The difference between Trinder’s reagent and TMB in the chromogenic substrate of POD/HRP enzyme

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Peroxidase POD or HRP is often used in enzyme-linked immunoassay to detect the biochemical indicators that can generate hydrogen peroxide, such as blood glucose testing, blood lipid testing, uric acid testing, etc., which can generate hydrogen peroxide, and POD/HRP enzyme color base The commonly used reagents are the new Trinder's reagent and TMB reagent. What is the difference between them?


In the presence of peroxidase, both Trinder's reagent and TMB can be oxidized by hydrogen peroxide (generated by the reaction of blood sugar, blood lipids, uric acid, etc.) to produce colored products. The concentration of the product and the amount of hydrogen peroxide produced and the amount to be measured The index content is proportional, so the colored product can be detected by a spectrophotometer to calculate the content of the index to be measured.


The difference between Trinder’s reagent and TMB in the chromogenic substrate of POD/HRP enzyme


Chromogenic new Trinder’s reagent color development principle:

First of all, the new Trinder's reagent of chromogen is similar to traditional chromogen phenol and aniline. Take N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline sodium salt ADPS as an example. Coupling reaction with 4-AA (4-aminoantipyrine) in the presence of hydrogen and POD, the N of aniline becomes a C=N double bond, and the amino group of 4-AA at the para position is combined to form a C=N double bond , Thus forming a quinone imine structure, similar to the C=O double bond of p-benzoquinone, the absorption wavelengths are all in the visible light region, thereby producing a color reaction. So the reaction here must involve 4-AA (or MBTH) to participate.


Chromogenic TMB reagent color development principle:

The chromogenic substrate TMB (TMBZ) is widely used in ELISA kits. It appears blue under the catalysis of horseradish peroxidase and turns yellow after adding stop solution. Measure the OD value with a microplate reader at 450nm wavelength. The antigen concentration is proportional to the OD value. The concentration of the antigen in the sample is calculated by drawing a standard curve. It does not need chromogen ADPS, TOOS, MAOS, etc. It needs to introduce another compound (such as 4-AA, MBTH) for color reaction, but TMBZ needs to perform color reaction under acidic conditions (HCl), which limits its application.


The new Trinder's reagent and TMB in the peroxidase substrate can be used in the kit. The principle is similar. The color reaction of TMB turns from blue to yellow after adding the stop solution. The measurement wavelength is 450nm, while Trinder's reagent develops color. The product is red, with an absorption wavelength above 540nm, and MADB and MAOS have reached 630nm, with stronger anti-interference ability. In addition to developing the chromogenic substrate for HRP, Desheng also has the luminescent substrate luminol and the direct luminescent reagent acridinium ester, which have higher sensitivity and more accurate detection.