intoduction to lumiscence introduction and principle of chemilumiscence different types of lumiscence detail of the electrochemilumiscence, working, principle, instrumentation, measurin. application in medical field difference between chemilumiscence and elecrochemiluminescence

1. Dr Anurag Yadav
Pg Resident
Father muller medical college
LUMINISCENCE

2. OUTLINE

3. History :
 Luminescence is a science closely related to
spectroscopy. Which is study of general law of
absorption and emission of radiation by matter.
 In 19th century (1852’) english Physicist G C
Stokes identified this phenomenon and
formulated a Law- Stoke’s Law.
 Wavelength of emitted light is greater than that of
the exiting radiation.

4. History :
 Wiedmann (1888) intoduced the term
Luminescence (weak glow).
 “ a phenomenon of certain kind of substance
emitting light on absorbing the various energies
without heat generation is called Luminescence.”

5. Definition
Luminescence, emission of light by certain material
when they are relatively cool.
‘also referred as the Cool-Body radiation’
 It can be caused by chemical reactions, electrical
energy, subatomic motions, or stress on a crystal.

6. Definition
Chemiluminescence Bioluminescence &
Electrochemiluminescence are type of luminescence in
which the excitation event is caused by a
(1) Chemical,
(2) Biochemical or
(3) Electrochemical reaction
& not by photoillumination

7.  Substances emitting luminescence are called as
Luminophors or Phosphors.

8. How Chemiluminescence is different
from Photoluminescence
 Energy is produced by a chemical reaction.
 Excitation is not required.
 Problems like - light scattering & source instability are
absent.
 High background due to unselective photoexcitation is
also absent.
 Light detection is by photomultiplier- which are relatively
cheaper.

9. Luminometry
 Luminometry is the technique used to measure
luminescence, which is the emission of radiation
in the energy range of visible light as a result of a
reaction

10. Types : Chemiluminescence
• Bioluminescence
• Electrochemiluminesc
ence
Crystalloluminescence
Electroluminescence
Mechanoluminescence
Photoluminescence:
• Fluorescence
• Phosphorescence
Radioluminescence
Thermoluminescence

11. Excitation event process
Chemicals Luminol Isoluminol
acridinium ester
Chemiluminescence
Biochemical Luciferin
aequorin
Bioluminescence
Electromagnetic Ruthenium
Tris (bipyridly) chelate
Electroluminescence
Photons inorganic phosphors Photoluminescence
TYPES LUMINESCENCE

12. Principle- Chemiluminescence
Chemiluminescence occurs when there is
emission of light when an electron returns from an
excited or higher energy level to a lower energy level.
Excitation event is caused by a chemical reaction.

13. Principle- Chemiluminescence
 Excitation of electrons to higher energy level caused by a
chemical reaction (oxidation of luminol/ isoluminol/
acridinium esters/ luciferin by hydrogen peroxide/oxygen)
 Excited electrons return to lower energy level, light
emission

14. Principle- Chemiluminescence
- Involves oxidation of an Organic compound
- Luminol
- Isoluminol
- Acridinium esters
- Luciferin
- An Oxidant
- H2O2
- Hypochlorite
- Oxygen
- Light is emitted from the excited product formed

15. Principle- Chemiluminescence
Occurs in the presence of a CATALYST:
- Enzymes eg: - Alkaline phosphatase
- Horseradish-peroxidase
- Microperoxidase
- Metal ions
- Metal complexes eg: - Cu2+ & Fe3+
- Phthalocyanine complex
- Hemin

16. Principle- Chemiluminescence
2H2O2 + LUMINOL + ENHANCER------------------> 2H2O + hv +
Oxidized Luminol
• Sensitive
• linear response
• low consumption of reagent & High
stability
• fast emission of light
• More than using radioisotopes
• short incubation period
• absence of toxicity
ADVANTAGES:

17. Principle- Chemiluminescence
2H2O2 + LUMINOL -------------------> 2H2O + hv + Oxidized Luminol
DISADVANTAGES:
Low photon yield
Limited sensitivity
Less application

18. Principle- Bioluminescence
Naturally occurring Chemiluminescence
phenomenon.
Best understood
LUCIFERIN + ATP + O2 ----------> Oxyluciferin +
Light
- Quantitative
- 1 photon of light per ATP consumed
- Sensitivity limited by photodetectors ability to
count photons

19. Electrochemiluminescence

20. Electrochemiluminescence
Differ from Chemiluminescence that the reactive
species that produce reaction are electrochemically
generated from the stable precursors at the surface of
electrode.
Successful system- Chemiluminescent properties
of Ruthenium complexes when they encounter free
radicles.

21.  The antigen-biotinylated monoclonal antibody-monclonal antibody
labeled with ruthenium complex form a sandwich complex
 After addition of streptavidin-coated microparticles the complex
becomes bound to solid phase via interaction of biotin and
streptavidin.
 Microparticles are magnetically captured onto the surface of the
electrode.
 Application of a voltage to the electrode induces chemiluminescent
emission which is measured by a photomultiplier.
Principle-
Electrochemiluminescence

22. Principle- Electrochemiluminescence
Ru(complex) 2 - TPA (Tripropylamine); forms a
redox couple, emitting the light signal.

25. Principle- Electrochemiluminescence

26. Principle- Electrochemiluminescence
Ru(complex) can be attached to
Proteins
Nucleic acids
Ligands
Variety of IA formats

27. INSTRUMENTATION
Luminometers are instruments used to measure
chemi & electrochem
1. Sample cell
Housed in a light tight chamber
2. Injector system
Used to add reagent to the sample cell
3. Detector
PMT
4. For ECLIA
Electrode is incorporated in reaction vessel

29. Application
Chemiluminescence:
Ultrasensitive(attomole –zeptomole detection)
Wide dynamic range eg:TSH
Automated IA & DNA probe assay systems
Alkaline phosphatase labels
Enhanced-luminol reaction for horseradish
peroxidase labels

30. Application
Electrochemiluminescence:
Immunoassay
Nucleic acid assay
Advantages:
Improved reagent stability
Simple reagent preperation
Enhanced sensitivity
Detection limits 200fmol/L
Dynamic range ranging 6order of magnitude

31. Chemiluminescence
Immunoassay in Routine Clinical
Chemistry
 Hormones – T3, T4, FT4 ,FT3 ,TSH ,Anti TPO, Cortisol , LH,
FSH, Prolactin
 Tumor Markers - CEA, CA 125 , AFP, PSA, CA 19.9, CA 15-3 ,
Beta-HCG
 IgE, Hepatitis C virus anibody, Endothelin-1, Granulocyte colony
stimulating factor.

32. Non-immunoassay Applications of
Chemiluminescence
 Quantification of tyrosine
 Lipid peroxides and hydroperoxides
 Oxygen derivatives released from activated polymorphonuclear
leukocytes
 Biosensor applications
 Forensic applications

33. Limitations
Light leaks, Light piping
High background luminescence
from assay reagents and
reaction
Extreme sensitivity of
Chemiluminescence
Stringent control –Reagent
purity
• Solvent purity
Efficient injectors for
Chemi & electrochem
Pulse pile up in PMT

34. Reference :
 Tietz - clinical chemistry and molecular
diagnostic.
 Kaplan – clinical chemistry.
 Keith Wilson - principles and techniques of
biochemistry and molecular biology.
 Internet sources.