Chapter 19 - Part 3

Section 19-6
Sensors Based on Luminescence

Quenching
Quantitative Chemical Analysis, Daniel C. Harris and Charles A. Lucy, © 2020 W. H. Freeman and Company 75
Luminescence Quenching
Luminescence quenching: process by which emission from Figure 19-20
an excited state molecule is decreased by energy transfer to
another molecule
The molecule that accepts the energy transferred from the

fluorophore is called a quencher. The quencher reduces
fluorescence by providing a nonradiative relaxation path.
Example of an O2 Sensor Based on Quenching
This fiber-optic sensor measures O2 in a fish tank by its

ability to quench the luminescence of Ru(II) embedded on
the tip of the fiber. A blue light-emitting diode provides
excitation energy.
Video: Amperometric and optical measurements of dissolved oxygen
Absorption, Emission, and Deactivation
• Molecule M absorbs light and is promoted to the excited state M*:
*
d [M ]
M + hv → M*
Rate = = ka[M]
dt
• Following absorption, M* can emit a photon and return to the
ground state. *
d [M ]
M → M + hv Rate = −
*
= ke [M* ]
dt
• Alternatively, the molecule can lose energy in the form of heat.
d[M* ]
M* → M + heat Rate = − = kd[M* ]
dt
Quantum Yield (1 of 2)
The quantum yield for a photochemical process is the fraction of absorbed
photons that produce a desired result.
• If the result occurs every time a photon is absorbed, then the quantum yield is
unity.
• The quantum yield is a number between 0 and 1.
The quantum yield for emission from M* is the rate of emission divided by the
rate of absorption.
photons emitted per second emission rate ke [M* ]

0 = = =
photons absorbed per second absorptionrate ka [M* ]
Quenching
• Another possibility is that the excited molecule can transfer energy to a
quencher (Q), promoting Q to an excited state (Q*).
d[M* ]
M* + Q → M + Q * Rate = − = kq[M* ][Q]
dt
d[M* ]
• The rate of appearance of M* is Rate = − = ka[M].
dt
• The rate of disappearance of M* is Rate = 𝑘e[M∗] + 𝑘d[M∗] + 𝑘q[M∗][Q].
• The system reaches a steady state in which the concentrations of M and M*
remain constant. Setting the rates equal to each other gives
𝑘a[M] = 𝑘e[M∗] + 𝑘d[M∗] + 𝑘q[M∗][Q]
Quantum Yield (2 of 2)
An expression for the quantum yield in the absence of a quencher ([Q] = 0):
ke [M* ] ke [M* ] ke
0 = = =
ka [M ] ke [M ] + kd [M ] + kq[M ][Q] ke + kd
* * * *
An expression for the quantum yield in the presence of a quencher ([Q] ≠ 0):
ke [M* ] ke
Q = =
ke [M ] + kd [M ] + kq [M ][Q] ke + kd + kq[Q]
* * *
Stern-Volmer Equation
• We measure the emission in the absence and in the
presence of a quencher.
• The relative yields are given by the Stern-Volmer
equation.
0 ke + kd + kq [Q]  kq 
= =1+ [Q]
Q ke + kd  ke + kd 
• If we measure relative emission (0 /Q )
as a function of quencher concentration and
0 /Q = I0 /IQ
plot this quantity versus [Q] we should
observe a straight line. I0 is the emission intensity in the absence of quencher
IQ is the intensity in the presence of quencher.
The fluorescence lifetime is a measure of the time a
fluorophore spends in the excited state before
returning to the ground state by emitting a photon
Luminescence Lifetime [1]. The lifetimes of fluorophores can range from
picoseconds to hundreds of nanoseconds.
Luminescence lifetime: time needed for the intensity to fall

to 1/e (= 37%) of its initial value, where e is the base of
natural logarithms
Fluorescence lifetime in the absence of a dynamic quencher:
1
0 =
ke + kd
Fluorescence lifetime in the presence of a dynamic quencher
1
Q =
ke + kd + kq [Q]
Stem-Volmer equation in terms of fluorescence lifetimes:
0
= 1 + kq0 [Q]
Q
[1] Lakowicz, J.R. (1999). Principles of Fluorescence Spectroscopy, 2nd Edition, Kluwer Academic/Plenum Publishers, New York.
A Luminescent Intracellular O2 Sensor (1 of 3)
Ru(II) complexes strongly absorb visible light and efficiently emit
light at significantly longer wavelengths than they absorb. They
are stable for long periods, and have a long-lived excited state
whose emission is quenched by collisions with O2.
Fluorescence in the
Fluorescence in the absence
presence of quencher (O2
of quencher (O2 had been
gas was bubbled through
removed by bubbling with
the solution for 30 s)
dry ice)
Color Plate 19 Fluorescence from 5 mM (bipyridyl)3RuCl2 in methanol

Ground state Ru(II) is a singlet, and the lowest excited state is a triplet.
O2 quenches the luminescence by providing a radiationless pathway by which the
excited-state triplet is converted into the ground-state singlet.
Figure 19-21
Oxygen levels within living cells can be determined by
implanting particles containing two dyes.
When illuminated with blue light, one dye emits green light
near 547 nm and the other emits orange light near 601 nm.
The green dye is not affected by O2, but the orange ruthenium
dye is affected.
As the O2 concentration increases, ruthenium fluorescence

intensity and lifetime both decrease.
Using the ratio of orange-to-green emission intensity corrects for variations in light intensity in a
microscope, allowing determination of the concentration of O2 in the vicinity of the beads.
Förster Resonance Energy Transfer (1 of 2)
Förster resonance energy transfer (FRET): nonradiative energy transfer due to dipole-
dipole interactions between molecules that are close (within 1 to 10 nm of each other)
but not touching
Energy is transferred from an excited-state donor (D*) to ground state acceptor (A).
D∗ + A → D + A∗
FRET is only observed when D* → D and A → A* are of similar energy. (The emission
spectrum of the donor must overlap the absorption spectrum of the acceptor.)
FRET decreases as the sixth power of the distance separating the donor and acceptor.
FRET is also called fluorescence resonance energy transfer
Förster Resonance Energy Transfer (2 of 2)
A protease enzyme cleaves proteins and peptides. Protease enzymes can be elevated in
cancer cells.
Figure 19-22
Protease enzymes can be detected
by reaction with a probe consisting of
a peptide bound to a fluorophore
(doxorubicin) and a quencher.
When no protease is present, the

probe remains intact ➔ low
fluorescence due to close proximity
of the quencher.
When protease is present, the probe is cleaved ➔ free fluorophore ➔ high fluorescence.
[Protease] ∝ rate of fluorescence increases
Example of an Upconversion Process
Fluorescence and phosphorescence generally occur at lower
energy than the excitation energy, because some excitation
energy is converted to heat rather than fluorescence.
Color Plate 20 shows the odd case: Low-energy green

photons from a laser are shined into a solution, and the
emission of high-energy blue photon fluorescence is
observed.
This upconversion, which creates high-energy photons from

low-energy photons, does not violate conservation of energy
because it requires two green photons to create one blue
photon.
Box 19-3 Upconversion (1 of 5)
Inside the cuvette is:
Solvent: an organic solvent that has been

purged of oxygen
Chromophore
(a) Absorbs green photons
(b) Transfers this energy to the fluorophore
Fluorophore
(a) Takes the energy of two photons
gathered by the chromophores
(b) Emits one photon of blue fluorescence
Step 1: Two chromophores each absorb one photon, and convert into a long-lived triplet state.
Step 2: Each chromophore gives its remaining energy away to a fluorophore.
Step 3: One excited fluorophore gives its energy to the other, promoting it from a triplet to a
higher singlet excited state.
Step 4: The fluorophore in the excited singlet state emits a photon of blue fluorescence to
return to the ground state.
Photobleaching
Photobleaching: irreversible loss of fluorescence due to the excited molecule chemically
reacting to produce a nonfluorescent product; a form of photoinstability
Photobleaching depends on:
• The number of excitation emission cycles (brighter excitation light, longer exposure ➔
more photobleaching)
• Lifetime (longer lifetime➔ more photobleaching)
• Spin state (triplet states photobleach more readily than singlet states)
High-quality fluorescent dyes are limited to 104 to 106 excitations before photobleaching.
Table 19-3 Classes of Luminescent Materials
Luminescent materials have been developed which have greater photostability or other
attractive photochemical features.
Property Dyes and proteins Lanthanide complexes Quantum dots Polymer dots
Excitation spectra narrow narrow very broad broad
Molar absorptivity moderate low high very high
Emission spectra broad narrow narrow very broad
Quantum yield variable high moderate moderate
Emission lifetime ns μs to ms 10’s of ns 100’s ps
Photobleaching common resistant resistant resistant
Lanthanide complexes have extremely long lifetimes, which are advantageous in immunoassays.
Polymer dots are nanoparticle aggregates of p-conjugated semiconducting polymers.
Video: Early detection of corrosion
Quantum Dots via fluorescence quenching
Quantum Dots
• Semiconductor nanocrystals (4- to 10-nm
diameter)
• Core/shell structure
• Example: Core/shell = CdSe/ZnS
Quantum dots have
• Higher molar absorptivity
• Higher photostability
• Narrower emission
than luminescent dyes and luminescent proteins.
Section 19-7
Immunoassays
Immunoassays
An important application of light absorption and emission is in immunoassays.
Immunoassays employ antibodies to detect an analyte.
An antibody is a protein produced by the immune system of an animal in

response to a specific foreign molecule called an antigen.
An antibody binds very strongly and specifically to its target antigen.
The formation constant for the antibody-antigen complex is large, whereas the
binding of the antibody to other molecules is weak.
Enzyme-linked immunosorbent assay, ELISA (1 of 2)
Figure 19-23
Antibody 1, specific for the analyte of interest (the
antigen), is bound to a polymeric support.
1. A sample containing the target analyte is incubated
with the polymer-bound antibody.
2. Wash to remove unbound analyte.
3. Treat with antibody 2, which binds to a different
region of the analyte. Antibody 2 is covalently
attached to an enzyme.
4. Wash to remove unbound antibody.
The fraction of the antibody sites that bind analyte is
proportional to [analyte] in the unknown.
Enzyme-linked immunosorbent assay, ELISA (2 of 2)
• The enzyme can transform a colorless Figure 19-24
reactant into a colored product or
convert a nonfluorescent reactant into a
fluorescent product.
• It can catalyze the same reaction many
times, amplifying the signal.
• The higher the [analyte] in the unknown,
the more enzyme that is bound, and the
greater the rate of the reaction.
• Sensitive to less than a nanogram of
analyte.
Immunoassays in Environmental Analysis
Figure 19-25 Immunoassay for Hg2+ in natural water. (a) Immunoassay procedure. (b) Standard curve.
Box 19-4 How Does a Home Pregnancy Test Work?
Time-Resolved Fluorescence
Figure 19-26
Problem: Organic chromophores such as fluorescein are plagued by
background fluorescence at 350−600 nm from solvent, matrix, and
particles ➔ LOD ~10−10 M.
Solution: time-resolved fluorescence with long lifetime fluorophores
Time-resolved fluorescence improves the LOD by:
• Using longer-lifetime fluorophores (e.g., lanthanide like Tb3+)
• Waiting to start collecting fluorescence until the background
fluorescence has died away (t > 100 μs after excitation)
LOD for time-resolved fluorescence is ~1 000 times better (~10−13 M).
Time-resolved fluorescence is also called time-gated fluorescence.
Terbium Complex: Long-Lived Fluorophore
Time-Resolved Fluorescence lmmunoassay
Antibody 1 is labeled with a long lifetime (ms) Figure 19-27
Terbium fluorophore (emits at 495 nm).
Antibody 2 is labeled with a short lifetime (ns)

quantum dot fluorophore (QD).
In the presence of antigen, both antibodies bind,

pulling the chelate and quantum dot close.
Förster resonance energy transfer occurs,

resulting in quantum dot luminescence at 660
nm with a long lifetime (ms) dictated by Tb3+.

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Section 19-6

Sensors Based on Luminescence

The molecule that accepts the energy transferred from the

Example of an O2 Sensor Based on Quenching

This fiber-optic sensor measures O2 in a fish tank by its

photons emitted per second emission rate ke [M* ]

Luminescence lifetime: time needed for the intensity to fall

Color Plate 19 Fluorescence from 5 mM (bipyridyl)3RuCl2 in methanol

As the O2 concentration increases, ruthenium fluorescence

When no protease is present, the

Color Plate 20 shows the odd case: Low-energy green

This upconversion, which creates high-energy photons from

Solvent: an organic solvent that has been

Quantum Dots via fluorescence quenching

Immunoassays employ antibodies to detect an analyte.

An antibody is a protein produced by the immune system of an animal in

An antibody binds very strongly and specifically to its target antigen.

Antibody 2 is labeled with a short lifetime (ns)

In the presence of antigen, both antibodies bind,

Förster resonance energy transfer occurs,

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