The invention discloses an HRP chemiluminescent substrate
system, a kit and a preparation method, the HRP chemiluminescent substrate
system is composed of a liquid A and a liquid B which are physically isolated and stored; the solution A is an alkaline buffer
system with the pH value of 9.0-9.6, and comprises isoluminol or a salt substrate thereof, a cationic surface
active agent CTAC and a
metal chelating agent
diethylene triamine pentaacetic acid; and the solution B is an acidic solution with the pH value of 4.5-5.5, and comprises an
oxidizing agent and a reinforcing agent N-(3-chloro-4-hydroxyphenyl)
acetanilide. According to the invention, the reinforcing agent N-(3-chloro-4-hydroxyphenyl)
acetanilide is placed in the acidic liquid B, when the acidic liquid B is mixed with the alkaline liquid A, the mixed system is maintained at a specific pH value of 8.3-8.6, and the reinforcing agent is rapidly dissociated into phenoxy anions; and carrying out in-situ capture and enrichment on a
micelle interface by CTAC cationic micelles with positive charges, which exist in the solution A in advance, through
electrostatic attraction. And in cooperation with a low-
ion-strength environment brought by no buffer salt in the liquid B, the system ensures the
high activity of the HRP
enzyme, realizes directional interface
catalysis of the
enhancer, and significantly improves the luminous efficiency and stability.