What factors may affect the effectiveness of using ADPS as a chromogenic substrate?

In the field of biological detection and diagnosis, the chromogenic substrate N-ethyl-N - (3-sulfopropyl) -3-methoxyaniline sodium salt (ADPS reagent) has become a key reagent in enzyme-linked immunosorbent assay (ELISA), biochemical analysis and other scenarios due to its high sensitivity and stability. However, its actual performance is often affected by multiple factors, leading to fluctuations in color intensity, background interference, or poor repeatability.

1, Substrate quality

Chemical purity: The presence of impurities may cause non-specific coloration or competitive inhibition. For example, incompletely reacted intermediates may interfere with the binding of enzymes to substrates, leading to a weakened color signal. Choosing high-purity (≥ 98%) ADPS raw materials is a prerequisite for ensuring effectiveness.

ADPS powder

Particle uniformity: ADPS usually exists in powder or crystalline form. If the particle size is not uniform, it may lead to differences in dissolution rate, affecting the concentration uniformity in the reaction system. Coarse particles may also clog microplates or instrument pipelines, and optimization through grinding or sieving is required.

Storage stability: ADPS is sensitive to light, humidity, and temperature. Long term exposure to strong light may trigger photolysis reactions, producing colorless or low color developing products; However, high humidity environments can cause it to absorb moisture and clump, affecting weighing accuracy. It is recommended to store separately under dark, dry, and low-temperature conditions to avoid repeated freezing and thawing.

2, Reaction conditions

PH value matching: The active center of the enzyme is highly sensitive to pH. If the pH of the buffer deviates from this range, the enzyme activity may decrease, resulting in insufficient ADPS color intensity. Phosphate, carbonate, and other buffer systems should be selected based on the characteristics of the target enzyme, and accurately calibrated using a pH meter.

Temperature control: Increasing the temperature can accelerate molecular motion and increase the collision frequency between enzymes and substrates, but exceeding the optimal temperature can cause enzyme denaturation and inactivation, resulting in a decrease in ADPS color signal.

3, Operational standardization

Dissolution and dilution: ADPS powder should be dissolved in deionized water or specialized buffer solution, avoiding the use of solutions containing metal ions or organic solvents. When dissolving, gently shake or stir to avoid producing bubbles due to violent shaking, which may result in lower actual concentration. Dilute according to a gradient and mix thoroughly to prevent excessive local concentration.

Termination solution selection: After the color reaction is completed, the termination solution should be added immediately to fix the color. If the termination is not timely, the color products may continue to decompose, resulting in a decrease in absorbance values; However, the pH or concentration deviation of the termination solution may alter the spectral characteristics of the product, affecting the accuracy of detection.

4, External interference factors

Sample interference: Hemoglobin, lipids, or drug metabolites in blood samples may inhibit enzyme activity through adsorption or chemical reactions, or directly bind to ADPS to form colorless complexes.

Reagent cross reaction: If there are compounds with structures similar to ADPS in the detection system (such as other aniline substrates), it may cause non-specific color development.

Container contamination: If the microplate or colorimetric dish is not thoroughly cleaned, it may retain enzymes or substrates from the previous batch, leading to false positive results.

The expired use of ADPS powder not only leads to distortion of experimental data, but also may cause safety hazards. Therefore, when purchasing and using ADPS powder, it is important to pay attention to the production date and shelf life, and scientifically handle expired ADPS powder. Hubei Xindesheng Material Technology Co., Ltd. specializes in producing new Trinder's reagents such as ADPS. If you have any related needs, please feel free to contact me at any time!

Product packaging

The expired use of ADPS powder not only leads to distortion of experimental data, but also may cause safety hazards. Therefore, when purchasing and using ADPS powder, it is important to pay attention to the production date and shelf life, and scientifically handle expired ADPS powder. Hubei Xindesheng Material Technology Co., Ltd. specializes in producing the new Trinder's reagents such as ADPS. If you have any related needs, please feel free to contact me at any time!