A stable water-soluble tetramethylbenzidine-2-hydroxypropyl-β-cyclodextrin inclusion complex and its applications in enzyme assays

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°C by forming a water-soluble inclusion complex with 2-hydroxypropyl-β-cyclodextrin (hp-β-CyD). Cyclic voltammetry and absorbency measurement were employed to characterize the TMB-hp-β-CyD complex. Well-defined cyclic voltammograms of TMB exhibited two oxidation waves which merged into a single wave with increasing hp-β-CyD concentrations. Cyclic voltammetry was then used to examine the effect of complexation with hp-β-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-β-CyD/horseradish peroxidase system to the respective oxidase enzymes with the formation of either a blue (absorption coefficient of 35,800 M^-1cm^-1 at 650 nm) or a yellow color (absorption co-efficient of 67,300 M^-1cm^-1 at 450 nm) as an indication of the metabolite concentration. These assays possessed a sensitivity limit below 10 μM and the results obtained were in excellent agreement with standard enzymatic assays when tested in various food and clinical samples.