Luminescence Definition Luminescence is emission of light by a substance not resulting from heat Types of luminescence Fluorescence Phosphorescence Chemiluminescence Bioluminescence ID: 237667
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Slide1
ChemiluminescenceSlide2
Luminescence
Definition
Luminescence
is emission of light by a substance not resulting from
heat
Types of
luminescence
Fluorescence
Phosphorescence
Chemiluminescence
Bioluminescence
Electrochemiluminescence
Slide3
Luminescence
Excitation
(
higher energy
level)
an
electron returns
from an excited or higher energy level to a
lower energy level
The
physical event of the
light emission
Slide4Slide5
Luminescence
Fluorescence & Phosphorescence
The luminescence
produced by certain
substances after absorbing radiant
energy.
The
excitation
event is caused by
photoillumination
Phosphorescence
is distinguished from fluorescence
in that it continues
(for second to minutes)
even after the radiation causing
it has ceased. Slide6
Chemiluminescence
the
emission
of light when an electron returns from an excited or higher energy level to a lower energy level.
The
excitation
event is caused by a
chemical reaction
Involves the
oxidation
of an
organic compound
Such as
luminol
,
isoluminol
,
acridinium
esters,
or
luciferin
by an
oxidant
(e.g., hydrogen peroxide,
hypochlorite,or
oxygen)
Light is emitted from the excited product formed in the oxidation reactionSlide7
Chemiluminescence
Reactions occur in the presence of
catalyst
s
Such as
Enzymes
(e.g., alkaline phosphatase, horseradish peroxidase, and
microperoxidase
),
Metal ions
or metal complexes (e.g., Cu
2+
and Fe
3+
phthalocyanine
complex), and
hemin
.Slide8
Bioluminescence
Bioluminescence is a special form of
chemiluminescence
found in biological systems.
In bioluminescence, an enzyme or a
photoprotein
increases the efficiency of the luminescence reaction.
Catalysts
Luciferase
Aequorin
The quantum yield (e.g., total photons emitted per total molecules reacting) is approximately 0.1% to 10% for
chemiluminescence
and 10% to 30% for bioluminescence.Slide9
Chemiluminescence
assays are
Ultrasensitive
attomole
to
zeptomole
detection limits
have
wide dynamic range
s.
Several orders of magnitude
They are now widely
used in
automated immunoassay
DNA probe assay systems
e.g.,
acridinium
ester and
acridinium sulfonamide labels and 1,2-dioxetane substrates for alkaline phosphatase labelsthe enhanced-Iuminol reaction for horseradish peroxidase labels
One zeptomole = 10-3 attomoles or 10-6 femtomoles.
The enzyme horseradish peroxidase (HRP), found in the roots of plant
horseradish,…Slide10
Electrochemiluminescence
the reactive species that produce the
chemiluminescent
reaction are electrochemically generated from stable precursors at the surface of an electrode.
A ruthenium (Ru
2+
),
tris
(
bipyridyl
) chelate is the most commonly used
electrochemiluminescence
label
Electrochemiluminescence
is generated at an electrode via an oxidation-reduction-type reaction with
tripropylamine
.Slide11
Ruthenium and TPA are oxidized at the surface of the electrode when voltage is applied. The TPA loses a proton, which reduces the ruthenium to an excited state, causing light to be emitted. Ruthenium is not consumed in the reaction, so this cycle can continue as long as TPA is present. Multiple excitation/emission cycles amplifies the light emitted and increase sensitivity. The emitted light is measured to determine concentration of
analytes
in sample
. (TAG: Label) Slide12
Electrochemiluminescence
The advantages
Improved reagent stability, simple reagent preparation, and enhanced sensitivity.
Detection limits of 200
fmol
/L and a dynamic range extending over six orders of magnitude can be obtained.Slide13
INSTRUMENTATION
Luminometers
are instruments used to
measure
chemiluminescence
and
electrochemiluminescence
.
The basic components
the sample cell
Housed in a light-tight chamber
the injection system
to add reagents to the sample cell
the
detector
A photomultiplier tube.
an electrode
(For electrochemiluminescence)at which the electrochemiluminescence is generated.Slide14
Limitations of
Chemiluminescence
Light leaks, light piping, and high background
luminescence from
assay reagents and reaction vessels (e.g., plastic
tubes exposed
to light) are common
factors
The extreme sensitivity of
chemiluminescence
assays requires stringent controls on the
purity
of reagents and the solvents (e.g., water) used to prepare reagent solutions.Slide15
Limitations
of
Chemiluminescence
Efficient capture of the light emission from reactions that produce a flash of light requires an efficient injector that provides adequate
mixing
when the triggering reagent is added to the reaction vessel
Chemiluminescent
and
electrochemiluminescent
assays have a wide linear range, usually several orders of magnitude, but very high intensity light emission can lead to
pulse pile-up
in
photomultiplier
tubes and this leads to a serious
underestimate
of the true light emission intensity.Slide16
A
,
Chemiluminescent
assay for horseradish
peroxidase label using
luminol
.
B
,
Chemiluminescent
assay for
an
alkalinephosphatase
label
using AMPPD
.
C
, Photometric assay for an alkalinephosphatase
label using acascade detection reaction. INT, p-iodonitrotetrazolium violet.3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl- 1,2-dioxetane (AMPPD)Slide17
Aequorin
is a
photoprotein
isolated from the hydrozoan
Aequorea
victoria
.Slide18