Chemiluminescence

Luminescence

• Definition• Luminescence is emission of light by a substance not resulting from heat

• Types of luminescence• Fluorescence • Phosphorescence• Chemiluminescence• Bioluminescence• Electrochemiluminescence

Luminescence

• Excitation (higher energy level)• an electron returns from an excited or higher energy level to a lower

energy level• The physical event of the light emission

Luminescence

• Fluorescence & Phosphorescence• The luminescence produced by certain substances after absorbing radiant energy.• The excitation event is caused by photoillumination

• Phosphorescence• is distinguished from fluorescence in that it continues (for second to minutes) even

after the radiation causing it has ceased.

Chemiluminescence

• the emission of light when an electron returns from an excited or higher energy level to a lower energy level.

• The excitation event is caused by a chemical reaction• Involves the oxidation of an organic compound

• Such as luminol, isoluminol, acridinium esters, or luciferin• by an oxidant

• (e.g., hydrogen peroxide, hypochlorite,or oxygen)

• Light is emitted from the excited product formed in the oxidation reaction

Chemiluminescence

• Reactions occur in the presence of catalysts• Such as

• Enzymes (e.g., alkaline phosphatase, horseradish peroxidase, and microperoxidase),• Metal ions or metal complexes (e.g., Cu2+ and Fe3+ phthalocyanine complex), and hemin.

Bioluminescence

• Bioluminescence is a special form of chemiluminescence found in biological systems.

• In bioluminescence, an enzyme or a photoprotein increases the efficiency of the luminescence reaction.

• Catalysts • Luciferase • Aequorin

• The quantum yield (e.g., total photons emitted per total molecules reacting) is approximately 0.1% to 10% for chemiluminescence and 10% to 30% for bioluminescence.

• Chemiluminescence assays are• Ultrasensitive

• attomole to zeptomole detection limits • have wide dynamic ranges.

• Several orders of magnitude

• They are now widely used in • automated immunoassay • DNA probe assay systems

• e.g., • acridinium ester and acridinium sulfonamide labels and 1,2-dioxetane substrates for alkaline

phosphatase labels• the enhanced-Iuminol reaction for horseradish peroxidase labels

One zeptomole = 10-3 attomoles or 10-6 femtomoles. The enzyme horseradish peroxidase (HRP), found in the roots of plant horseradish,…

Electrochemiluminescence

• the reactive species that produce the chemiluminescent reaction are electrochemically generated from stable precursors at the surface of an electrode.

• A ruthenium (Ru2+), tris(bipyridyl) chelate is the most commonly used electrochemiluminescence label

• Electrochemiluminescence is generated at an electrode via an oxidation-reduction-type reaction with tripropylamine.

Ruthenium and TPA are oxidized at the surface of the electrode when voltage is applied. The TPA loses a proton, which reduces the ruthenium to an excited state, causing light to be emitted. Ruthenium is not consumed in the reaction, so this cycle can continue as long as TPA is present. Multiple excitation/emission cycles amplifies the light emitted and increase sensitivity. The emitted light is measured to determine concentration of analytes in sample. (TAG: Label)

Electrochemiluminescence

• The advantages • Improved reagent stability, simple reagent preparation, and enhanced

sensitivity.• Detection limits of 200 fmol/L and a dynamic range extending over six orders

of magnitude can be obtained.

INSTRUMENTATION

• Luminometers• are instruments used to measure chemiluminescence and

electrochemiluminescence.

• The basic components• the sample cell

• Housed in a light-tight chamber• the injection system

• to add reagents to the sample cell• the detector

• A photomultiplier tube.• an electrode (For electrochemiluminescence)

• at which the electrochemiluminescence is generated.

Limitations of Chemiluminescence

• Light leaks, light piping, and high background luminescence from assay reagents and reaction vessels (e.g., plastic tubes exposed to light) are common factors

• The extreme sensitivity of chemiluminescence assays requires stringent controls on the purity of reagents and the solvents (e.g., water) used to prepare reagent solutions.

Limitations of Chemiluminescence

• Efficient capture of the light emission from reactions that produce a flash of light requires an efficient injector that provides adequate mixing when the triggering reagent is added to the reaction vessel

• Chemiluminescent and electrochemiluminescent assays have a wide linear range, usually several orders of magnitude, but very high intensity light emission can lead to pulse pile-up in photomultiplier tubes and this leads to a serious underestimate of the true light emission intensity.

A, Chemiluminescent assay for horseradish peroxidase label using luminol.B, Chemiluminescent assay for an alkalinephosphatase label using AMPPD.C, Photometric assay for an alkalinephosphatase label using acascade detection reaction. INT, p-iodonitrotetrazolium violet.

3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl- 1,2-dioxetane (AMPPD)

• Aequorin• is a photoprotein isolated from the hydrozoan Aequorea victoria.