Introduction to Chemiluminescence Immunoassay (CLIA) and its Principle -part 3

4 CLIA Chemiluminescent Substrate Types 


4.1 Luminol

Luminol, with the chemical structure simple, easy synthesis, stable properties, non-toxic, environmentally friendly, and good water solubility, is one of the most widely used chemiluminescent reagents.

CAS number: 521-31-3 Luminol

Luminol's chemiluminescent properties were first discovered by Albrechi HO in 1928. He found that Luminol in an alkaline solution, when hydrogen peroxide is added, exhibits a faint luminescence. On this basis, with the addition of suitable oxidants and catalysts, its chemiluminescent intensity can be significantly increased. Catalysts or oxidants such as hydrogen peroxide, horseradish peroxidase, iron salts, manganese salts, transition metal ions, amino acid complexes, etc., can enhance the chemiluminescence of luminol. Therefore, according to the principle of chemiluminescent detection analysis, luminol can be used for the analysis and detection of the above substances, as these substances affect the chemiluminescent rate of luminol, thereby affecting its chemiluminescent intensity. Luminol and its derivative IsoLuminol are commonly used chemiluminescent substances in CLIA. The luminescence of luminol substances is based on an oxidation reaction. In an alkaline solution, luminol can be oxidized and luminesce by many oxidants, with hydrogen peroxide being the most commonly used. Early luminol systems were mainly used for the determination of inorganic substances and small organic biomolecules. However, due to the decrease in luminescent intensity after labeling, its sensitivity was affected. In the 1980s, researchers found that the addition of certain phenols and their derivatives, amines and their derivatives, and phenylboronic acid derivatives to the luminescent system significantly affected luminescence. Under the action of these substances, the luminescent intensity of luminol can be increased by 1000 times, and the "background" luminescence and luminescence time observed when oxidants and luminescent agents act alone are significantly reduced. The use of these enhancers has made this system widely used in the analysis of proteins and nucleic acids. The chemical luminescence mechanism of luminol is generally considered to have been thoroughly studied. However, the intermediate products of its oxidation have not yet reached a unified conclusion. It has only been determined that the final luminescent substance is the amino-substituted phthalic acid salt ion. The chemical luminescence mechanism of luminol is shown in the figure.

Novel Luminol-like Chemiluminescent Reagents

Common luminescent reagents include IsoLuminol Isothiocyanate (ILITC), N-(4-aminobutyl)-N-ethyl IsoLuminol (ABEI), and 4,5-diamino phthaloyl hydrazide (DPH), which have been applied in many fields. Due to the low luminescent intensity of luminescent reagents containing the luminol part and the difficulty of synthesis, there are not many reports of related novel derivatives. In recent years, the research trend of this type of novel derivative luminescent reagents mainly includes two types: one is to carry out relevant structural modifications based on the original luminescent reagents to discover more superior luminescent reagents. Palmioli based on ABEI for structural modification, synthesized a novel IsoLuminol luminescent reagent ABEI lactone . Compared with general equivalent reagents, luminescent reagent 1 has better stability and reactivity, and its synthesis route is simple and efficient. Another is to introduce different aromatic rings into the amino position of luminol to increase its chemical luminescence intensity and obtain novel luminol derivatives. Deshmukh synthesized luminol derivatives containing hydroxyphenyl benzimidazole parts, and researchers in two other articles reported novel compounds containing phenanthro imidazole or mononitrogen. The application of such luminescent reagents is still in its early stages, but it is believed that these derivatives are likely to be candidates for chemiluminescent probes.


4.2 Acridinium Ester

Acridinium ester is an important type of chemiluminescent reagent with a high quantum yield. Its chemical luminescence efficiency is higher, usually five times or more, than that of luminol. In addition, the chemical luminescence process of acridinium ester reacts quickly, has low background, and can luminesce in the presence of sodium hydroxide and hydrogen peroxide. During the oxidation reaction, the complex is decomposed, which does not affect the luminescence of free acridinium ester. In addition, acridinium ester chemiluminescent reagents have good stability and are easy to store. Acridine is a type of chemiluminescent reagent with a very high quantum yield. Its molecular structure is composed of at least two parts: the luminescent group (emitter) and the leaving group. The most widely used are acridinium ester and acridinium sulfonamide.

1,2-Dioxetane Chemiluminescent Substances

1,2-Dioxetane is a type of chemically initiated electron exchange luminescence (CIEEL) reagent. It is a four-membered heterocyclic system, and the four-membered ring has tension energy, allowing it to release a large amount of energy during the reaction to meet the energy requirements for the chemiluminescent reaction. Therefore, it is an efficient type of chemiluminescent reagent. 

Since the first synthesis of such compounds by Kopecky and Mumford in 1969, more than 100 types of 1,2-dioxetane compounds have been prepared. Among them, a representative one is 3-(2'-spiroadamantylketone)-4-methoxy-4-(3″-phosphorylphenyl)-1,2-dioxetane (AMPPD). The chemical luminescence mechanism of 1,2-dioxetane compounds (using AMPPD as an example) is as follows: AMPPD, catalyzed by alkaline phosphatase (ALP), forms a highly unstable intermediate m-AMPD; m-AMPD rapidly decomposes into one molecule of adamantane ketone and one molecule of excited-state peroxy anion benzoic acid methyl ester (m-MOB); during the return of the excited-state m-MOB to the ground state, photons are emitted.


Research on the luminescence of 1,2-dioxetane has been widely applied. For instance, the AMPPD reagent uses particle chemiluminescence technology, with the latest magnetic particles coated with antibodies and alkaline phosphatase (ALP) labeled antigens (antibodies). After the antigen-antibody reaction, alkaline phosphatase binds to the particles (the binding amount of alkaline phosphatase is proportional to the substance to be tested in the patient's serum). There is a magnetic field on both sides of the reaction tube, and the antigen-antibody complex with magnetic particles is adsorbed on both sides of the tube, while other free components are aspirated away. Finally, the chemiluminescent substrate is added, and the luminescent intensity of the reaction is detected through a photomultiplier tube. In addition, CSPD and CDP-Star are also chemiluminescent reagents synthesized after AMPPD, with performance superior to AMPPD.


Chemiluminescent Immunoassay (CLIA) has the advantages of strong specificity, high sensitivity, simple operation, high throughput, and high automation. It is widely used in the field of human medicine and has gradually become the main trend to replace enzyme immunoassay, radioimmunoassay, and fluorescence assay technologies. With the rapid development of magnetic bead separation technology, monoclonal antibody technology, and even immunologic analysis technology with uniform energy transfer of chemiluminescence, CLIA is moving towards convenience, high throughput, and full automation.