Application of a dsDNA imprinted polymer to detection for bacterial toxin

分子印迹聚合物（MIPs）是一种有吸引力的研究应用材料，因为它们具有对目标分子的人工识别腔。我们以2-乙烯基- 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MIP，并将其应用于电泳凝胶基质中。首先，研究了VDAT类似物作为一种新的功能单体与dsDNA的相互作用，以提高靶dsDNA对MIP凝胶的特异性。由此筛选出一些候选化合物作为新的功能单体，并将其应用于MIP中。为了实现检测设备的小型化、无标签样品和实时检测，我们展示了一种新的dsDNA印迹聚合物凝胶检测系统，该系统以VDAT为功能单体。该系统采用了微型凝胶基质。在电泳过程中，通过MIP凝胶洗脱的dsDNA在凝胶边缘用荧光分子染色，并通过载体溶液携带到荧光检测器上。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，具有目标序列的dsDNA的洗脱时间应该延长。在最佳测量条件下，在λDNA 564bp片段印迹聚合物凝胶中证实了λDNA 564bp片段洗脱时间的延长。并对该系统在细菌dsDNA检测中的应用进行了研究。然而，没有检测到目标细菌的dsDNA。细菌dsDNA（34bp）比λDNA （564bp）片段更小。这表明DNA的大小对靶dsDNA的检测有影响，因为MIP凝胶中结合位点周围的官能团数量随着靶dsDNA大小的增加而增加。我们计划在该系统中研究目标dsDNA的大小与检出限的关系。

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.