The document discusses the principles and applications of chemiluminescence, focusing on its use in immunoassays and various biochemical analyses. It outlines the mechanisms of chemiluminescence, enhanced chemiluminescence, and electrochemiluminescence, as well as the types of substrates and enzymes employed. Additionally, the document addresses the benefits, limitations, and specific applications of chemiluminescence in fields such as forensic science and clinical diagnostics.

1. Chemiluminescence
DIPESH TAMRAKAR
M.SC. CLINICAL BIOCHEMISTRY
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2. OVERVIEW
• Luminescence
• Chemiluminescence IMMUNOASSAY
• Enhanced Chemiluminescence IMMUNOASSAY
• Electro Chemiluminescence IMMUNOASSAY
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3. LUMINESCENCE
Luminescence is described as the emission of light from a
substance as it returns from an electronically excited state to ground
state.
The various forms of luminescence (bioluminescence,
chemiluminescence, photoluminescence) differ in the way the
excited state is reached.
For example,
Photoluminescence is simply fluorescence; the excitation is
initiated by light at a particular wavelength.
Bioluminescence is characterized by the use of a bioluminescent
compound, such as luciferin and firefly luciferase.
Chemiluminescence is light produced by a chemical reaction.
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4. PRINCIPLE:
• Luminescence means that light is emitted by a substance when it
returns from an excited state to a ground state.
• There are different types of luminescence and the various forms
differ in the way that they achieve the excited state.
• For chemiluminescence it is light produced by a chemical reaction.
This chemiluminescent substance can be excited by an oxidation
reaction forming an intermediate.
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5. • It is when this immediate return to a stable ground state happens,
that a photon is released and this is detected by the luminescent
signal instrument.
• The particular luminescence indicates the presence of the antigen.
• The amount of the particular biological molecule, which is being
looked for and is present in the sample, is based on the
luminescence observed.
• There are different types of substrate that are used for
chemiluminescence, with the most popular types being luminol or
its derivatives.
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6. TYPES OF LUMINESCENCE
Excitation event Process
Chemicals Luminol, Isoluminol,
acridinium ester
Chemiluminescence
Biochemical Luciferin, aequorin Bioluminescence
Electromagnetic Ruthenium, Tris
(bipyridly) chelate
Electroluminescence
Photons inorganic phosphors Photoluminescence
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7. • Chemiluminescent immunoassays are variations of the standard
ELISA where an enzyme converts a substrate to a reaction product
that emits photons of light instead of developing a visible color.
• The chemiluminescent substance is excited by the oxidation and
catalysis forming intermediates.
• When the excited intermediates return back to their stable ground
state, a photon is released, which is detected by the luminescent
signal instrument.
CHEMILUMINESCENCE
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8. Emission of light with limited emission of heat (luminescence), as the result of
a chemical reaction.
[A] + [b] → [◊] → [products] + light
[A], [b]: reactants [◊]: Excited intermediate
For example, if [A] is luminol and [B] is hydrogen peroxide in the presence of
a suitable catalyst we have:
Luminol + H2O2 →3-APA[◊] →3-APA + light
Where:
3-APA is 3-aminophthalate
3-APA[◊] is the excited state producing light as it decays to a lower energy
level.
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9. • Both bioluminescence and chemiluminescence are widely used for
immunoassays as “luminescence”.
• Luminescent assays are very sensitive and have a wide dynamic
range.
• It is believed that luminescence is the most sensitive detection
method currently in use due to the ability of signal multiplication
and amplification.
• Luminescent reactions are measured in Relative Light Units (RLU)
that are typically proportionate to the amount of analyte present in
a sample.
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10. • Chemiluminescence differs from fluorescence or phosphorescence
in that the electronic excited state is the product of a chemical
reaction rather than of the absorption of a photon
• It is the antithesis of a photochemical reaction, in which light is
used to drive an endothermic chemical reaction
• Light is generated from a chemically exothermic reaction.
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12. • The widely used enzymes for luminescent immunoassays are also
AP and HRP.
Suitable Substrates For Peroxidase
1. Luminal
(3-aminophthal
hydrazide)
2. Polyphenols and
acridine esters
i.3. Luciferin
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13. i. Luminal:
• The most popular substrate used for immunoassays is luminal or
its derivatives such as isoluminol (4-aminophthalhydrazide).
• It is the most suited for clinical diagnostic tests due to its
properties when used in an enhanced luminescence system.
• Commercially available luminol is provided with an enhancer
(phenols, naphthols, aromatic amines, or benzothiazoles) that acts
as an enzyme protector and allows the reaction to proceed for
many minutes without substantial decay in light output.
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14. • Luminol oxidation reaction is carried out in an alkaline buffer,
peroxidase enzymes and reactive oxygen species [peroxide anion
(O2-), singlet oxygen (1O2), hydroxyalkyl radical (OH •), peroxide
hydrogen (H2O2)], to generate excited state intermediates.
• When they return to the ground state, a wavelength of 425 nm is
emitted. Typically, light emission stabilizes in less than 2 minutes,
and sustained emission lasts for approximately 20 minutes or
more.
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15. 2. Polyphenols and acridine esters
• Polyphenols are actually a class of substrates that include
pyrogallol, purpurogallin, gallic acid, and umbelliferone.
• All polyphenols are known for their excellent signal to noise ratio
and extremely rapid light decay.
• Polyphenol and acridine ester substrates can only be used in
conjunction with luminescent detectors equipped to handle
“flash” reactions.
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16. 3. Luciferin
• The reaction of peroxidase with luciferin is considered
bioluminescence.
• In this reaction, peroxidase replaces the in vivo enzyme,
luciferase.
• The other substrates are chemiluminescent compounds.
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17. Working principle of Diasorin Liaison CLIA for TSH
• sandwich chemiluminescence immunoassay.
• A specific mouse monoclonal antibody is coated on the magnetic particles (solid phase);
another monoclonal antibody is linked to an isoluminol derivative (isoluminol-
antibody conjugate).
• During the incubation, TSH present in samples binds to the solid phase monoclonal
antibody, and subsequently the antibody conjugate reacts with TSH already bound to
the solid phase.
• After incubation, the unbound material is removed with a wash cycle. Subsequently,
the starter reagents are added and a flash chemiluminescence reaction is thus induced.
• The light signal, and hence the amount of isoluminol-antibody conjugate, is measured
by a photomultiplier as relative light units (RLU) and is indicative of TSH
concentration present in samples
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18. Application of Chemiluninescence
Chemiluminesence Immunoassay
DNA Hybridization Detection
Western Blotting
Forensic Science
Food Analysis
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19. CHEMILUMINESENCE IMMUNOASSAY
Provides a sensitive, high throughput alternative to conventional
colorimetric methodologies
Principle: -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 (serum)
HRP labelled antibody conjugate
Test antigen: sandwich between solid phase ab and
enzyme labelled ab
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21. Incubate for 1 hr at 37° C
Remove unbound enzyme labeled Ab
Chemiluminescence reagent added
Read Relative Light Unit with luminometer
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22. • USES
Hormones: insulin, thyroxin, estradiol, testosterone,
progesterone
Vitamin: vit B12, vit D
Tumor markers: bone morphogenic protein-2, carcino
embryonic antigen (CEA), alpha fetoprotein (AFP)
Human beta chorionic gonadotropin
C-reactive protein
Tumor necrosis factor
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23. Benefits of CLIA
• ultra-sensitive and can detect small amounts of the biological
molecule.
• wider dynamic range, as well as having a linear relationship
between luminous intensity and the concentration of the
measured substance.
• Enhancer, which acts as an enzyme protector can allows the
reaction to occur for a longer period of time
• The enhanced reaction gives an intense light emission for a
prolonged period of time, as well as the substrate only needing to
be added minutes before detection.
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24. Forensic science
Chemiluminescence is used by criminalists to detect traces of
blood at crime scene
Solution: luminol powder (C8H7O3N3), hydrogen peroxide, and a
hydroxide (eg. KOH) sprayed where blood might found
Tiny amount of iron from Hb in blood serves as catalyst for the
chemiluminescence reaction proceeding luminol to glow
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25. Limitations
• Light leaks from assay reagent & reaction vessels
• Ultra sensitive – stringent controls on purity of reagents
• High intensity light emission leads to pulse pileup in
photomultiplier tubes leads to underestimation
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26. Enhanced Chemiluminescence
• Enhanced chemiluminescence is a common technique for a variety
of detection assays in biology.
• A horseradish peroxidase enzyme (HRP) is tethered to an antibody
that specifically recognizes the molecule of interest.
• This enzyme complex then catalyzes the conversion of the enhanced
chemiluminescent substrate into a sensitized reagent in the vicinity
of the molecule of interest, which on further oxidation by hydrogen
peroxide, produces a triplet (excited) carbonyl, which emits light
when it decays to the singlet carbonyl.
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27. • It allows detection of minute quantities of a biomolecule (Proteins in
femtomole quantities)
• Enhanced luminescence is characterized by the following desirable
features:
• Intense light emission
• Prolonged light emission, low background
• No preincubation step, and
• Substrate that can be added several minutes prior to detection.
• As long as commercial preparations of luminol are used, control of
the reaction pH is not a concern.
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28. • As mentioned, luminol based chemiluminescence is well suited for
microplate-based immunoassays; in addition, this system is also
recommended for DNA probe assays.
• AMPPD (3-(2'-spiroadamantane)-4- methyl-4-(3'-
phosphoryloxyphenyl-1, 2- dioxetane, disodium salt) is the
substrate most commonly used with alkaline phosphatase.
• A similar substrate, AMPGD (3-(2'-spiroadamantane)-4-methoxy-4-
(3'-ß-D-galactopyranosyloxyphenyl-1,2-dioxetane), is routinely used
with ß-galactosidase.
• Both substrates are compatible with commercially available
enhancers.
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29. Working principle of Vitros ECI for TSH estimation
• TSH present in the sample reacts with a biotinylated antibody (mouse
monoclonal anti-whole TSH) and a horseradish peroxidase (HRP)-
labeled antibody conjugate (mouse monoclonal anti-TSH β-subunit).
• The antigen-antibody complex is captured by streptavidin on the wells
& unbound materials are removed by washing.
• The bound HRP conjugate is measured by a luminescent reaction.
• A reagent containing luminogenic substrates (a luminol derivative and
a peracid salt) and an electron transfer agent, is added to the wells.
• The HRP in the bound conjugate catalyzes the oxidation of the luminol
derivative, producing light.
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30. • The electron transfer agent (a substituted acetanilide) increases the level
of light produced and prolongs its emission. The light signals are read
by the system.
• The amount of HRP conjugate bound is directly proportional to the
concentration of TSH present.
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31. Avidin-Biotin Interaction
• biotin label is stable and small, it rarely interferes with the function
of labeled molecules enabling the avidin-biotin interaction to be
used for the development of robust and highly sensitive assays.
• Advantages of using avidin-biotin systems:
• The avidin-biotin complex is the strongest known non-covalent
interaction (Kd = 10-15M) between a protein and ligand.
• The bond formation between biotin and avidin is very rapid,
and once formed, is unaffected by extremes of pH, temperature,
organic solvents and other denaturing agents.
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33. Electrochemiluminescence
• Electrochemiluminescence or electrogenerated
chemiluminescence (ECL) is a kind of luminescence produced
during electrochemical reactions in solutions.
• In electrogenerated chemiluminescence, electrochemically
generated intermediates undergo a highly exergonic reaction to
produce an electronically excited state that then emits light upon
relaxation to a lower-level state.
• This wavelength of the emitted photon of light corresponds to the
energy gap between these two states
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34. • ECL excitation can be caused by energetic electron transfer (redox)
reactions of electrogenerated species.
• Such luminescence excitation is a form of chemiluminescence
where one/all reactants are produced electrochemically on the
electrodes.
• ECL is usually observed during application of potential (several
volts) to electrodes of electrochemical cell that contains solution of
luminescent species (polycyclic aromatic hydrocarbons, metal
complexes, Quantum Dots or Nanoparticles) in aprotic organic
solvent (ECL composition).
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35. • The excitation energy is obtained from recombination of oxidized
and reduced species.
• In aqueous medium, which is mostly used for analytical applications,
simultaneous oxidation and reduction of luminescent species is
difficult to achieve due to electrochemical splitting of water itself
so the ECL reaction with the coreactants is used.
• In the later case luminescent species are oxidized at the electrode
together with the coreactant which gives a strong reducing agent
after some chemical transformations (the oxidative reduction
mechanism).
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36. Application
• ECL proved to be very useful in analytical applications as a highly
sensitive and selective method.
• As an analytical technique it presents outstanding advantages over
other common analytical methods due to its versatility, simplified
optical setup compared with photoluminescence (PL), and good temporal and
spatial control compared with chemiluminescence (CL).
• Enhanced selectivity of ECL analysis is reached by variation of
electrode potential thus controlling species that are oxidized/reduced
at the electrode and take part in ECL reaction (see electrochemical
analysis).
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37. • It generally uses Ruthenium complexes, especially [Ru (Bpy)3]
regenerating with TPrA (Tripropylamine) in liquid phase or liquid–
solid interface.
• It can be used as monolayer immobilized on an electrode surface or
as a coreactant or more commonly as a tag and used in HPLC,
Ru tagged antibody based immunoassays,
Ru Tagged DNA probes for PCR,
NADH or H2O2 generation based biosensors,
oxalate and organic amine detection
and many other applications and can be detected from
picomolar sensitivity to dynamic range
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38. • Photon detection (which releases a photon at ~620 nm) is done with
photomultiplier tubes (PMT) or silicon photodiode or gold coated
fiber-optic sensors.
• The importance of ECL techniques detection for bio-related
applications has been well established.
• ECL is heavily used commercially for many clinical lab
applications
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40. Summery:
CLIA
• High sensitivity & specificity
• No background signal (less
interfering)
• Measures light production
• Expensive
• Higher detection range
• Rapid
• No stop solution
ELISA
• Comparatively low
• Presence of interfering factors
• Measures color complex formation
• Cheaper
• Detection range is low
• Time consuming
• Need stop solution
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