Chemiluminescence : Principle, Instrumentations and Applications

1. Prepared by-
Vedshree Raole
PQA, M.PHARM 1

2. • Introduction
• Principle
• Instrumentation
• Applications
Contents

3.  What happens to the EM energy subjected on a sample?
After the absorption of ultraviolet-visible light, the excited
molecules species are extremely short-lived and deactivated
occurs due to:
a. Internal collisions(internal conversion)
b. Cleavage of chemical bonds, initiating photochemical reaction
c. Re-emission as light( luminescence)
Re-emission of energy as luminescence occurs from molecules
in which the electron system is shielded from normal
deactivation processes so that complete deactivation by
collisions is discouraged.
Introduction

4. • Fluorescence
– Some energy is lost through various processes (e.g.
non-radiative transitions) and then light is given off.
• Phosphorescence
– The molecule transitions from an excited triplet state
to an lower energy singlet state and gives off light.
Non-radiative transitions intervene.
10-5 to 10-8 s fluorescence
10-4 to 10 s phosphorescence
10-14 to 10-15 s
- Excitation of e- by absorbance of hn.
- Re-emission of hv as e- goes to ground state.
- Use hn2 for qualitative and quantitative analysis

5. • Luminescence
- A spontaneous emission of radiation
from an electronically excited species (or
from a vibrationally excited species) not in
thermal equilibrium with its environment.
If the excitation energy is obtained from
chemical energy of reaction the process is
termed as Chemiluminescence
A + B  C*  C + hn

6. Criteria for a “successful” chemiluminescent
reaction
1. Sufficient excitation energy provided (by the chemical
reaction) for red emission (λ = 600 nm) 47.6 kcal/mol for
blue emission (λ = 450 nm) 63.5 kcal/mol
• The liberation of this much energy usually comes from
bond cleavage or electron transfer.
2. Formation of products capable of forming an excited
state multiple bonds, conjugation, aromatic systems.
3. Presence of an emitter (doesn’t have to be a product)
multiple bonds, conjugation, aromatic systems
4. Rapid kinetics (of the chemical reaction)
rate is more important than high yield (φCL)
φCL = φrxn(rxn) × φf(fluor.) << 10% (typical)
5. Reaction coordinate system must favor formation of an
excited state over ground state of product(s)

7. • Chemiluminescent reactions can be grouped
into three types:
1. Chemical reactions using synthetic compounds
and usually involving a highly oxidized species
such as a peroxide are commonly termed
chemiluminescent reactions.
2. Light-emitting reactions arising from a living
organism, such as the firefly or jellyfish, are
commonly termed bioluminescent reactions.
3. Light-emitting reactions which take place by
the use of electrical current are designated
electrochemiluminescent reactions.

8. • Chemiluminescent and bioluminescent reactions usually
involve the cleavage or fragmentation of the O-O
bond an organic peroxide compound.
• Peroxides, especially cyclic peroxides, are prevalent in
light emitting reactions because the relatively weak
peroxide bond is easily cleaved and the resulting
molecular reorganization liberates a large amount of
energy.
• Some electron transfer reactions ( such as with aromatic
radical ions from rubrene or p-benzoquinone or from metal
complexes such as tris(2,2’-bipyridyl) ruthenium(II) ) result
in CL emission without bond cleavage or rearrangement, so
those systems can be recycled, as in common with some
types of electrogenerated chemiluminescence.
• In order to achieve the highest levels of sensitivity, a
chemiluminescent reaction must be as efficient as
possible in generating photons of light. Each
chemiluminescent compound or group can produce no
more than one photon of light.

10. Luminol and peroxidase
before adding H2O2
Chemiluminiscence after
addition H2O2

11. Luciferin + O2
Luciferase
O C
O O
C R2
R1
Spontaneous
CO2 + O C*
R2
R1
Light

13. • Light emitted by the chemical reaction can e
detected and measured by various devices such as
photomultiplier tube, photographic films,
photocells and multiple channel detectors.
• The attractiveness of Chemiluminescence as an
analytical tool is the simplicity of detection. The
fact that a chemiluminescent process is, by
definition, its own light source means that assay
methods and the instruments used to perform them
need only provide a way to detect light and record
the result.
• Luminometers need consist of only a light-tight
sample housing and some type of photo-detector.
Instrumentation

14. • Microwell plates :- Light emitted in chemiluminescent
reactions is isotropic - it is emitted equally in all
directions. If a chemiluminescent assay were conducted
in the wells of a trans-parent microwell plate, light
would radiate out not only vertically, in the direction
of the detector, but also laterally in the direction of
other wells. Light is easily transmitted through the inter-
well gaps and throught the plate material itself, a
phenomenon termed light piping. Relatively bright
wells will introduce significant interference in adjacent
wells and beyond.
• For this reason,
CHEMILUMINESCENCE SHOULD NEVER BE MEASURED
IN CLEAR PLATES.
• Opaque microwell plates and strips are commercially
available from several suppliers. They come in two
kinds - white and black.

15. • Photomultiplier tubes have traditionally been the
workhorse detector in luminometers. Their advantages
include good sensitivity, a broad dynamic range and
applicability over a reasonably broad spectral range.
PMTs are known for their very low dark currents leading
to excellent signal to noise for low intensity samples.
• PMT based systems operate in two basic modes, single
photon counting and current sensing. There are examples
of hybrid systems which are single photon counting to a
light level in the low millions of photons/second and then
switch to current sensing above that level.
Photomultiplier-based Detection

16. • PMT single photon counting systems are capable of
exquisite sensitivity. Use of this type of detector is
the method of choice for low light detection and
quantitation as in, for example, detecting the
ultraweak luminescence associated with
phagocytosis. The greater sensitivity comes at a
cost however. Sample housings must be very light-
tight. Moderate light levels saturate the detector;
high levels can cause physical damage.
• PMT current sensing systems are also capable of
excellent sensitivity and will often read higher light
levels than single photon counting systems without
damage.

17. Photodiodes are capable of recording higher light intensities than
photomultiplier tube detectors. This facet makes them an excellent
choice for applications where high light intensities are to be
measured. However, the inherent dark current in solid state detectors
is generally much higher than that of photomultiplier tubes. One
method of mitigating this problem is to cool the solid state detector
via a Peltier or other thermoelectric cooler. Dark currents in solid
state detectors drop dramatically with temperatures in the 0 to -30
degree celsius range. Cooled detectors can then be used to integrate
the light intensity for one to hundreds of seconds without the signal
being overwhelmed by dark current.CCD and other solid state
detectors, although , possess several inherent advantages.
1. Solid state detectors typically offer a "flatter" optical response over the
visible range. Luminescent reactions emitting red and even near
infrared light can be detected with enhanced sensitivity.
2.Camera systems allow imaging of variety of objects. Virtually any kind
of sample or container can be accommodated ranging from
microwell plates and test tubes to bacterial or cell cultures in Petri
dishes, electrophoresis gels and blotting membranes.
Solid State Detection

18. 3. Single PMT systems must have the sample position well
defined before it can be read. A sample tube has to be
brought to a reproducible position to be read
repeatably. Microwell plate PMT readers rely upon the
standard spacing of microwells and will generally move
the plate around to a precalculated position so the
wells can be read one by one. Camera systems have
the advantageof being able to read a sample without
knowing its position in advance, as in the example of a
band on a blot. The camera imaging system gives
positioning information along with sample intensity.
4. CCD camera systems allow imaging of numerous
objects simultaneously. In the present era of 96, 384
and higher number well plates, parallel data collection
is no longer a luxury. Solid state camera imaging
systems have the potential to permit imaging and
quantitation of entire plates in one pass.

19. APPLICATIONS
Chemiluminescence immunoassay
DNA hybridization detection
Western blotting
Forensic science
Food analysis

20. 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
CHEMILUMINESENCE IMMUNOASSAY

22. Monoclonal antibody coated well
Test specimen (serum)
HRP labelled antibody conjugate
Test antigen: sandwich between solid phase
Ab and enzyme labelled Ab

23. Incubate for 1 hr at 37° C
Remove unbound enzyme labeled Ab
Chemiluminescence reagent added
Read relative light unit with luminometer

24. • The widely used enzymes for luminescent
immunoassays are AP(Alkaline
phosphatase) and HRP(Horseradish
peroxidase) and pyruvate kinase(in
bioluminescence)
The suitable substrates for luminescent
immunoassays include
(i) luminol (ii) acridine esters (iii)Isoluminol
(iv)Luciferin (v)AMPPD

25. • USES
Hormones: insulin, thyroxin, estradiol,
testosterone, progesterone
Vitamin: Vit B12
Tumor markers: bone morphogenic
protein-2, carcino embryonic antigen
(CEA), alpha fetoprotein (AFP)
Human beta chorionic gonadotropin
C-reactive protein
Tumor necrosis factor

26. DNA hybridization detection
Southern blotting Involves DNA separation,
transfer and hybridization
Hybridization - Process of forming a double-
stranded DNA molecule between a single-
stranded DNA probe and a single-stranded
target DNA

27. 3.The restriction
fragments present in
the gel are denatured
with alkali and
transferred onto a
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.

28. 4. The filter is incubated
under hybridization
conditions with a specific
HRP labelled DNA probe.
• The probe hybridizes to
the complementary DNA
restriction fragment.
• Detection reagent
containing H2O2 &
luminol is added onto the
membrane

30. • USES
Identifying DNA in crime case
Paternal Dispute
Classify DNAs of various organism

31. Steps in southern blotting
1.The DNA to be
analyzed, such as the
total DNA of an
organism, is digested
to completion with a
restriction enzyme.
2.The complex mixture
of fragments is
subjected to gel
electrophoresis to
separate the fragments
according to size.

32. Western blotting
Western blotting (or protein
immunoblotting) is a technique widely used
to detect specific proteins in samples of
tissue homogenate, cell lysates, cell culture
supernatants or body fluids.

33. Western blotting

35. 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, luminol to glow

37. A trail of blood made visible with the use of the
reagent luminol.

38. Food analysis
Organophosphorous most popular
pesticide
Most commonly used
organophosphorous: QUINALPHOS (O,O
diethyl-o-quinoxalinly phosphorothioate)

39. Quinalphos +H2O2 peroxophosphonate
Peroxophosphonate+ luminol 3aminophthalate
anion*
3-aminophthalate* 3-aminophthalate
+
observed emission
Food analysis

40. Clinical Utilities

41. • Pesticide Analysis
Other Applications
Determination of nitrogen monoxide
NO + O3 → NO2* + O2
NO2* + → NO2 + hn ( = 600 – 2800 nm)
Determination of sulfur
4H2 + 2SO2 → S2* + 4H2O
S2* → S2 + hn ( = 384 and 394 nm)

42. • 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
Limitations

43. 1. Estimation of glipizide by CL:
• Luminol + K3Fe(CN)6 3-aminophthalate ion* (3-
AP*)
3-AP* 3-AP hv (λmax = 425 nm)
• GP + dissolved oxygen + NaOH
superoxide radical + other reaction product
• Superoxide radical + luminol + K3Fe(CN)6 (3-AP*)
• 3-AP* 3-AP + hv (λmax =425 nm)
Other Applications

44. • The fluorescence spectra of luminol,luminol–
K3Fe(CN)6, and luminol– K3Fe(CN)6–GP reaction
were scanned in the range of 280– 650 nm, using
an RF5301 fluorescence spectrophotometer. It had
been reported that 3-aminophthalate ion(3-AP),an
oxidized product of luminol, peaking at 425nm
was known as the emitter in the luminol–
K3Fe(CN)6 system. The results obtained were all
found to have the same maximum emission
appearing at 425nm; the luminophor was
confirmed to be 3AP. This indicated that the
luminant in the luminol– K3Fe(CN)6–GP system
was still 3-AP.
• The CL reaction of luminol with superoxide radical
can be catalyzed by potassium ferricyanide in
alkaline solution.

45. 2. Estimation of Atropine
• A sensitive and fast chemiluminescence (CL)
method has been described for the direct
determination of atropine. The method is
based on the quenching effect of atropine on
the CL signals of the luminol– H2O2 reaction
catalyzed by hemin in an alkaline medium.
Hemin could strongly enhance the
chemiluminescence of the luminol–H2O2
system. The luminophor of the luminol–
H2O2–hemin CL system was identified as the
excited state 3-aminophthalate anion. The CL
from luminol–H2O2–hemin system is strongly
inhibited by the presence of atropine.

47. 3. Estimation of Vitamin B12

48. 4. Estimation of Estrogen:
Adsorption of H2O2 on the surface of gold
nanoparticles, we can deduce there must
be competitive adsorption on the surface
of gold nanoparticles between estrogens
and H2O2, which leads to inhibition of CL.
Estrogens can, on the other hand, react with
hydrogen peroxide or oxygen-related
radicals generated from H2O2, which might
consume the oxidant and inhibit CL
intensity.

49. • It has been found that gold nanoparticles
(nano- Au) enhance the chemiluminescence
(CL) of the luminol-hydrogen peroxide system
and that estrogens inhibit these CL signals in
alkaline solution. CL spectra, UV–visible
spectra, X-ray photoelectron spectra (XPS),
and transmission electron microscopy (TEM)
were used to investigate the mechanism of the
CL enhancement. On the basis of the
inhibition, a flow-injection CL method has
been established for determination of three
natural estrogens.
• This method has been used for analysis of
estrogens in commercial tablets and in urine
samples from pregnant women.

51. 5. DNA hybridization by electro-chemiluminescence
Quantum Dots ECL in aqueous solution and SWASV*
were employed for DNA detection. Through the
biotin–avidin-system, avidin–QDs bind tightly to
the biotin-modified cDNA oligonucleotides. By
utilizing the ECL property of QDs, the DNA
hybridization event was converted into a
detectable ECL signal. On the other hand, by
detecting cadmium content in the bound QDs, the
target DNA was indirectly detected through the
SWASV assay. It provided a relatively simple, time-
saving and multi-approach for DNA detection.
*SWASV :square wave anodic stripping voltammetric technique.

53. 6. TOPICAL COMPOSITIONS AND METHODS
OF DETECTION AND TREATMENT
A topical composition includes a nanoemulsion
of a plurality of hydrophobic particles having
a hydrophilic coating therein. The
hydrophobic particles are derived from the
same or different hydrophobic material and
each hydrophobic particle has a melting point
below the melting point of the respective
hydrophobic material. The nanoemulsion
further includes one or more pharmaceutically
active agents and/or one or more
chemiluminescent disease-detecting systems.

54. • The nanoemulsion can also include one or more
chemiluminescent disease-detecting systems incorporated
therein for detecting a disease or condition in a host. For
example, hydrogen peroxide (H202) is a reactive oxygen
metabolic by-product that can serve as a key regulator for a
number of oxidative stress related states. Hydrogen
peroxide is believed to be over-produced by cells at the
early stages of most diseases such as asthma, infammatory
arthritis, athero sclerosis, diabetic vasculopathy,
osteoporosis, and a number of neurodegenerative diseases.
• overproduction of hydrogen peroxide can occur in the
development of damage caused to skin by exposure to
ultraviolet radiation. Thus, detecting low levels of hydrogen
peroxide in the skin could serve as an early Warning
indicator for skin cancer. The topical composition of the
present invention can therefore be used as a simple, all-
purpose diagnostic tool for detecting diseases.

55. • chemical reaction that produces
chemiluminescence. In general, the
mechanism is that first the phenyl oxalate
ester and hydrogen peroxide (H202) react to
form a peroxy acid ester and phenol; and
then the peroxy acid ester decomposes to
form more phenol and a highly energetic
intermediate, presumed to be a cyclic
compound containing a four-membered ring
dimer of CO2.As the cyclic dimer decomposes
into two CO2 molecules, it gives up its energy
to a waiting dye molecule, Which then
fluoresces.(fig.1)

56. • the chemical reaction taking place includes a
solution of a phenyl oxalate ester (commonly
bis(2,4,5-trichlorophenyl-6-
carbopentoxyphenyl)oxalate (CPPO), a fluorescent
dye that determines the color of light, and
hydrogen peroxide (H2O2). The hydrogen
peroxide reacts With the phenyl oxalate ester
producing carbon dioxide, a phenol and, most
importantly, releasing energy. This energy is
absorbed by the dye, exciting electrons in the dye’s
molecules to a higher energy level.
• Once at the higher energy level, the electrons
immediately lose the energy they absorbed and fall
to lower energy levels. As the electrons fall back to
loWer energy levels, the energy that is lost is
transformed into electromagnetic radiation, some
of Which is visible light.(fig. 2)

58. 7. A simple and rapid flow injection (FI) method for the
determination of retinyl acetate is reported based on its
enhancing effect on the luminol-periodate chemiluminescence
(CL) system in an alkaline medium.
8. A simple and rapid flow-injection method is reported for the
determination of retinol, its derivatives and a-tocopherol by its
enhancement effect on a potassium permanganate–formaldehyde
chemiluminescence system in an acidic medium.
9. A novel chemiluminescence (CL) system was evaluated for the
determination of hydrogen peroxide, glucose and ascorbic acid
based on hydrogen peroxide, which has a catalytic-cooxidative
effect on the oxidation of luminol by KIO(4). Hydrogen peroxide
can be directly determined by luminol-KIO(4)-H(2)O(2) CL
system. The present method provides a source for H(2)O(2),
which, in turn, coupled with the luminol-KIO(4)-H(2)O(2) CL
reaction system. The CL was linearly correlated with glucose
concentration. Ascorbic acid was also indirectly determined by the
suppression of luminol-KIO(4)-H(2)O(2) CL system.

59. 10. The proposed CL-FIA system has been applied to the
determination of sulphadiazine (a sulphonamide
mainly used in the treatment of urinary tract infections
for human and veterinary use) using bis[2,4,6-
trichlorophenyl]oxalate (TCPO) as CL precursor, H2O2
as oxidant, imidazole as a catalyst and fluorescamine as
the fluorescent derivatizing agent.
• The use of the PO-CL reaction in micellar medium,
coupled to a FIA manifold has been proposed as an
alternative detection system for quality control of
sulphonamides in pharmaceutical samples. The method
implies the off-line formation of a fluorescent
derivative (fluorophore) with fluorescamine and the
subsequent oxidation of TCPO by H2O2 using
imidazol as catalyst in alkaline medium, in presence of
the fluorophore, whose CL emission is proportional to
the sulphonamide concentration.

60. • Development of a novel luminol
chemiluminescent method catalyzed by gold
nanoparticles for determination of estrogens
Yongxin Li & Ping Yang & Po Wang & Lun Wang.
• Luminol–K3Fe(CN)6 chemiluminescence system
for the determination of glipizide Xin Chena, Li-
LiXingb, Yu-HaiTangb,n, Guang-BinZhang.
• Quantum dot-based DNA hybridization by
electroc-hemiluminescence an anodic stripping
voltammetry Haiping Huang, Jingjing Li, Yanglan
Tan, Jinjun Zhou and Jun-Jie Zhu*
Refereces

61. • Analytical Applications of Flow Injection
Chemiluminescence for the Determination of
Pharmaceuticals–A Review Amir Waseema*,
Mohammad Yaqoobb and Abdul Nabib.
• Vitamin A Determination in Milk Samples Based on the
Luminol-Periodate Chemiluminescence System Lubna
Rishi1, Mohammad Yaqoob1, Amir Waseem2,* and
Abdul Nabi1.
• Evaluation of luminol–H2O2–KIO4 chemiluminescence
system and its application to hydrogen peroxide,
glucose and ascorbic acid assays Yanxiu Zhou ,Tsutomu
Nagaoka,Feng Li,Guoyi Zhu
• Flow injection methods for the determination of
retinol and α-tocopherol using lucigenin-enhanced
chemiluminescenceMohammad Asgher,Mohammad
Yaqoob,A. Waseem,Abdul Nabi

62. • Chemiluminescence determination of sulphadiazine in
drugs by flow injection analysis using the peroxyoxalate
reaction in micellar medium. Giuseppe Lattanzio a, Ana
M. Garc´ıa-Campa˜na b,∗, Jorge J. Soto-Chinchilla
b,Laura G´amiz-Gracia b, Steffano Girotti a.
• Chemiluminescence Determination of Atropine using
Luminol-Hemin-H2O2 System Seyed Naser Azizi*,
Mohamad Javad Chaichi, Maryam Heidarpour.
• Chemiluminescence-based detection: principles and
analytical applications in flowing streams and in
immunoassays W.R.G. Baeyens a,*, S.G. Schulman b, A.C.
Calokerinos c, Y. Zhao a, A. Ma Garcı´a Campan˜a d, K.
Nakashima e, D. De Keukeleire f

63. • Chemiluminescent flow-through sensor for 1,10-
phenanthroline based on the combination of molecular
imprinting and Chemiluminescence Jin-Ming Lin* and
Masaaki Yamada.
• Chemiluminescence Analyses of Biological Constituents
using Metal-Complex Catalysts -A Review,Tadashi HARA
and Kazuhiko TSUKAGOSHI.
• Effect of Catlyst on Luminol - Hydrogen Peroxide–Water
Chemiluminescence System V.K. Jain*

64. • Topical compositions and methods of
detection and treatment Jennifer L. Sample,
Chevy Chase, MD (US); Julia B. Patrone,
Ellicott City, MD (US); Jason J. Benkoski,
Ellicott City, MD (US); James C. Crookston,
Falls Church, VA (US); Huong Le, Olney, MD
(US); Jennifer L. Breidenich, Atlanta, GA (US);
Lisa A. Kelly, Ellicott City, MD (US)(PATENT
2012)