Mv. Cattaneo et Jht. Luong, A STABLE WATER-SOLUBLE AMETHYLBENZIDINE-2-HYDROXYPROPYL-BETA-CYCLODEXTRIN INCLUSION COMPLEX AND ITS APPLICATIONS IN ENZYME ASSAYS, Analytical biochemistry, 223(2), 1994, pp. 313-320
3,3',5,5'-Tetramethylbenzidine (TMB), a hydrophobic and noncarcinogeni
c chromogen with a high absorption coefficient widely used in solid-ph
ase assays involving labeled horseradish peroxidase was rendered solub
le (up to 40 mM) and more stable for at least 2 months at 22-24 degree
s C by forming a water-soluble inclusion complex with 2-hydroxypropyl-
beta-cyclodextrin (hp-beta-CyD). Cyclic voltammetry and absorbency mea
surement were employed to characterize the TMB-hp-beta-CyD complex. We
ll-defined cyclic voltammograms of TMB exhibited two oxidation waves w
hich merged into a single wave with increasing hp-beta-CyD concentrati
ons. Cyclic voltammetry was then used to examine the effect of complex
ation with hp-beta-CyD on the oxidation potential of TMB and provided
evidence of a 1:1 complex between TMB and the cyclodextrin molecule wi
th a formation constant of 1.6 M(-1). Enzyme assays for D-glucose, lac
tate, and glutamate were performed by coupling the TMB-hp-beta-CyD/hor
seradish 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 co-efficient 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 mu M and the resu
lts obtained were in excellent agreement with standard enzymatic assay
s when tested in various food and clinical samples. (C) 1994 Academic
Press, Inc.