Compounds that participate in energy transfer in luminescence reaction and ultimately release energy in the form of emitting photons
Luminescent agents refer to compounds that participate in energy transfer in the luminescence reaction and ultimately release energy in the form of emitting photons. According to the above luminescence characteristics, luminescent agents can be divided into fluorescein, bioluminescent agent and chemiluminescent agent.There are three commonly used chemiluminescent agents, namely, the luminescent agent of the luminescent substrate of the enzymatic reaction, the direct chemiluminescent agent, and the electrochemiluminescent agent.
Chinese name
Chemiluminescent agent
Discipline
Chemistry
Type
Photoluminescence, bioluminescence and chemiluminescence
When a substance reverts from the electronic excited state to the ground state, the energy released is shown as lightlaunch, called luminescence.According to the excitation energy of excited molecules, luminescence can be divided into three types: photoluminescence, bioluminescence and chemiluminescence.
Common Luminescent Agents
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Luminescent Substrates for Enzymatic Reactions
Luminescent substrate of enzymatic reaction refers to a kind of luminescent substrate that emits light through enzymatic degradation. Enzymes commonly used in chemiluminescent enzyme immunoassay includeHorseradish peroxidase(HRP) andalkaline phosphatase（AP）。The luminescent substrates of HRP are luminol or its derivatives and p-hydroxyphenylacetic acid.The luminescent substrates of AP are 3 - (2-spiramantane-4-methoxy-4-methyl-4 - (3-phosphateoxy) - phenyl-1,2-dioxyethane (AMPPD) and 4-methyl umbelliferone phosphate (4-MUP, fluorescent substrate).
(1) Luminol or its derivatives.The oxidation reaction of luminol is carried out in alkaline buffer, usually using 0.1mol/L pH8.6Tris buffer as the substrate.It should be noted that: first, luminol and H2O2 can also slowly spontaneously emit light without HRP catalysis, which will cause blank interference in the final light intensity measurement, so it is advisable to prepare two bottles of reagent solutions respectively, and mix them immediately before use;Secondly, HRP luminescence enhancers such as some phenol reagents (such as o-iodophenol) or firefly luciferase can enhance HRP catalyzed luminol oxidation reaction and extend the luminescence time to improve the luminescence sensitivity.
(2) . p-hydroxyphenylacetic acid (HPA).P-hydroxyphenylacetic acid (HPA) is oxidized by HRP or oxidized dimer (fluorescent substance) in the presence of H2O2, and emits 450 nmfluorescence, available fluorophotometermeasure。
（3）.AMPPD. Under alkaline conditions, AMPPD is enzymatically hydrolyzed by ALP to produce a fairly stable AMP-D anion, which has a decomposition half-life of 2-30 minutes and emits persistent light with a wavelength of 470 nm. Its intensity reaches a peak at 15 minutes, and remains relatively stable within 15-60 minutes.
（4）4-MUP。 4-MUP is catalysed by ALP to produce 4-methyl umbelliferone. Under the action of 360 nm excitation light, it emits 448 nm fluorescence, which is measured with a fluorescence photometer.
The direct chemiluminescent agent does not need the catalysis of enzyme, and only needs to change the pH of solution and other conditions to emit light, such as acridine ester (AE)hydrogen peroxideIt can emit light in dilute alkali solution.
Electrochemiluminescence agent
Electrochemiluminescence agent refers to the substance that emits light through electrochemical reaction on the electrode surface.The chemiluminescent agent ruthenium [Ru (bpy) 3] 2+(Figure 16-7) and the electron donor tripropylamine (TPA) can lose an electron on the positive electrode surface at the same time, resulting in an oxidation reaction.The divalent [Ru (bpy) 3] 2+is oxidized to trivalent, becoming a strong oxidant. After losing electrons, TPA is oxidized to cation free radical TPA+, which is very unstable and can spontaneously lose a proton (H+) to form free radical TPA,As a strong reducing agent, it can deliver a high-energy electron to the trivalent [Ru (bpy) 3] 3+to form an excited [Ru (bpy) 3] 2+.The excited tripyridine ruthenium is unstable, and soon emits a photon with a wavelength of 620nm, returning to the ground state of tripyridine ruthenium.This process can be carried out repeatedly on the surface of the electrode, generating many photons to enhance the optical signal.
