Inuyama Yasuhiro, Nakamura Chikashi, Oka Tomohiro, Yoneda Yasuhiro, Obataya Ikuo, Santo Noriaki, Miyake Jun
Biotechnology Research Laboratory, Towa Kagaku Co. Ltd., 6-5 Funairi-machi, Hiroshima 730-0841, Japan.
Biosens Bioelectron. 2007 Apr 15;22(9-10):2093-9. doi: 10.1016/j.bios.2006.09.016. Epub 2006 Oct 30.
The purposes of this study are to construct a simple dioxin detection system using peptides that bind to dioxin, and to test the system on real environmental samples. In this method, dioxin and N-NBD-3-(3',4'-dichlorophenoxy)-1-propylamine (NBD-DCPPA) are competitively bound to the peptides synthesized on beads. The fluorescence intensity of the bead decreases with increasing dioxin concentration. The concentration of dioxin is determined by measuring the fluorescence intensity using a fluorescence microscope equipped with a CCD camera. The fluorescence microscope system was equipped with a motor-driven stage and could be used with 96-well microplates and analytical software that automatically measured the fluorescence intensity of the bead images in the wells. Dioxin detection conditions, reaction temperature, number of beads and concentration of the organic solvent were optimized. About 0.5 nM (150 pg mL(-1)) of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TeCDD) could be detected under the optimized conditions. Environmental soil samples were subjected to the detection system using the peptide beads. Although the results obtained correlated poorly with the toxicity equivalency quantity (TEQ) concentration obtained by a GC/MS method, our method is robust enough as a prescreening method to detect at least 250 pg-TEQ g(-1), the survey level for soil as stipulated in the law concerning special measures against dioxins in Japan.
本研究的目的是构建一个使用与二噁英结合的肽的简单二噁英检测系统，并在实际环境样品上测试该系统。在该方法中，二噁英和N - NBD - 3 - (3',4'-二氯苯氧基)-1 - 丙胺（NBD - DCPPA）竞争性地与珠子上合成的肽结合。随着二噁英浓度的增加，珠子的荧光强度降低。通过使用配备电荷耦合器件（CCD）相机的荧光显微镜测量荧光强度来确定二噁英的浓度。荧光显微镜系统配备了电动载物台，可与96孔微孔板和自动测量孔中珠子图像荧光强度的分析软件一起使用。优化了二噁英检测条件、反应温度、珠子数量和有机溶剂浓度。在优化条件下，可以检测到约0.5 nM（150 pg mL(-1)）的2,3,7,8 - 四氯二苯并 - p - 二噁英（2,3,7,8 - TeCDD）。使用肽珠对环境土壤样品进行检测系统测试。尽管获得的结果与通过气相色谱/质谱（GC/MS）方法获得的毒性当量（TEQ）浓度相关性较差，但我们的方法作为一种预筛选方法足够稳健，能够检测至少250 pg - TEQ g(-1)，这是日本关于二噁英特别措施法规定的土壤调查水平。
