Chemiluminescence involves the emission of light from a chemical reaction without the generation of significant heat. It has various applications including chemiluminescent immunoassays, DNA hybridization detection, and analysis in forensics and food. The three major chemiluminescent technologies are those using acridinium ester labels, horseradish peroxidase labels, and alkaline phosphatase labels. Chemiluminescence provides advantages such as wide dynamic range and high sensitivity and has been used in clinical testing, research, and cellular and gene-based assays.

1. CHEMILUMINESCENCE AND
ITS APPLICTIONS
Presented By- Dr. Y L S

2. OVERVIEW
• Luminiscence, types and principle
• Chemiluminescence immunoassay
• Enhanced Chemiluminescence immunoassay
• Electro Chemiluminescence immunoassay
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3. HISTORY
• WEIDMANN (1888) introduced the term Luminescence(weak glow)
• Defined it as
A phenomenon of certain kind of substance emitting light on absorbing
the various energies without heat generation .
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4. LUMINESCENCE
• Luminescence is described as emission of cold light at lower
temperature .
• It is emitted from a substance as it returns from an electronically
excited state to ground state.
• Various forms of Luminescence:
• 1)Chemiluminescence
• 2)Electroluminescence
• 3)Bioluminescence
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6. BIOLUMINESCENCE
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7. PRINICIPLE OF CHEMI LUMINESCENCE
• Luminescence is described as emission of light from a substance as it
returns from an electronically excited state to ground state.
• For chemical luminescence it is light produced by a chemical reaction
• The substance can be excited by oxidation reaction.
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9. CHEMILUMINESCENCE
• Emission of light with limited emission of heat as a result of chemical
reaction
• A + B C (PRODUCT + LIGHT)
• A,B are reactants
• C= excited intermediate
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10. • For example A is luminol and B is hydrogen peroxide in the presence
of suitable catalyst we get
• Luminol + H2O2 =3 APA ( ) =3-APA + LIGHT
• Where
• 3-APA is aminophthalate
• 3 –APA ( ) Is the excited state producing light as it decays to a lower
energy level
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11. CRITERIA FOR SUCCESSFUL
CHEMILUMINISCENT REACTION
• Sufficient excitation energy provided for chemical reaction for red
emissionis 47.6 kcal/mol and blue emission is 63.5kcal/mol.
• The liberation of this much energy usually comes from bond
cleavage or electron transfer.
• Formation of products capable of forming an excited state multiple
bonds ,conjugation.
• Rapid kinetics( rate is imp than high yield)
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12. • Luminescent reactions usually involves the cleavage or fragmentation
of O-O bond an organic peroxide compound.
• Peroxides are prevalent in light emitting reaction because the relative
weak bond in easily cleaved and liberates large amount of energy.
• In order to achieve highest level of sensitivity a reaction must be as
efficient as possible in generating photons of light.
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13. • CL REACTION CAN BE GENERATED BY TWO BASIC MECHANISMS
In a DIRECT REACTION, two reagents, usually a substrate and an
oxidant in presence of some co-factor, react to form a product, then some
fraction of the product will be formed in an electronically excited state
which can subsequently relax to the ground state with emission of a photon.
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14. • INDIRECT REACTION is based on a process of transference of energy of the
excited species to a fluorophore.
• This process makes it possible for those molecules that are unable to be directly
involved in CL reactions to transfer their excess of energy to a fluorophore that in
turn is excited, releasing to its ground state with photon emission.
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15. • CHEMILUMINESCENT METHODS
• direct—using luminophore markers
• indirect—using enzyme markers.
• Either method may be competitive or non-competitive.
• In direct chemiluminescent methods, the luminophore markers used are
acridinium and ruthenium esters.
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16. • The enzymatic markers used in indirect methods .
• alkaline phosphatase with adamantyl 1, 2-dioxetane aryl phosphate
(AMPPD) substrate and horseradish peroxidase with luminol or its
derivatives as substrate.
• Synthesizing molecules such as AMPPD and isoluminol base molecule
derivatives are more stable and result in light emission with a
characteristically elevated quantum yield.
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17. • The addition of an enhancer (e.g. ferrocyanide, metallic ions) further
boosts the electronic activation, ultimately leading to extremely
elevated analytic sensitivity (mol−16 per litre).
• certainly superior to those attainable by other immunoassay methods
such as RIA, immune enzymatic (ELISA) and fluoro immune
enzymatic (FEIA) methods etc.
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18. APPLICATIONS OF CHEMILUMINESCENCE
• Chemiluminescence immunoassay
• DNA hybridization detection
• Forensic science
• Food analysis
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19. CHEMILUMINESCENCE IMMUNOASSAY
• Provides a sensitive high through output alternative to conventional
colorimetric methodologies.
• PRINCIPLES
• Same as ELISA
• Uses chemiluminescent substrate, hydrogen peroxide ,enhancers
• Stopping reagent is not required
• Incubation period is small
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20. PROCEDURE
• MONOCLONAL ANTIBODY COATED WELL
• TEST SPECIMEN
• HRP LABELLED ANTIBODY CONJUGATE
• TEST ANTIGEN; SANDWICH BETWEEN SOLID PHASE ANTIBODY
AND ENZYME LABELLED ANTIBODY
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21. • INCUBATE FOR 1 HR AT 37C
• REMOVE UNBOUND ENZYME LABELLED ANTIBODY
• CHEMILUNESCENCE REAGENT ADDED
• READ RELATIVE LIGHT UNIT WITH LUMINOMETER
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23. labels
• Reagents required for reactions that produce CL may be coupled to
Abs or antigens (Ags) and used as labels for IA.
• This category involves labels that are consumed in the CL analytical
reaction (e.g., luminol derivatives, acridinium esters).
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24. • Luminol is the best known and one of the most efficient CL reagents.
It is coupled to ligands via reactions involving the amino group.
• However, the resulting conjugates have lower CL efficiencies than the
parent compounds. Labels derived from isoluminol have been more
successful.
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25. • SOLID-PHASE MATERIALS
 Commonly used solid-phase are 96-well microtitration plates prepared
with Polystyrene.
 For the purposes of IA, the microplates are pre-coated with capturing
protein like Ab to allow analyte immobilization.
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26. • IMMUNOASSAY
• Chemiluminescent immunoassay test kits and automated immunoassay
analyzers have been developed.
• The analysers routinely measured in a clinical chemistry, immunology,
toxicology, virology and endocrinology laboratories for the assessment
of :
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27. Clinical utilities
Thyroid Tumor markers Anemia Harmones Drug monitoring
Anti TPO
Anti TG
Free T3
Free T4
Total T3
Total T4
TSH
AFP
CA 125H
CA 19-9
CEA
PSA
Serum HER 2 NEU
Ferritin
Vitamin b12
CARDIAC
BNP
CKMB
D DIMER
FSH
LH
HcG
Progesterone
Prolactin
Testosterone
Carbamazepine
Digoxin
Gentamycin
Phenytoin
Theophylline
Valproic acid
Vancomycin
Cyclosporine
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28. The three major chemiluminescent
technologies
1. Acridinium ester and sulphonamide labels in chemiluminescent
immunoassays
2. Chemiluminescent detection techniques for horseradish peroxidase labels
3. Chemiluminescent detection techniques for alkaline phosphate labels in
enzyme immunoassays.
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29. NUCLEIC ACID ASSAYS
• The first major success of chemiluminescence in nucleic acid testing
with non separation chemiluminescent hybridization protection assay
for detecting specific DNA or RNA sequences.
• This type of chemiluminescent test is now widely used test for
infectious agents (e.g. chlamydia)
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30. CLINICAL RESEARCH
• In clinical research sensitivity, dynamic range and diversity of
chemiluminescent assays has lead to a vast range of applications notably in
 Immunoassays
 Protein and nucleic acid blotting
 Microarray- based assays
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31. • Monitoring reactive oxygen species and as detection reactions for
substances separated by HPLC
• Capillary electrophoresis ( CE) and
• Flow- injection analysis.
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32. RESEARCH IMMUNOASSAY
• A large number of research immunoassays and emerging clinical tests have been
developed in chemiluminescent format e.g.
 cytokines (IL-4, IL-5, IL-6, IL-10)
 Endothelin-1
 Epidermal growth factors
 50 granulocyte colony- stimulating factor
 Interferon
 vascular endothelial growth factor c
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33. • The luminescent oxygen channeling immunoassay (LOCI) exploits the
in situ production of a chemiluminescent compound due to single
oxygen transfer between a donor and acceptor antibody coated
microbead (250nm diameter) brought in to contact as a result of
specific binding with the test agent.
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34. • This assay has been adopted for competition assays
• Interaction assay( ligand/receptor, protein/protein, protein/ DNA)
• Enzyme assays (e.g protease, kinase, helicase)
• Immunoassays( thyrotropin, hepatitis B surface antigen, digoxin).
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35. • The LOCI- type assay is also used for nucleic acid assays, example
• Single nucleotide polymorphism typing
• Nucleic acid amplification techniques such as the polymerase chain reaction
• Lassa fever virus( reverse transcriptase polymerase chain reaction method)
papilloma virus in cervical scrapes.
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36. BLOTTING TECHNIQUES
• WESTERN BLOTTING for proteins using gel electrophoresis
 Southern blotting for DNA
 Northern blotting for RNA.
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37. GENE- BASED ASSAYS
• A successful research application for chemiluminescence is detecting the
expressed products of the reporter genes developed as alternatives to the
traditional methods like detecting chloramphenicol acetyl transferase (CAT)
gene.
• It is also used to detect and quantitate expression products of genes for placental
alkaline phosphatase(PLAP), galactosidase(GAL), and glucuronidase(GUS).
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38. CELLULAR CHEMI LUMINESCENCE
• The cellular chemi luminescence studies includes investigations /detecting-
• human spermatozoa
• defects in the production of reactive oxygen intermediates
• response of cells (e.g. PMN-leukocytes, neutrophils) to drugs and different agents
• Complement receptors
• Polyunsaturated fatty acids ,lectins, endotoxins, H PYLORI lipopolysacharides
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39. Advantages and limitations of CLIA technology
• The key advantages of chemiluminescent analytical methods
• Wide dynamic range
• High signal intensity
• Absence of interfering emissions (i.e. high specificity)
• Rapid acquisition of the analytical signal
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40. • high stability of reagents and their conjugates
• Low consumption of reagents
• Random access
• Reduced incubation time and full compatibility with immunology
assay protocols (homogenous or non-homogenous).
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41. • DISADVANTAGES OF CLIA
• Limited Ag detection
• High costs
• Limited tests panel
• Closed analytical systems.
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42. CHEMILUMINESCENT ASSAYS AND FOR ANALYSIS IN BODY FLUIDS
• Urinary N-acetyl-d-glucose aminidase
• Cholesterol
• Vitamin C
• Blood spot screening tests for phenyl ketonuria (PUK)
• Galactossemia and maple syrup urine disease.
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43. • Chemi luminescence is also useful in detection reactions for
components of mixtures separated by techniques such as
• HPLC
• Capillary electrophoresis
• Flow injection.
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44. DNA HYBRIDIZATION
• STEPS IN HYBRIDIZATION
• Process of forming double stranded DNA molecule between a single
stranded DNA probe and single stranded targeted DNA.
• The restriction fragments present in the gel are denatured with alkali
and transferred on to nitrocellulose filter or nylon membrane by
blotting
• This procedure preserves the distribution of the fragments in the gel
creating a replica of the gel on the filter paper.
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45. • The filter is incubated under hybridization conditions with a HRP
labelled DNA probe.
• The probe hybridize to the complimentary DNA restriction fragment
• Detection reagent containing H2O2and luminol is added on to the
membrane.
• USES:
• Identifying DNA in crime case
• Paternal dispute
• Classify DNA of various organism
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47. FORENSIC SCIENCE
• It is used by criminalists to detect traces of blood at crime scene.
• Solution luminol powder ,hydrogen peroxide are sprayed where blood
might be found.
• Tiny amount of iron from Hb in blood serves as catalyst for the
chemiluminiscence reaction, luminol to glow.
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49. FOOD ANANLYSIS
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Peroxophoshonate +
luminol
3 aminophthalate
anion
Quianalphos + H2O2 Peroxophoshonate
3 aminophthalate 3
aminophthalate+
emission

50. OTHER APPLICATIONS
• GAS ANALYSIS-to determine small amount of impurities and poisons in
air
• Other compounds such as ozone, S compounds , fluxes of -oxide
compounds .
• Lightening objects, chemiluminescent kites, glow stick, emergency
lightenings
• Children toys
• COMBUSTION ANALYSIS-certain radical species (CH+ AND OH+) gives off
radiation at specific wavelengths .
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51. ENHANCED CHEMILUMINESCENCE
• It is common technique for variety of detection assays
• A horse radish peroxide enzyme is tethered to antibody that
specifically recognises the molecule of interest.
• This enzyme complex then catalyse the conversion of enhanced
substrate into sensitised reagent .
• Which on further oxidation by H2O2 produces a triplet carbonyl
which emits light when it reduced to single carbonyl
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52. • It allows the minute quantities of biomolecules detection in femto
litres
• It is characterised by following features
• Intense light emission
• Prolong emission of light no pre incubation step
• Substrate that can be added several minutes prior to detection
• As long as commercially preparations of luminal are used control of
reaction PH is not a concern
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54. ELECTROCHEMICAL LUMINESCENCE
• In this process electro chemically generated intermediates undergo
highly exogonic reaction to produce electronically excited state .
• Emits light upon relaxation to lower state.
• ECL excitation can be caused by redox reaction of electron generated
species.
• It is observed during application of potential to electrodes of
electrochemical cell that contains solution of luminescent species
(metal complexes, quantum dots) in aprotic organic solvents.
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55. • The excitation energy is obtained from recombination of oxidised and
reduced species .
• The luminescent species are oxidised at electrode together with co
reactant which gives strong reducing agent after some chemical
transformation.
• It generally uses ruthenium complexes regenerating with TPrA
(tripropylamine) in liquid phase
• Photon detection is done in photo multiplier tubes or silicon photo
diodes.
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56. • It is highly sensitive and specific method.
• It present with outstanding advantage over other methods
• versatility
• simplified optical setup compared with photo luminescence
• good temporal control.
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59. LIMITATONS
• Light leaks from light pumpings
• Reagent/solvent purity
• High background luminescence from reagents and reactions
• Extreme sensitivity of chemiluminescence
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60. CONCLUSION
• Chemiluminescent methods are now well established in routine
clinical analysis and as tools in clinical and biomedical research.
• Many clinical laboratories preform routine immunoassay and nucleic
acid testing and research applications.
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61. References
• Campbell, A K. Chemiluminescence. Principles and applications in biology and
medicine. Germany: N. p., 1988. Web.
• C. Dodeigne, L. Thunus, R. Lejeune, Chemiluminescence as diagnostic tool. A
review, Talanta, Volume 51, Issue 3, 2000, Pages 415-439.
• ixia Zhao, Li Sun, Xiaogang Chu, Chemiluminescence immunoassay,TrAC Trends
in Analytical Chemistry, Volume 28, Issue 4,2009,Pages 404-415.
• L.J Kricka,Clinical applications of chemiluminescence,Analytica Chimica Acta,
Volume 500, Issues 1–2, 2003, Pages 279-286.
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