Chemiluminescent Presentation l4

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4/4/2012 CHEMICAL PATHOLOGY WORK


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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 reactionsusing synthetic compounds and usually involving a highlyoxidized species such as a peroxide.

Bioluminescent reactions: Light-emitting reactions arisingfrom a living organism, such as the firefly or jellyfish.

Electrochemiluminescent reactions: Light-emittingreactions 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 NaHCO33. 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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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 of1L.

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) forabout 60 minutes.

6.Rinse and flick the microtiter wells withwashing solution and final 1 time with distilled

water.

7.Strike the wells sharply onto absorbent paperto remove residual water droplets.

8.Dispense Chemiluminescence substratesolution into each well. Then gently mix for 5seconds.



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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 isdirectly 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 canbe reliably detected. The analyte can be labeled with somedetectable tag, such as a chemiluminescent compound or anenzyme. The analyte can also be detected by a specific

binding reaction with an affinity binding partner having alabel.

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 thischemiluminescent 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 distributionof 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 thatare 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 thecolumn.



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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.


CHEMILUMINESCENCE APPLICATIONS


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CHEMILUMINESCENCE APPLICATIONS

Clinical chemistry Drug analysis

Diagnostic tool in medicine Biomedical research

Radical scavenging and antioxidant effect in

oxidative stress...

Analytical chemistry High sensitive trace analysis

Detector for HPLC

Capillary electrophoresis,

Supercritical fluids,

Environmental chemistry Agricultural analysis

Air analysis

Water analysis

Soil analysis

Food chemistry Quality control

Oxidation and deterioration

Olive oil

Residue determination



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ADVANTAGES

Rapid measurement

Wide measuring range

Controlled reactions and stability

High Sensitivity and Linearity over a Broad ConcentrationRange

Good Reproducibility and High Specificity for the analyte

Measurements Possible in Turbid or Colored Samples, Even atExtreme 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 ofnimesulide in pharmaceutical dosage forms by second

order derivative UV spectrophotometry. J. Pharm.

Biomed. Anal., 22, 175-182.

4. T. Nieman, Chemiluminescence: Theory andInstrumentation, Overview, inEncyclopedia of Analytical

Science, Academic Press, Orlando, 608-613 (1995).



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Chemiluminescent Presentation l4