Enzymatic oxidation reactions, based on the hydrogen peroxide and peroxidase reactions, have been utilized over several decades for diagnostic analyses because of their high selectivity and sensitivity. To determine the concentration of a specific element in a biological sample, the specificity of the reaction is necessary. A large number of oxidases have been found and used in detection systems. In most cases, the enzymatic reaction of an oxidase generates hydrogen peroxide, and the concentration of hydrogen peroxide indicates the concentration of a substrate in a reaction solution. Therefore, the concentration of substrate can be determined by measuring the amount of hydrogen peroxide. The amount of hydrogen peroxide is determined by spectrophotometer using an oxidative chromogenic dye and peroxidase. Since peroxidase is one of the most commonly used enzymes for enzyme immunoassays (EIA), several oxidative chromogenic dyes are available for staining tissue sections and membrane filters. It can also be utilized for microplate assays. The stability of these oxidative chromogenic dyes, however, is sometimes low due to the oxidation by oxygen. Dojindo offers stable and water-soluble oxidative chromogenic dyes for the hydrogen peroxide (H2O2)-peroxidase assay. The table on page 197 indicates the characteristics of water-soluble aniline derivatives (New Trinder’s Reagents) that are used for hydrogen peroxide detection.
Tetramethylbenzidine (TMBZ) is a commonly utilized oxidative chromogenic dye for peroxidase detection on a membrane filter or an EIA plate. Peroxidase catalyses the reduction of hydrogen peroxide to generate two water molecules. TMBZ is the electron source for this reduction. Since the oxidized forms of TMBZ have intense colors, peroxidase can be determined colorimetrically. Horseradish peroxidase (HRP) is widely applied in EIA and diagnostic analyses. HRP (brown color) reacts with hydrogen peroxide to form Compound I (yellowishgreen color). Then Compound I turns back to HRP by one electron oxidoreduction via Compound II (red color). Most of the chromogenic dyes are easily oxidized with activated oxygen by metals or light. TMBZ is a stable oxidative chromogenic dye utilized for microplate assays. Colorless TMBZ solution is turned blue or greenish-blue by the hydrogen peroxide and peroxidase oxidation reaction. Sulfuric acid (1-2 M) is sometimes added to the assay solution to enhance the color development of oxidized TMBZ. Oxidized TMBZ solution turns from blue to yellow when sulfuric acid is added, and its molar absorptivity is 2 to 3 times that of the blue oxidized TMBZ.
Hydrogen Peroxide Detection
Enzymatic oxidation reactions are highly selective and sensitive enough to measure a specific substrate in complicated mixtures such as biological samples. Due to their selectivity, oxidases have been used in various assay systems established for diagnostic analyses. There are two steps to determining the amount of substrate: 1) the oxidation reaction of the substrate with oxidase, and 2) the detection of hydrogen peroxide generated by the oxidation reaction. Aniline and phenol compounds have been used as oxidative chromogenic reagents for hydrogen peroxide detection. However, they are not stable enough to prepare assay solutions or test papers. New Trinder’s Reagents, which are aniline analogs, have propylsulfonic acid (PS) or hydroxypropylsulfonic acid (OS) attached to an amino group of their aniline structure. These PS or OS groups give aniline water solubility and markedly improved its stability. Trinder’s reagent requires a coupling reagent such as 4-aminoantipyrin or methylbenzothiazolinonehydrazone (MBTH), to produce a stable dye. Dojindo offers various New Trinder’s Reagents for the best combination with oxidase-substrate reactions.
- Reductive Chromogenic Dyes
Code Product name Unit size C440 Reductive Chromogenic Dye
100 mg M009 Reductive Chromogenic Dye
N011 Reductive Chromogenic Dye
T012 Reductive Chromogenic Dye
1 g W201 Reductive Chromogenic Dye
W202 Reductive Chromogenic Dye
100 mg W203 Reductive Chromogenic Dye
100 mg W204 Reductive Chromogenic Dye
100 mg W209 Reductive Chromogenic Dye
10 mg W217 Reductive Chromogenic Dye
- Electron Mediators
Code Product name Unit size A543 Amine Reactive Electron Mediator
10 mg M003 Electron Mediator
100 mg M470 Electron Mediator
- Oxidative Chromogenic Dyes
- Trinder Reagents