The meaning of detection wavelength of different substrates of enzymes such as Trinder's reagent
Release time:
2021-01-31
In biochemical diagnosis, the substrate used in enzyme-linked immunoassay is a key factor in determining the indicators to be tested, and often determines the accuracy and sensitivity of the test. After Trinder's reagent, NADP, PNPG and other substrates react to produce colored products, the determination can be achieved by detecting the light absorption at the maximum absorption wavelength of the product.
For different substrates, the maximum absorption wavelength of the color product is different. If the sample to be tested is more complicated, and one of the components is close to the detected wavelength, it is easier to cause interference, which requires the reaction of the substrate. The maximum absorption wavelength is known.
Detection wavelength of different substrates of enzymes such as Trinder's reagent
Detection wavelength of different substrates:
1. NADH and NADPH have characteristic absorption peaks at 340nm, while NAD+ and NADP+ have characteristic absorption peaks at 340nm, while NAD+ and NADP+ have no characteristic absorption peaks at 340nm. Use their coupled dehydrogenase (tool enzyme) to react According to the change of absorbance at 340nm, the concentration or activity of the substance can be measured. The corresponding enzymes are glutamate dehydrogenase, lactate dehydrogenase, malate dehydrogenase, glucose dehydrogenase, steroid dehydrogenase and so on.
2. Trinder's reagent can be coupled with 4-AA and MBTH to produce colored products under the action of peroxidase. The maximum absorption wavelength of the product and the set detection wavelength are relatively high, such as ADPS, ADOS, TOOS, and ALPS at 540- 560nm, DAOS, HDAOS are about 590nm, and MAOS and MADB are relatively high, reaching 630nm. The commonly used TMB in ELISA kits is 450nm, which is much lower than Trinder's reagent.
3. Other chromogens such as p-NP p-nitrophenol phosphate and p-NA p-nitrophenol are also used in the detection of alanine aminotransferase and acid phosphatase. p-NA has a characteristic absorption peak at 405 nm.
It should be noted that the detection wavelength here is the absorption wavelength of the substrate or the product after the reaction, which is not consistent with the color of the reagent or solution, but is complementary. For example, the absorption wavelength of the MAOS product is in the orange or red-yellow wavelength band at 630nm, and its solution is blue-green. The ADPS product has a maximum absorption wavelength of 540nm in the green band, and the solution is purple-red.
In addition, in addition to the chromogenic substrate, the enzyme substrate also has recently popular luminescent substrates, such as luminol and isoluminol. The difference is that luminol emits light by itself, and the detection wavelength is the wavelength of the emitted light. , And the detection wavelength of Trinder's reagent is the absorption wavelength, which needs to be distinguished.
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