Luminescence Kinetics of Acridine Ester DMAE-NHS
The luminescence of the acridinium ester DMAE-NHS is flash. The luminous intensity reaches the maximum at 0.4s. The luminous intensity decreases rapidly in the first 2s, and then decreases slowly. The luminous half-life is about 0.9s. The luminescence dynamics of DMAE-NHS is not affected by the concentration of DMAE-NHS. Changing the concentration of DMAE-NHS only affects the size of the luminous intensity, but does not affect the time and half-life of the maximum luminous intensity.
Luminous intensity of acridinium ester DMAE-NHS
The dynamic detection range of DMAE-NHS is 1.25×10-11~1.25×10-17mol, and the luminescence intensity is 6.11×1018cps/mol or 1.03×107cps/ng. When the TSH monoclonal antibody is labeled with DMAE-NHS and the molecular ratio of DMAE-NHS to Ab is 2.4, the experimentally measured luminescence intensity of DMAE-NHS-Ab is 1.8×1019cps/molAb or 1.125×105cps/ngAb. The dynamic detection range of DMAE-NHS-Ab is 1.0×10-12~1.0×10-18mol.
The effect of luminescence initiating reagent on the luminescence intensity of DMAE-NHS
There are three formulations of luminescence starting reagents commonly used for acridine ester luminescence:
The formula of the two substances is H2O2 and NaOH;
The formula of the three substances is HNO3 (some use HCL instead), H2O2 and NaOH;
The formula of the four substances is to add a surfactant in addition to the above three substances. Such as CTAC, TritonX-100, Tween-20, etc. to enhance luminescence.
The concentration and volume of HNO3, H2O2, and NaOH used in the luminescence start reagent are different, and the luminescence intensity of DMAE-NHS is different. The luminescence intensity is the largest when the following composition is used, namely (1) 100μl 0.1mol/LHNO3+0.1%H2O2 .25mol/LNaOH.
Adding surfactants can increase the luminous intensity of DMAE-NHS. Three surfactants: CTAC, TritonX-100 and Tween-20 have been tested for their influence on the luminous intensity of DMAE-NHS, and the effect of 2% TritonX-100 has been enhanced. Well, it can increase the luminous intensity of DMAE-NHIS by 4.5 times, and 2% Tween-20 and 0.2% CTAC can increase the luminous intensity of DMAE-NHS by 3.5 and 2.8 times, respectively. Adding TritonX-100 or Tween-20 can increase the luminous intensity of DMAE-NHS without causing an increase in the background, but when CTAC is added, the background increases a lot, so CTAC is not suitable as an enhancer.
Thermal stability of acridinium ester DMAE-NHS
The thermal stability of DMAE-NHS decreases with increasing pH and temperature. At room temperature (20°C), DMAE-NHS is stable in PB buffers with pH 5.8, 7.0, and 8.0, and the luminescence activity is respectively after 16 days. Decrease by 1.6%, 3.6% and 8.3%. At 37°C, the luminescence activity of DMAE-NHS in aqueous solutions at pH 5.8, 7.0, and 8.0 decreased by 8.9%, 22%, and 42%, respectively, after 16 days.
Hydrolysis of acridinium ester DMAE-NHS
The reason why the luminescence activity of DMAE-NHS in aqueous solution decreases with time is due to hydrolysis, which is beneficial to hydrolysis in an alkaline environment, and increasing temperature is also beneficial to hydrolysis, that is, the hydrolysis rate of DMAE-NHIS increases with the pH value of the solution. The temperature rises and speeds up. Under 4°C or freezing conditions, DMAE-NHS is stable in a 0.1mol/L PB buffer with a pH of 7.0. After half a year of storage, its luminescence activity does not decrease.
Advantages of Desheng Acridine Ester DMAE-NHS
Compared with traditional acridine esters, DMAE-NHS has higher luminous efficiency, better thermal stability, and stronger hydrophilicity. The chemiluminescence reagent has high photon yield and low background. It is compatible with proteins, antigens, and antibodies. After the coupling, the luminous efficiency is almost unaffected, making it an excellent chemiluminescence immunoassay labeling reagent.