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京都大学0048新制・課程博士博士(工学)甲第13839号工博第2943号新制||工||1435(附属図書館)UT51-2008-C755京都大学大学院工学研究科物質エネルギー化学専攻(主査)教授 垣内 隆, 教授 小久見 善八, 教授 江口 浩一学位規則第4条第1項該当Doctor of EngineeringKyoto UniversityDA

Dopamine as a polymerizable reagent for enzyme-linked immunosorbent assay using horseradish peroxidase

We demonstrate that dopamine can be used as a reagent for colorimetric enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase (HRP). Dopamine was able to be polymerized in the presence of HRP and H2O2, and black polydopamine was obtained after the enzymatic reaction. Because of the black color, the absorbance was significantly changed in the whole range of the visible light region. Here, an indirect competitive ELISA based on the polymerization of dopamine was performed to detect a fluoroquinolone antibiotic, enrofloxacin. The antibiotic is commonly used in livestock farming. The anti-antibiotics antibody was produced from egg yolk from chicken hens. In the visible range, sufficient absorbance changes of ∼0.4∼0.5 and a low background level for the ELISA response were obtained, and the 50 % inhibitory concentration value at 450 nm was determined to be 26 ppb. The performance of the indirect competitive ELISA based on the polymerization of dopamine was compared to that based on the oxidation of catechol because dopamine has a catechol skeleton. By the complex of HRP and H2O2, catechol can be oxidized to o-benzoquinone having a maximum absorption wavelength of 420 nm. It was shown that the absorbance change in the case of polydopamine was about 2.5 times higher than that of catechol, where the background levels were similar. This confirms that the polymerization of dopamine significantly enhanced the photosignal