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High-sensitivity ECL kit and preparation method thereof

A high-sensitivity, kit-based technology, applied in the direction of chemical reaction of materials for analysis, chemiluminescence/bioluminescence, etc., can solve the problems of poor reagent stability, low sensitivity, easy attenuation, etc., and achieve simple components, low cost, The effect of strong luminous intensity

Pending Publication Date: 2021-02-05
武汉菲恩生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The luminol-hydrogen peroxide-horseradish peroxidase luminescence system is a commonly used chemiluminescence analysis system, but when it is directly used for the determination of biological macromolecules, the sensitivity is low and it is easy to attenuate. common defect
In addition, there are also problems such as poor reagent stability and high background after the luminescent agent and oxidant are mixed.

Method used

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  • High-sensitivity ECL kit and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] A kind of highly sensitive ECL test kit of the present invention comprises luminescent agent, oxidant, enhancer and stabilizer, and enhancer is 100mM p-bromophenol dissolved in DMSO, and luminescent agent is luminol or luminol containing 10mmol / L The pH 9.5 buffer solution of derivatives and 1mM EDTA, the oxidizing agent is a citric acid buffer solution with a final concentration of 10mmol / L hydrogen peroxide and 1mM EDTA at a pH of 5.0, and the volume ratio of luminescent agent, oxidant and enhancer is 100:100 : 1 used after mixing, the stabilizer is added to the oxidant, the stabilizer is 0.1% ProClin 300.

[0023] The background value of the luminescent system mixture in Configuration Example 1 did not increase after being left for 1 day, and the signal value was stable.

[0024] On the other hand, its preparation method comprises the following steps,

[0025] S1 Preparation of luminescent agent: dissolve luminol with 0.1M NaOH solution, then add to 50mM Tris buffer...

Embodiment 2

[0029] Wherein, compared with Example 1, the luminescent agent is replaced with a pH 9.5 buffer containing 0.5mmol / L luminol or luminol derivatives and 1mM EDTA, and the oxidizing agent is replaced with a peroxide containing a final concentration of 0.5mmol / L. Hydrogen and 1 mM EDTA in citrate buffer at pH 5.0, other conditions unchanged.

Embodiment 3

[0031] Wherein, compared with Example 1, no stabilizer is added in the oxidizing agent, and other conditions remain unchanged.

[0032] It should be noted that the main component of p-bromophenol is 4-BOP, which is called 4-bromophenol in Chinese. It is precipitated from chloroform or ether as tetragonal bipyramidal crystals. It is an intermediate in organic synthesis and is often used as a disinfectant, pesticide and pharmaceutical raw material. It is low in cost and easy to obtain.

[0033] The present invention utilizes p-bromophenol as the principle that luminol is oxidized by hydrogen peroxide to produce an intermediate of α-hydroxyperoxide, and then decomposes to release light energy, while p-bromophenol can improve the oxidation of luminol by hydrogen peroxide. The efficiency of electron transfer in the process, so that more light is emitted per unit time, which is manifested by the increase of luminous intensity; and because of the increased luminous intensity, it can ...

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Abstract

The invention provides a high-sensitivity ECL kit, which comprises a luminescent agent and an oxidant, wherein the luminescent agent is a luminol solution, and the oxidant is a hydrogen peroxide solution. The high-sensitivity ECL kit further comprises a reinforcing agent, wherein the reinforcing agent is a p-bromophenol solution, the luminescent agent, the oxidant and the reinforcing agent are mixed according to a volume ratio of 100:100:1, and the reinforcing agent is 100 mM p-bromophenol dissolved by DMSO. According to the high-sensitivity ECL kit, p-bromophenol is used as the reinforcing agent, and the electron transfer efficiency of p-bromophenol in the reaction process of chemiluminiscence of luminol oxidized by hydrogen peroxide is greatly improved, so that the luminous efficiency inunit time is improved, fluorescent light with enough intensity can be emitted by using a smaller amount of enzyme to realize development and color development, and the purposes of prolonging the plateau period of luminescence and maintaining high luminous intensity for a long time are achieved.

Description

technical field [0001] The invention relates to the technical field of chemiluminescence immunoassay, in particular to a high-sensitivity ECL kit and a preparation method thereof. Background technique [0002] Chemiluminescence analysis is a new type of labeling analysis technology for detecting trace substances. It is applied in the field of immunology and mainly determines the content of antigen or antibody according to the light radiation generated by chemical reaction. Due to its advantages of high sensitivity, wide linear range, simple instrument, convenient operation, no radiation hazard and easy automation, it has become an important direction of labeling immunization in recent years. Among them, chemiluminescence immunoassay with horseradish peroxidase (HRP) and alkaline phosphatase (AP) as markers is the main development trend. [0003] Luminol is a luminescent substance that was widely used in chemiluminescence in the early days. Its chemical name is 3-aminophthal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/76
CPCG01N21/76
Inventor 刘少华
Owner 武汉菲恩生物科技有限公司
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