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CN-121975909-A - HRP chemiluminescent substrate system, kit and configuration method

CN121975909ACN 121975909 ACN121975909 ACN 121975909ACN-121975909-A

Abstract

The invention discloses an HRP chemiluminescent substrate system, a kit and a configuration method, wherein the HRP chemiluminescent substrate system consists of a liquid A and a liquid B which are physically isolated and stored, the liquid 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 surfactant CTAC and a metal chelating agent diethylenetriamine pentaacetic acid, and the liquid B is an acidic solution with the pH value of 4.5-5.5 and comprises an oxidant and an enhancer N- (3-chloro-4-hydroxyphenyl) acetanilide. In the invention, the reinforcing agent N- (3-chloro-4-hydroxyphenyl) acetanilide is placed in an acidic B solution, when the acidic B solution is mixed with an alkaline A solution, the mixed system is maintained at a specific pH value of 8.3-8.6, the reinforcing agent is rapidly dissociated into phenolic oxyanions, and the phenolic oxyanions are immediately captured in situ by pre-existing positively charged CTAC cation micelles in the A solution through electrostatic attraction and are enriched at micelle interfaces. The system realizes directional interface catalysis of the reinforcing agent while ensuring high activity of HRP enzyme by matching with low ionic strength environment brought by buffer salt-free solution B, and remarkably improves luminous efficiency and stability.

Inventors

  • XU KE
  • LIU JUN
  • ZHOU YUE
  • Song Juanyan

Assignees

  • 东方伊诺(苏州)医疗科技有限公司

Dates

Publication Date
20260505
Application Date
20251218

Claims (6)

  1. 1. An HRP chemiluminescent substrate system consists of a liquid A and a liquid B which are physically isolated and stored, and is characterized in that: 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 surfactant CTAC and a metal chelating agent diethylene triamine pentaacetic acid; the solution B is an acidic solution with the pH value of 4.5-5.5 and comprises an oxidant and an enhancer N- (3-chloro-4-hydroxyphenyl) acetanilide; when the solution A and the solution B are mixed, the pH value of the system is maintained in the range of 8.3-8.6.
  2. 2. The HRP chemiluminescent substrate system of claim 1 wherein the concentration of cationic surfactant CTAC in solution a is 0.2% -0.3% and the molar ratio of CTAC to enhancer in solution B is controlled to be between 20:1 and 40:1.
  3. 3. The HRP chemiluminescent substrate system of claim 1 wherein the solution a comprises Tris-HCl buffer pair at a concentration of 0.1-0.2M and the metal chelator DTPA is at a concentration of 50-150 μmol/L.
  4. 4. The HRP chemiluminescent substrate system of claim 1 wherein the pH of solution B is adjusted by a volatile mineral acid or dilute nitric acid and the oxidizing agent is carbamide peroxide or hydrogen peroxide.
  5. 5. An HRP chemiluminescent kit comprising liquid a and liquid B of any of claims 1-4, wherein liquid a is packaged in a first container and liquid B is packaged in a second container protected from light.
  6. A method of formulating an hrp chemiluminescent substrate system, the method comprising the steps of: dissolving Tris base in pure water, regulating the pH value to 9.0-9.6, sequentially adding a metal chelating agent DTPA and a cationic surfactant CTAC, heating and stirring until the CTAC is completely dissolved to form micelle stock solution; Adding an acid regulator into the pure water, pre-regulating the pH value to be in an acid range of 4.0-5.0, adding an enhancer N- (3-chloro-4-hydroxyphenyl) acetanilide, stirring until the mixture is dissolved, and finally adding an oxidant, and fixing the volume to obtain a solution B.

Description

HRP chemiluminescent substrate system, kit and configuration method Technical Field The invention relates to the technical field of immunoassay, in particular to an HRP chemiluminescent substrate system, a kit and a configuration method. Background Chemiluminescent immunoassay is widely used due to its high sensitivity, with the horseradish peroxidase (HRP) -catalyzed luminol-hydrogen peroxide luminescent system being the dominant technology. In order to increase the detection sensitivity and signal duration, it is often necessary to add enhancers. The strategy commonly employed in the prior art (e.g., CN106990100 a) is to dissolve a phenolic enhancer (e.g., p-iodophenol) in an alkaline buffer along with the luminescent substrate, and place the oxidizing agent alone in an acidic solution. The strategy of placing the traditional reinforcing agent in alkaline solution has the obvious technical defects that firstly, the stability of the reinforcing agent is poor, novel high-efficiency reinforcing agents such as N- (3-chloro-4-hydroxyphenyl) acetanilide contain amide bonds and phenolic hydroxyl groups, and the amide bonds are easy to hydrolyze or the phenolic hydroxyl groups are easy to be oxidized by air when the reinforcing agent is stored in the alkaline solution for a long time, so that the color of the solution is deepened, the background is raised, and the sensitization capacity is greatly attenuated with time. Second, the reaction microenvironment is not matched. In the prior art, nonionic surfactants (such as Tween-20) are mostly used, negative-charge enhancer ions cannot be effectively adsorbed, and in order to maintain the pH, high-concentration buffer salt is usually added into A, B liquid, so that the ionic strength of the mixed liquid is too high, the enzyme activity of HRP is inhibited, and the detection rate of a low-value sample is limited. Disclosure of Invention The invention aims to overcome the defects in the prior art, and provides an HRP chemiluminescent substrate system with high sensitivity and high stability, a kit and a configuration method. In order to achieve the aim, the HRP chemiluminescent substrate system consists 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 surfactant CTAC and a metal chelating agent diethylene triamine pentaacetic acid, wherein the solution A does not contain an enhancer; The solution B is an acidic solution with the pH value of 4.5-5.5, contains an oxidant and an enhancer N- (3-chloro-4-hydroxyphenyl) acetanilide, and does not contain buffer salt components. When the solution A and the solution B are mixed, the pH value of the system is maintained in the range of 8.3-8.6, and the enhancer is deprotonated at the mixing moment and is captured in situ by micelles formed by the CTAC. Further, the concentration of the cationic surfactant CTAC in the solution A is 0.2-0.3%, and the molar ratio of CTAC to the enhancer in the solution B is controlled between 20:1 and 40:1. Further, the solution A contains Tris-HCl buffer pair with the concentration of 0.1-0.2M, and the concentration of the metal chelating agent DTPA is 50-150 mu mol/L. EDTA is not contained in the reaction system. Further, the pH value of the solution B is regulated by volatile inorganic acid or dilute nitric acid, and the oxidant is carbamide peroxide or hydrogen peroxide. An HRP chemiluminescent kit comprises the liquid A and the liquid B, wherein the liquid A is packaged in a first container, and the liquid B is packaged in a second container which is protected from light. A method of formulating an HRP chemiluminescent substrate system, the method comprising the steps of: dissolving Tris base in pure water, regulating the pH value to 9.0-9.6, sequentially adding a metal chelating agent DTPA and a cationic surfactant CTAC, heating and stirring until the CTAC is completely dissolved to form micelle stock solution; Adding an acid regulator into the pure water, pre-regulating the pH value to be in an acid range of 4.0-5.0, adding an enhancer N- (3-chloro-4-hydroxyphenyl) acetanilide, stirring until the mixture is dissolved, and finally adding an oxidant, and fixing the volume to obtain a solution B. The HRP chemiluminescent substrate system, the kit and the configuration method have the beneficial effects that: (1) In the invention, the reinforcing agent N- (3-chloro-4-hydroxyphenyl) acetanilide is placed in an acidic B solution, when the acidic B solution is mixed with an alkaline A solution, the mixed system is maintained at a specific pH value of 8.3-8.6, the reinforcing agent is rapidly dissociated into phenolic oxyanions, and the phenolic oxyanions are immediately captured in situ by pre-existing positively charged CTAC cation micelles in the A solution through electrostatic attraction and are enriched at micelle interfaces. The syste