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Principles of Fluorescence Spectroscopy

Chemistry Department

XMU

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Chapter Five

Quenching of

Fluorescence

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Quenching of fluorescence

5.1 Introduction

5.2 Stern-Volmer equation

5.3 Modified Stern-Volmer equation

5.4 factors influencing quenching

5.5 quenching mechanisms

5.6 Application

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5.1 introduction Fluorescence Quenching

Any processes decreasing the fluorescence intensity

Excited-state reactions

Molecular rearrangements

Ground-state complex formation

Collision

Quencher

Any species causing the decrease in the fluorescence

General quencher

Specific quencher

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Dynamic quenching and static quenching Dynamic quenching

Collision quenching

** QMMhvM QA

relaxation (10-12 s)

S0

S1

S1

hvA hvF knr

Q

Q

kq[Q]

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Dynamic quenching

nrkΓ

1][

1

QkkΓ qnr

In general, without permanent change in the fluorophore

Diffusion control

No changes in the absorption spectrum

Decreasing the lifetime

Change into

Effected by viscosity of solvent

100 F

F

Intensify with temperature increasing

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Static quenching

*21 MQMQQM AA hvorhv MQ* → MQ +

MQ* → MQ + hvF2

1*

1 FA hvMMhvM

relaxation (10-12 s)

S0

S1

S1

hvA1 hvF1 knr

M

relaxation (10-12 s)

S0

S1

S1

hvA2 hvF2 knr

MQ

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Static quenching

Changing the absorption spectrum

How about excitation spectrum? Change? Or not change?How about excitation spectrum? Change? Or not change?

Depend on MQ emitting or not

nrkΓ

1No change in the lifetime

How about the effect of temperature?How about the effect of temperature?

Depend on the thermodynamic properties of M and MQ

Forming a new species

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Quenchers

oxygen

halogens

Causing ISC

Aromatic and aliphatic amines

Forming excited charge-transfer complexes

Carboxyl groups

Nitroxides

Nitromethane and nitro compounds

Heavy atoms

more……

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5.2 Stern-Volmer eqution

][10 QKF

FSV

F0 and F

[Q] Concentration of quencher

Fluorescence intensities in the absence and presence of quencher, respectively

KSV Stern-Volmer quenching constant, given by kq

0

KD dynamic quenching

KS static quenching

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Dynamic quenching01

dt

dS

]][[][][][ 1110 SQkSkSΓSk qnrA ][

][][ 0

1 QkkΓ

SkS

qnr

A

For steady-state measurement

In the presence of quencher

In the absence of quencher

][][][ 110 SkSΓSk nrA

nr

A

kΓ

SkS

][][ 0

01

relaxation (10-12 s)

S0

S1

S1

hvA hvF knr

Q

Q

kq[Q]

nr

qnr

kΓ

QkkΓ

S

S

][

][

][

1

01

][1

][1

0 Qk

QkΓ

k

q

nr

q

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Dynamic quenching

][1][

][0

1

01 QkS

Sq

][1][

][0

0

1

01 QkF

F

S

Sq

nrkΓ

10

For quantitative measurement

Stern-Volmer equation

][

1

QkkΓ qnr Because

][1 00 Qkq

Stern-Volmer equation

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Dynamic quenching

][1 00 Qk

F

Fq

kq Bimolecular quenching constant

Typically, 11010 mol-1 L s-1

0 Lifetime in the absence of quencher

kq = f(Q)k0

f(Q) Quenching efficiency

k0The diffusion-controlled bimolecular rate constant

))((1000

40 QFQF DDRR

Nk

R Molecular radius

D Diffusion coefficients N Avogadro’s number

(RF+RQ) collision radius

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Example

Oxygen quenches The fluorescence of tryptophan

25°C, O2, Dq = 2.5 10-5 cm2/s;

tryptophan, DF = 0.66 10-5 cm2/sThe collision radius

R = ( RF + Rq ) = 5 Å

k0 =1.2 1010 mol-1 L s-1

Measured KD = 32.5 mol-1 L

Given = 2.7 ns,

Thus kq =1.2 1010 mol-1 L s-1

Thus

f(Q) = kq/k0 = 1

How to measure?How to measure?

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The effect of lifetime on quenching

20 F

F

KD =kq0According to Stern-Volmer equation

Typically, kq = 11010 mol-1 L s-1

Typical fluorescence lifetime 0 = 10-8 s

Thus, KD = 102 mol-1 L

When LmolK

QD

/01.01

][ %50

Typical phosphorescence lifetime 0 = 10-3 s

Thus, KD = 107 mol-1 L

20 F

FWhen LmolK

QD

/101

][ 7%50

What do these calculations suggest? What do these calculations suggest?

Using oxygen as the quencher

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Static quenching

MQ

M*

M + Q

hvF

(MQ)*

KS

]][[

)][(

QM

MQKS

)][(][][ 0 MQMM

]][[

][][ 0

QM

MMKS

][

][][][ 0

M

MMQKS

][1][

][ 0 QKM

MS

According to F = Kc

][1][

][ 0 QKF

FSThus

May or may not fluoresce

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Stern-Volmer constant

KD

KS

= kq0 Related with lifetime, controlled by diffusion

Formation constant

Increasing with temperature increasing

T↑

1.0

[Q]

F0/F

T↑

1.0

[Q]

F0/F T↑

1.0

[Q]

F0/F

Exothermal reactionEndothermal reaction

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Combined dynamic and static quenching

Violation of Stern-Volmer equation

1.0

[Q]

F0/FSuggest the combination of dynamic and static quenching

M*

MQM + Q

hvF

(MQ)*kq[Q]

dyanmic quenching

static quenching

KS

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F*

FQ

Correction to Stern-Volmer equation

0][

][

F

Ff s ][1

][

][1 0 QKF

F

f Ss

)/()[Q]

(0*

*

nrqnr,F

FD kΓ

Γ

kkΓ

Γ

Φ

Φf

F

FQ

the fraction of fluorescence, due to static quenching

F0

hv

F*

Q*

the fraction of fluorescence, due to dynamic quenching

)[Q]

(qnr

nr

kkΓ

kΓ

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Correction to Stern-Volmer equation

][1)[Q]

(1

0 QkkΓ

kkΓ

f qnr

qnr

D

DS ffF

F

0

F*

FQ

F

FQ

F0

hv

F*

Q*

fs

fS.fD])[1])([1(

110

QKQK

ffF

F

DS

DS

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Correction to Stern-Volmer equation

])[1])([1(0 QKQKF

FDS

]/[)1(][)( 0 QF

FQKKKKK SDSDapp

Kapp apparent Stern-Volmer constant

][1

][])[(1 2

QK

QKKQKK

app

SDSD

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Correction to Stern-Volmer equation

][)(]/[)1( 0 QKKKKQF

FSDSD

]/[)1( 0 QF

F

[Q]

KDKS

KD+KS

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Modified Stern-Volmer equation in interpreting “sphere of action”

)1000/)]exp([])[1(/0 NQQKFF D

Where is the volume of the sphere.

The radius of the sphere is slightly larger than the sum of the radii of the fluorophore and the quencher.

There exists a high probability that quenching will occur before these molecules diffuse apart.

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Example 1

Oxygen quenching of tryptophan

0/

x F0/F

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Example 2

Acrylamide( 丙烯酰胺） quenching of N-acetyl-L-tryptophan-amide(N- 乙酰 -L- 色氨酸酰胺）

F0/F

F0/FQ

■ 0

/

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Example 3

From JRL. P.245

Acrylamide quenching of dihydroequilenin (DHE ，二氢马萘雌甾酮 ) in buffer containing 10% sucrose （蔗糖） at 11°C

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Example 4

10-methylacridinium chloride quenching of guanosine-5’-monophosphate （鸟嘌呤核苷 -5‘- 单磷酸）

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Example 4

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5.4 factors influencing quenching

Adenine (A) Guanine (G)腺嘌呤 鸟嘌呤

Base

Suger-phosphoatebackboog

Cytosine (C) Thymine (T) Uracil (U)胞嘧啶 胸腺嘧啶 尿嘧啶

Adenine (A) Guanine (G)腺嘌呤 鸟嘌呤

Base

Suger-phosphoatebackboog

Cytosine (C) Thymine (T) Uracil (U)胞嘧啶 胸腺嘧啶 尿嘧啶

Adenine (A) Guanine (G)腺嘌呤 鸟嘌呤

Base

Suger-phosphoatebackboog

Cytosine (C) Thymine (T) Uracil (U)胞嘧啶 胸腺嘧啶 尿嘧啶

Steric effect

NCH2CH3 Br

NCH2CH3 Br

Example 1

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The ethidium bromide-DNA complex

Oxygen quenching of ethdium bromide fluorescence

Why smaller than 11010?

Why smaller than 11010?

NCH2CH3 Br

NCH2CH3 Br

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Example 2

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F

I

O2

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Example 1

Copolymer 1 1% tryptophan + 99% glutamic acid

Copolymer 2 3% tryptophan + 97% lysine

At neutral pH glutamic acid nagatively charged

Lysine positive charged

What happens to the fluorescence of tryptophan in the presence of oxygen and iodide, respectively?

What happens to the fluorescence of tryptophan in the presence of oxygen and iodide, respectively?

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Example 1

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Example 2

氯化十二烷基三甲铵

十二烷基硫酸钠

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Micro-environment

Fluorescence quenching of trypsinogen 胰蛋白酶原荧光猝灭

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Different in micro-environment

1.0

[Q]

F0/F

Downward-curving Stern-Volmer plot

Partial quenching

Fin

Fex I

F0 = Fin,0 + Fex,0

In the absence of quencher

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Modified Stern-Volmer equation in interpreting the difference in micro-environment

][10, QKF

FD

ex

ex

0,0,

0, ][1 inD

exinex F

QK

FFFF

In the presence of quencher

Total fluorescence

0,0,

0,0,0 ][1 inD

exinex F

QK

FFFFFF

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Deriving modified equation

][10,

0,0 QK

FFFFF

D

exex

][1

][ 0,0,0,

QK

FFQKF

D

exexDex

][1

][0, QK

QKF

D

Dex

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Deriving modified equation

][1][

0,

0,0,0

QKQK

F

FF

F

F

D

Dex

inex

0,0,

0,

inex

exex FF

Ff

Let

Then

exDex fQKfF

F 1

][

10

Modified Stern-Volmer equation

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Deriving modified equation

F

F

0

1/[Q]

1 /(KDfex)

1 / fex

exDex fQKfF

F 1

][

10

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Example

Iodid

e qu

ench

ing of tryp

toph

an fl

uorescen

ce in lysozym

e(

溶菌酶）

Denatured protein

Native protein

Native protein

1/fex = 1.5, fex = 0.66

Denatured protein

1/fex = 1.0, fex = 1.0

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example

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Localization of membrane-bound fluorophores

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Localization of membrane-bound fluorophores

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5.5 quenching mechanisms

Nonradiative energy transfer, due to dipole-dipole interaction of donor and acceptor

5.5.1 Due to energy transfer

D* +A D+ A*

hvA hvF

D

D donor

A acceptor

Rate of energy transfer depends

Overlap of spectra

Relative orientation

Distance between A and D

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Principle of energy

JNrn

kK

D

DT 642

2

128

)10(ln9000

Rate of energy transfer

D quantum yield of D

D lifetime of D

r distance between A and D

n refrcative index

N Avogadro’s number

k orientation factor

J overlap integral

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Overlap integral

dFvd

v

vvFJ AD

AD

0

4

04

)()()()(

)(DF The corrected fluorescence intensity of D in -d, the total intensity normalized to unity

)( A The extinction coefficient of A at

The unit is (mol / L)-1 cm3

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Orientation factor

k = 2/3

randomize by rotational diffusion prior to energy transfer

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Förster distance

R0

relaxation (10-12 s)

S0

S1

S1

hvA hvF knrA(S0)

A(S1

)

TK

D1

The distance between A and D when

DnrT kΓK

1

R0 > r, energy transfer decay dominate

R0 < r, usual radiative and nonradiative decay dominate

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Förster distance

254

260 108.8

n

JkR D

60 )(1

r

RK

DT

6

1

rKT Rate of energy transfer

Efficieney of energy transfer DnrDT

T

kΓK

KE

,

D

DA

FFE 1

1)/(

)/(6

0

60

rR

rRE

(in cm)

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Example

聚脯氨酸

- 萘

D

丹磺酰

A

n =1-12, r = 12- 46Å

0% transfer

100% transfer

Excited D, measure F of A

no DF290= 2.3I0bcAA(A+ED)

A’= A+ED

D

AAE

'

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Example

A

AD

A

DA

F

F ,

n =1, 100% transfer, the ratio of absorbance = the ratio of emission

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example

1)/(

)/(

0

0

x

x

rR

rRE

01 lglg)1lg( RxrxE

x = 5.9±0.3

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Sensitized fluorescence and phosphorescence

)()()()( 1001 SASDSASD

)()()()( 1001 TASDSASD

)()()()( 1001 SASDSATD

D(S1

)

D(S0

)

D(T1)A(T1)

A(S1)

A(S0)

)()()()( 1001 TASDSATD

Sensitized fluorescence

Sensitized phosphorescence

For D, quenched

For A, sensitized

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ExampleSelf-association of ATPase （腺苷三磷酸酶） molecules in lipid vesicles

Mg2+-Ca2+-ATPase labeled individually with IAEDANS and IAF

Donor Acceptor

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Intra-molecule energy transfer

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Intra-molecule energy transfer

Trp

HSA

Tyr:Trp=18:1

R0 = 14Å

Comparable to the diameter of most proteins

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5.5.2 duo to photochemical reaction

FF

hv

vhhv

RPRR A

**

荧光猝灭所涉及的光化学反应类型光化学反应荧光猝灭是指沿着激发态超平面发生的反应，导致激发态聚集数减少而引起的荧光猝灭。单分子光化学反应

绝热光化学反应 产生另一种形态的发光分子，其发射波长不同于原来的发光体

TICT

非绝热光化学反应 产生另一种具有稳定基态的物质，不伴随光子的发射

F

hv

hv

PPRR A **光分解

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双分子光化学反应

F

RHhv

hv

RDHDD A 22*

激发态分子与其他分子发生反应，生成了稳定的或不稳定的新物质

光氧化还原 生成稳定的基态物质

激基二聚体 不具有稳定的基态

FF

Ahv

vhhv

AAAAA A

2*)(*

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光化学反应的机制

R*

R

P*

P

S0

S1

hvF

hv’F

Excited state hypersurface

TICT

Dual fluorescence

R*

R

P*

P

S0

S1

hvFhv’F

Photoreaction

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光化学反应的机制R*

R

P*

P

S0

S1

hvF

P’

Maurice R. Eftink, Fluorescence quenching: theory and application in “Topic in fluorescence spectroscopy” V2 principles, ed. By J.R.Lakowicz, p 53-120.

Herbert C. Cheung, Resonance energy transfer, in “Topic in fluorescence spectroscopy” V2 principles, ed. By J.R.Lakowicz, p 128-171.

Maurice R. Eftink, Fluorescence quenching: theory and application in “Topic in fluorescence spectroscopy” V2 principles, ed. By J.R.Lakowicz, p 53-120.

Herbert C. Cheung, Resonance energy transfer, in “Topic in fluorescence spectroscopy” V2 principles, ed. By J.R.Lakowicz, p 128-171.

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5.6 Application

Molecular beacons

A D

Q F

Target DNA

ssDNA binding protein

Denaturing reagent

QF

F

Q

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Molecular beacons

Hairpin probe show no fluorescence, do not need separation

Key type, almost no background fluorescence

Specific probe

Reference

Xiaohong Fang, Tianwei Heffery, John Perlette, Weihong Tan (University of Florida, USA) Kemin Wang (Hunan University, P.R. CHINA), Anal. Chem. 2000, Dec.1 747A-753A

Reference

Xiaohong Fang, Tianwei Heffery, John Perlette, Weihong Tan (University of Florida, USA) Kemin Wang (Hunan University, P.R. CHINA), Anal. Chem. 2000, Dec.1 747A-753A