靶标（如PAEs）筛选适配体的报道却很少。这项工作描述了一种基于磁珠的方法，旨在筛选针对PAEs的特定类别DNA适配体。合成了氨基功能化的邻苯二甲酸二丁酯（DBP-NH2）作为锚定靶标，并将其固定在环氧活化的琼脂糖磁珠上，使邻苯二甲酸酯基团展示在固定基质表面，从而筛选出特定类别的结合物。我们通过定量聚合链反应结合磁分离来测定候选适配体的解离常数。通过竞争分析来确定适配体对其他PAEs的相对亲和力和选择性，在竞争分析中，将候选适配体预先结合到附着有DBP-NH2的磁珠上，然后在与测试的PAEs或其他潜在干扰物质孵育后释放到上清液中。采用竞争分析是因为它为没有用于表面固定的官能团的PAEs之间提供了便捷的亲和力比较。最后，我们展示了一种电化学适配体传感器的制备，并将其用于超灵敏和选择性检测邻苯二甲酸二（2-乙基己基）酯。该方案为其他疏水性小分子的适配体发现提供了思路。
