chemiluminescent presentation l4
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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.
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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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GO AND READ U WILL
KNOW MORE
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