Mechanism of luminescence
In the luminescence reaction, before the electronically excited state intermediates are formed, the non-luminescent substituents attached to the acridine ring are separated from the acridine ring, that is, the non-luminescent part is separated from the luminescent part, so its luminous efficiency is basically not taken into account. The influence of substituting structure.
Hydrolysis mechanism
Acridine esters and acridine amides are both stable in acidic solutions (pH less than 4.8). These compounds and their protein conjugates are stored at room temperature for 4 weeks, and their light quantum yield does not decrease; the freeze-dried products are in- It can be stored for more than one year at 20°C. But when the pH is greater than 4.8 (especially in alkaline solutions), acridine esters and acridine amide compounds will reduce their stability due to partial hydrolysis, the hydrolysis mechanism. In the aqueous solution, when it is attacked by OH-, a pseudobase is formed. The pseudobase continues to break the ester bond under the action of OH-, and finally N-methylacridone is formed. The hydrolysis process is a dark reaction process without luminescence.
difference:
Different resistance to hydrolysis
The degree of hydrolysis of acridine compounds in aqueous solution increases with the increase of pH value; it increases with the increase of temperature. The stability of most acridine amide compounds is higher than that of acridine esters. The reason is that the bond level of CN bond and CO bond is different. CN bond is larger than CO bond; acridine amide compounds are more resistant to hydrolysis than acridine. Esters.
Different thermal stability
For acridine compounds, the stability of the acridine ring and the phenol ring or benzenesulfonyl ring is different if the substituents are not connected and the different substituents are connected. Usually acridine ring or phenol ring or benzenesulfonyl ring is attached to acridine ester or acridine sulfonamide with methyl and other substituents. Due to the large steric hindrance, the thermal stability increases, and when electron withdrawing groups are attached , Due to the favorable nucleophilic substitution reaction, the stability is reduced.
Same point:
No catalyst needed
The chemiluminescence of acridine esters or acridine sulfonamide compounds 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 or an enhancer, thereby reducing The background is illuminated, which improves the signal-to-noise ratio and reduces interference.
Easy to connect with protein
As luminescent markers for chemiluminescence immunoassays, this class of compounds also has other advantages, such as rapid concentration of light release, high luminous efficiency, high luminous intensity, easy to connect with proteins, 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℃, so acridine ester or acridine sulfonamide is a very effective chemiluminescent label.
Luminous type
This type of compound emits a flash type. After adding the luminescence starting reagent, the emitted light intensity reaches the maximum at about 0.4s, and the half-life is about 0.9s. Acridinium ester or acridine sulfonamide compounds are used in CLIA. The usually used system is the Acridiniumester/H2O2 system, that is, the antibody or antigen is labeled with acridinium ester or acridine sulfonamide, and HNO3+H2O2 and NaOH are used as luminescence initiating reagents. Some add TritonX-100, CTAC, Tween-20 and other surfactants to the luminescence starting reagent to enhance luminescence.