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Application of a dsDNA imprinted polymer to detection for bacterial toxin

双链DNA印迹聚合物在细菌毒素检测中的应用

基本信息

批准号：
18350080
负责人：
MINOURA Norihiko
金额：
$ 11.15万
依托单位：
Tokyo University of Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18350080/
关键词：
Molecular imprinting Polymer gel Electrophoresis dsDNA DNA sequence Molecular recognition Template Verotoxin 分子認織ペロ毒素

项目摘要

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.

分子印迹聚合物（MIPs）是一种对目标分子具有人工识别空腔的材料。以2-乙烯基-4，6-二氨基-1，3，5-三嗪（VDAT）为功能单体，制备了一种可识别dsDNA序列的MIP，并将其应用于电泳凝胶基质中。为了实现检测装置的小型化、样品的无标记化和真实的实时检测，我们以VDAT为功能单体，构建了一种新型的双链DNA印迹聚合物凝胶检测体系。在该系统中使用微型尺寸的凝胶基质。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。在该系统中，通过MIP凝胶中结合位点的捕获效应，与非印迹聚合物凝胶相比，具有靶序列的dsDNA的洗脱时间应被延长。在最佳测定条件下，λ DNA 564 bp片段印迹聚合物凝胶洗脱时间明显延长，并应用于细菌双链DNA的检测。但未检测到目的细菌dsDNA。细菌dsDNA的大小（34 bp）比λDNA的大小（564 bp）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的大小而增加。我们正计划研究目标双链DNA的大小与检测限之间的关系。