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What is chemiluminescence?
It is the generation of electromagnetic radiation in the form of visible light by the release of light from a chemical reaction. The light can, in principle, be emitted in the ultraviolet, visible or infrared region, those emitting visible light are the most common. They are also the most interesting and useful.
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Types of chemiluminescent reactions

Chemiluminescent reactions (CL):Chemical reactions using synthetic compounds and usually involving a highly oxidized species such as a peroxide. Bioluminescent reactions: Light-emitting reactions arising from a living organism, such as the firefly or jellyfish. Electrochemiluminescent reactions: Light-emitting reactions which take place by the use of electrical current.

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PRINCIPLE
The basic principle consists of light-producing reaction involves the excitation of a molecule from its ground state into an excited state. The energy for this excitation is generated through a chemical reaction. Upon returning to ground state, a photon of light is emitted from the molecule with a characteristic wavelength and frequency, and energy equal to the transition taking place.

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Contd
The intensity of the emitting light is proportional to the amount of enzyme present and is directly related to the amount of the analyte in the sample

A chemiluminescent reaction occurs between the compound, luminol and several other chemicals.
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Contd
Therefore, most chemiluminescent reactions (CL) use oxygen, hydrogen peroxide, or similar potential oxidants. As a principle in CL reactions, at least two reagents, A and B react to form a product C, some fraction of which is present in an electronically excited state, C*, which may subsequently relax to the ground state emitting a photon: A+B C* C + photon of light
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PROCEDURES
PROCEDURE FOR REAGENT PREPARATION

Materials and reagents 1. Sodium Carbonate 10 hydrate Na2CO3H2O 2. Sodium Bicarbonate NaHCO3 3. Luminol (5-amino-2, 3-dihydrophthalazine-1, 4-Dione) 4. Ammonium Carbonate Monohydrate (NH4)2CO3H2O 5. Copper (II) Sulfate 5 hydrate CuSO45H2O 6. Erlenmeyer Flasks 7. Glass beaker

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CHEMICAL PATHOLOGY WORK

PROCEDURE FOR REAGENTS PREPARATION 1. Add 500mL of deionized water to a 1L Erlenmeyer flask labeled solution A. 2. Add 10.7g of sodium carbonate to solution A. Stir. 3. Add 0.2g luminol to solution A. Stir. 4. Add 24.0g of Sodium Bicarbonate to solution A. Stir. 5. Add 0.5g of Ammonium Carbonate to solution A. Stir. 6. Add 0.4g of Copper Sulfate to solution A. Stir.

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Cont
7. Add Deionized water to Solution A flask to a final volume of 1L. 8. Add 950mL of deionized water to a 1L Erlenmeyer flask labeled solution B 9. Add 50mL 3% hydrogen peroxide to solution B. Stir. 10. Pour equal amounts (about 100mL) of Solution A and Solution B into separate beakers. 11. Dim the lights and then mix the two solutions in the beakers together.

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ASSAY PROCEDURE
Materials

Antibody-coated microtiter wells. Set of Reference Standards Enzyme Conjugate Reagent. Wash Buffer. Chemiluminescence Reagent A. Chemiluminescence Reagent B.

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Procedure
1. Secure the desired number of coated wells in the holder. 2. Dispense standards, specimens, and controls into appropriate wells. 3. Dispense Enzyme Conjugate Reagent into each well. 4. Thoroughly mix. It is very important to have complete mixing in this step.

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Cont
5.Incubate at room temperature (18-22C) for about 60 minutes. 6.Rinse and flick the microtiter wells with washing solution and final 1 time with distilled water. 7.Strike the wells sharply onto absorbent paper to remove residual water droplets. 8.Dispense Chemiluminescence substrate solution into each well. Then gently mix for 5 seconds.
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9.Read wells with a chemiluminescence microwell reader 5 minutes later. (Between 5 and 20 min after dispensed the substrates). 10.After a solution of chemiluminescent substrate is then added and read relative light units (RLU) in a Luminometers.The intensity of the emitting light is proportional to the amount of enzyme present and is directly related to the amount of the hormone in the sample. By reference to a series of standards assayed in the same way, the concentration of the analyte in the unknown sample is quantified.
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Microwell Plates

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SENSITIVITY
Chemiluminescent assays are very sensitive. Sensitivity refers to the lowest level at which something can be reliably detected. The analyte can be labeled with some detectable tag, such as a chemiluminescent compound or an enzyme. The analyte can also be detected by a specific binding reaction with an affinity binding partner having a label. Example: The mean TSH values based on 160 random normal adult blood samples, is 1.6 (0.4-7.0) IU/ml. The minimum detectable concentration of TSH by this chemiluminescent assay is estimated to be 0.2 IU/ml.
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DETECTORS

1. Liquid chromatography detectors

High-performance liquid chromatography (HPLC) is a form of liquid chromatography to separate compounds that are dissolved in solution. It permits the separation, detection and estimation of a wide range of compounds with a high degree of specificity.

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The compounds analysed are water soluble ,non-volatile and thermal unstable. The separation is achieved by competitive distribution of the sample between mobile phase and stationary phase.
The most used is the ultraviolet detector at fixed or variable wavelength but fluorimetric and electrochemical detectors are also available.

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2. Gas chromatography detectors Gas chromatography is a chromatographic technique that can be used to separate organic compounds that are volatile. A gas chromatograph consists of a flowing mobile phase, an injection port, a separation column containing the stationary phase, a detector, and a data recording system. The organic compounds are separated due to differences in their partitioning behavior between the mobile gas phase and the stationary phase in the column.

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Figure

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CLINICAL UTILITY
The chemiluminescence techniques have been applied to a great variety of analytes and samples analysis in clinical field.

The following figure summarises the other important applications of CL methods in several fields.

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CHEMILUMINESCENCE APPLICATIONS
Clinical chemistry

Drug analysis Diagnostic tool in medicine Biomedical research Radical scavenging and antioxidant effect in oxidative stress...
High sensitive trace analysis Detector for HPLC Capillary electrophoresis, Supercritical fluids, Agricultural analysis Air analysis Water analysis Soil analysis Quality control Oxidation and deterioration Olive oil Residue determination

Analytical chemistry

Environmental chemistry

Food chemistry

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ADVANTAGES
Rapid measurement Wide measuring range Controlled reactions and stability

High Sensitivity and Linearity over a Broad Concentration Range

Good Reproducibility and High Specificity for the analyte Measurements Possible in Turbid or Colored Samples, Even at Extreme pH
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 Moderate Running Costs Low sample volume Measurement of chemiluminescence is not a ratio measurement in the way fluorescence and absorption or colors are. Most samples have no 'background' signal, i.e. they do not themselves emit light. No interfering signal limits sensitivity.

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DISADVANTAGES
Provides Limited Structural Information Limited Sample Throughput High Purchase Price for Detectors.

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REFERENCES
1. Baker&Silvertons :Introduction to medical laboratory technology 7th edition. 2. Albrecht, H.O. (1999). Chemiluminescence of aminophthalic hydrazide. Z. Phys. Chem. 136, 321. 3. Altinoz, S. and Dursun, O.O. (2000). Determination of nimesulide in pharmaceutical dosage forms by second order derivative UV spectrophotometry. J. Pharm. Biomed. Anal., 22, 175-182. 4. T. Nieman, Chemiluminescence: Theory and Instrumentation, Overview, in Encyclopedia of Analytical Science, Academic Press, Orlando, 608-613 (1995).
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GO AND READ U WILL KNOW MORE

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