Stabilization method of enzyme and chromogenic substrate in Trinder reaction

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In the field of in vitro diagnostics, enzyme colorimetry is a common method for quantitative or qualitative testing of target components, and it is widely used in China, such as the detection method of the color reaction of the enzyme HRP catalyzed by the color substrate TOOS in the Trinder reaction. Because of the participation of enzymes, it is very valuable to improve the stability of the enzyme activity color substrate.


Some chromogenic substrates with high molar absorbance of reaction products, such as TOOS, TOPS, etc., can be used in wet chemical methods as well as dry chemical methods: the required reaction reagents (TOOS, 4-AAP) and the required enzymes (HRP) Etc.) It is fixed on the membrane carrier, and the sample is added dropwise to generate H2O2 and then release new ecological oxygen, oxidize the color substrate, and indirectly determine the target component through the amount of the reaction product. Enzymes are highly specific and highly efficient in catalysis. However, they have poor stability under the influence of temperature, pressure, and light in dry chemical methods. In wet chemical methods, enzymes are easily inactivated when they are in a liquid state or at low concentrations.


Chromogenic substrate TOOS powder


On the other hand, most chromogenic substrates, such as TOOS, MAOS, TMB, etc., are also easily oxidized slowly, so their solutions cannot be exposed to the air for a long time. There are many ways to improve the stability of biologically active enzymes and chromogenic substrates:

1. Adding enzyme solution protective agent can make the activity of various enzymes such as ALT, AST, LDH, ALP, CK in aqueous solution or serum matrix stable for 2 weeks at room temperature, but the effect on dry chemical test paper is not significant.

2. The color-developing substrate stabilization method uses surfactants and flavonoid pigment substances with an alkyl group of 8 to 16 carbon atoms, but the formula is complicated, the reagents are difficult to buy, and the protective agent interferes with the test results.

3. There is a protective agent that is more reducible than the color-developing substrate, but the added protective agent contains primary amino groups and often causes non-specific reactions.

4. The use of azo dyes as stabilizers for the color-developing substrate has a good stabilizing effect and does not cause interference, but the maximum absorption wavelength of the dye is sometimes close to that of the color-developing agent, which makes the blank control a bit higher.

5. A polymer and its composition protective agent, which can improve the stability of enzymes and chromogenic substrates. Applicable chromogenic substrates include TOOS, 4-AA, TOPS, MAOS, etc., suitable for HRP, CHO, etc. Enzyme, suitable for the detection of glucose, creatinine, uric acid, cholesterol, triglyceride and other indicators.


It can be seen that various existing protective agents for enzymes or chromogenic substrates, some of which are defective, such as high cost, biased detection, and only suitable for wet chemical methods, etc., thus improving the stability of enzymes and chromogenic substrates The method needs further research. Desheng is a manufacturer of chromogenic substrates and enzymes. It is recommended that you pay more attention to the use of chromogenic substrates and enzymes.