Application of a dsDNA imprinted polymer to detection for bacterial toxin

分子印迹聚合物(MIP)是一种极具研究应用价值的材料，因为它们对目标分子有一个人工识别的空腔。以2-乙烯-4，6-二氨基-1，3，5-三嗪(VDAT)为功能单体，制备了识别特定dsDNA序列的分子印迹聚合物(MIP)，并将其应用于凝胶基质中。为了实现检测装置的小型化、样品的无标记和实时检测，我们以VDAT为功能单体，构建了一种新型的dsDNA印迹聚合物凝胶双链DNA检测系统。该系统采用了微米级凝胶基质。电泳过程中通过MIP凝胶洗脱的dsDNA在凝胶边缘被荧光分子染色，并通过载体溶液被带到荧光检测器。在该体系中，由于MIP凝胶中结合位点的捕获作用，与非印迹聚合物凝胶相比，dsDNA与目标序列的洗脱时间应该延长。在最佳测定条件下，λDNA 564bp片段在印迹聚合物凝胶中的洗脱时间延长，并将该体系应用于细菌λ的检测。然而，没有检测到目标细菌的dsDNA。细菌双链DNA的大小(34bp)比λ的564bp小。这表明DNA大小对检测目标dsDNA有一定的影响，因为随着目标dsDNA大小的增加，MIP凝胶中结合位点周围的官能团数目也增加。我们计划在该系统中研究目标dsDNA大小与检测限之间的关系。

Molecularly imprinted polymers(MIPs) are materials that are attractive for research applications because they have an artificial recognition cavity for the target molecule. We prepared a MIP for recognizing a specific dsDNA sequence by using 2-vinyl-4, 6-diamino-1, 3, 5-triazine(VDAT) as a functional monomer and used it in an electrophoretic gel matrix.Firstly, interaction between VDAT analog as a new functional monomer and dsDNA was investigated to improve the specificity of target dsDNA to MIP gel. From the result, some candidate compounds were selected as new functional monomer and application of these compounds to MIP is now in progress.To achieve a miniaturization of detection device, a label-free sample, and a real time detection, we demonstrate a novel dsDNA detection system with dsDNA-imprinted polymer gel by using VDAT as a functional monomer. The miniature-sized gel matrix was used in this system. The dsDNA eluted through the MIP gel during electrophoresis was stained with fluorescence molecule at an edge of the gel and was carried to fluorescence detector by carrier solution. In this system, elution time of dsDNA with the target sequence should be elongated in comparison with the non-imprinted polymer gel by the capture effect of the binding sites in the MIP gel. Under optimum measurement condition, the elongation of elution time on λDNA 564bp fragment was confirmed in a λDNA 564bp fragment-imprinted polymer gel.Application of this system to a bacterial dsDNA detection was also investigated. However, the target bacterial dsDNA was not detected. The DNA size of bacterial dsDNA(34bp) is smaller in comparison with the λDNA 564bp fragment. It suggests that there is an influence of size of DNA on detection of target dsDNA, since the number of functional groups surrounding the binding sites in MIP gel increases with increasing the size of target dsDNA. We are planning to study the relationship between size of target dsDNA and detection limit in this system.