The chemiluminescence assay is becoming more and more popular due to its superb detection sensitivity. Commonly used are luminol, isoluminol, and acridine esters. In chemical analysis, chemiluminescence reactions are used to detect analytes directly covalently labeled with chemiluminescence compounds. Triggering a chemiluminescent label for a luminescent reaction generates a signal for the detection of the analyte. The advantage of this method is that it is simpler and clearer, and avoids the need for larger, relatively "sticky" labels. However, the factors for selecting the detection reaction, whether it is an enzyme label or a direct label, and how to determine the light intensity must be considered before designing the reaction.
Enzyme labeling or direct labeling? The common method of generating chemiluminescence is to use labeled enzymes to catalyze the chemiluminescence reaction. Each label can initiate multiple chemiluminescence reactions, so usually only one or a few enzyme labels/analytes need to be incorporated. The chemiluminescent compound is supplied in excess as an enzyme substrate to ensure saturation kinetics. The light intensity is a linear function of the amount of labeled enzyme. Intensity, the emission rate of photons per second, is the product of the catalytic conversion of the substrate and the lifetime of the luminescent compound.
The chemiluminescence intensity/time distribution graph includes an initial rising period and extending the luminescence until the plateau or false plateau. If there is no stable plateau value, it indicates that the substrate is exhausted or the enzyme is inactivated. The detection of chemiluminescence produced by enzymes provides great flexibility in the measurement process. The light intensity at any point in time passing through the high point may be related to the amount of enzyme. If the speed is a single-point or multi-point slope type measurement problem, you can measure it in the ascending part. In order to obtain the highest sensitivity, however, there are not many suitable chemiluminescent compounds. Appropriate candidates must first have certain functions to allow connection with other molecules. More importantly, once the tag is triggered, it should emit all the light in the shortest possible time.
When chemiluminescence is gradually emitted over a period of time, the signal intensity (photons/second) decreases, and it is best to determine the optimal number of chemiluminescence labels on the species to be detected based on experience. Even if theoretically more tags should provide more photons, if the tags are spaced too close together, a quenching effect will occur. The surface area and the number of derivatizable groups (usually amino or mercapto groups) provide further restrictions. In practice, up to 10-20 labels can be bound to one macromolecule. In addition, the more labels on the surface of the binding molecule, the more likely the label will interfere with its binding properties.
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