Cattaneo M V, Luong J H
Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec.
Anal Biochem. 1994 Dec;223(2):313-20. doi: 10.1006/abio.1994.1590.
3,3',5,5'-Tetramethylbenzidine (TMB), a hydrophobic and noncarcinogenic chromogen with a high absorption coefficient widely used in solid-phase assays involving labeled horseradish peroxidase was rendered soluble (up to 40 mM) and more stable for at least 2 months at 22-24 degrees C by forming a water-soluble inclusion complex with 2-hydroxypropyl-beta-cyclodextrin (hp-beta-CyD). Cyclic voltammetry and absorbency measurement were employed to characterize the TMB-hp-beta-CyD complex. Well-defined cyclic voltammograms of TMB exhibited two oxidation waves which merged into a single wave with increasing hp-beta-CyD concentrations. Cyclic voltammetry was then used to examine the effect of complexation with hp-beta-CyD on the oxidation potential of TMB and provided evidence of a 1:1 complex between TMB and the cyclodextrin molecule with a formation constant of 1.6 M-1. Enzyme assays for D-glucose, lactate, and glutamate were performed by coupling the TMB-hp-beta-CyD/horseradish peroxidase system to the respective oxidase enzymes with the formation of either a blue (absorption coefficient of 35,800 M-1 cm-1 at 650 nm) or a yellow color (absorption coefficient of 67,300 M-1 cm-1 at 450 nm) as an indication of the metabolite concentration. These assays possessed a sensitivity limit below 10 microM and the results obtained were in excellent agreement with standard enzymatic assays when tested in various food and clinical samples.
3,3',5,5'-四甲基联苯胺（TMB）是一种疏水性、无致癌性的显色剂，具有高吸收系数，广泛用于涉及标记辣根过氧化物酶的固相分析。通过与2-羟丙基-β-环糊精（hp-β-CyD）形成水溶性包合物，TMB可溶解（浓度高达40 mM），并在22-24℃下至少2个月内更稳定。采用循环伏安法和吸光度测量对TMB-hp-β-CyD复合物进行表征。TMB明确的循环伏安图显示出两个氧化波，随着hp-β-CyD浓度的增加，这两个氧化波合并为一个单一的波。然后使用循环伏安法研究与hp-β-CyD络合对TMB氧化电位的影响，并提供证据表明TMB与环糊精分子形成1:1复合物，形成常数为1.6 M-1。通过将TMB-hp-β-CyD/辣根过氧化物酶系统与各自的氧化酶偶联，对D-葡萄糖、乳酸和谷氨酸进行酶分析，形成蓝色（在650 nm处吸收系数为35,800 M-1 cm-1）或黄色（在450 nm处吸收系数为67,300 M-1 cm-1）作为代谢物浓度的指示。这些分析的灵敏度极限低于10 microM，在各种食品和临床样品中测试时，所得结果与标准酶分析结果非常一致。
