Comparison of the performance of four major chemiluminescent substrates
Chemiluminescence is a phenomenon of light radiation accompanied by substances in the process of chemical reactions. The simplest chemiluminescence reaction consists of two key steps: excitation and radiation. For example, the two substances A and B undergo a chemical reaction to produce substance C. The energy released by the reaction is absorbed by the molecules of substance C and transitions to the excited state C*. The excited C* generates light radiation (luminescence) during the process of returning to the ground state. .
The first luminescent substrate used was used in conjunction with horseradish peroxidase to emit light. Without adding enhancer, its luminous system is flash type and the signal is weak. The advantages are: low substrate synthesis difficulty, and currently the most widely used.
Advantages: In an alkaline environment, the degree of non-enzymatic hydrolysis of AMPPD is low, that is, the background is low; AMPPD has good thermal stability, in pH=7.0 water, the decomposition half-life at 30°C is 142h, and the activation energy is 21.5kcal/mole; the enzymatic luminescence of AMPPD is glow type, the wavelength is 470nm, the intensity reaches the peak at 15min, the light signal intensity remains the same within 15-60min, the change is small, even after 12h it can still be measured correctly Results: Adding enhancers such as polyvinyl chloride benzyl dimethyl ammonium (BDMQ) or BSA, etc., can significantly enhance the luminous intensity of AP enzymatic hydrolysis of AMPPD, the enhancement factor is 100-100000 times. The overall performance is stronger than luminol.
Advantages: Chemiluminescence does not require a catalyst, and can emit light in a dilute alkaline solution with H2O2. It has many advantages, especially without a catalytic process and no enhancer, thereby reducing background luminescence and improving signal-to-noise ratio , Less interference. As a luminescent marker for chemiluminescence immunoassay, this kind of compound also has other advantages, such as rapid concentration of light release, high luminous efficiency, high luminous intensity, easy to connect with protein, and the photon yield does not decrease after the connection, and the label is stable It can be stored for several months at 2-8°C, so acridine ester or acridine sulfonamide is a very effective chemiluminescent marker.
Disadvantages: produces unstable, intermittent, and flickering luminescence; its detection method requires in-situ sampling and time integration measurement, that is, install a sampler at the detector position, and ensure that the two processes of adding luminescent agent and detection are synchronized get on.
Advantages: Fast detection time, controllable reaction conditions, wide detection range, high precision and high sensitivity, and low sample detection volume.
Disadvantages: The flow colorimetric cell used in the instrument is a potential problem with cross-contamination; it is too sensitive to environmental factors and other non-specific reactions; the detection instrument is expensive.
In chemiluminescence analysis, the luminescence intensity of acridine ester is influenced by various factors, such as reaction medium, temperature, time, and excitation light source energy. To achieve good detection results, it is necessary to comprehensively consider and optimize these factors. Meanwhile, attention should be paid to controlling and standardizing experimental conditions to ensure accurate and reliable results. Thoroughly studying these influencing factors will help promote the development of chemiluminescence analysis methods.