Download Excitation Transfer between Conjugated Polyelectrolytes and

Department of Physics, Chemistry and Biology
Master’s Thesis
Excitation Transfer between Conjugated Polyelectrolytes and
Triplet Emitter Confined in Protein Nanowires
Chorpure Thinprakong
August 15, 2010
LITH-IFM-A-EX--10/2321—SE
Linköpings universitet Institutionen för fysik, kemi och biologi
581 83 Linköping
Departmant of Physics, Chemistry and Biology
Excitation Transfer between Conjugated Polyelectrolytes and
Triplet Emitter Confined in Protein Nanowires
Chorpure Thinprakong
August 15, 2010
Supervisor
Niclas Solin, IFM
Examiner
Olle Inganäs, IFM
Avdelning, institution
Division, Department
Semiconductor Materials
Department of Physics, Chemistry and Biology
Linköping University
Språk
Language
Svenska/Swedish
Engelska/English
________________
Rapporttyp
Report category
Licentiatavhandling
Examensarbete
C-uppsats
D-uppsats
Övrig rapport
Datum
Date
2010-08-15
ISBN
ISRN: LITH-IFM-A-EX--10/2321--SE
_________________________________________________________________
Serietitel och serienummer
Title of series, numbering
ISSN
______________________________
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URL för elektronisk version
http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-58566
Titel:
Title
Energi överföring mellan konjugerade polyelectroter och Iriplett emittrar inbyggda i protein - nano trådar
Excitation transfer between conjugated polyelectrolytes and triplet emitter confined in protein nanowires
Författare
Author
Chorpure Thinprakong
Sammanfattning
Abstract
Phosphorescent metal complexes can be incorporated into amyloid-like fibrils, and these fibrils can be decorated with
conjugated polyelectrolytes (CPEs). In this study, fac-tris[2-phenylpyridinato-C 2 ,N]irdium(III) complexes
[Ir(piq) 3 ] were used as the phosphorescence emitter and Sodium -poly(3-thiophene acetic acid) (PTAA-Na)
compounds were used as CPEs. Herein we study the energy transfer processes between the iridium complexes and the
CPEs. To investigate these mechanisms, the analysis of the emission quenching and time-resolved measurements were
done. Our measurements show that energy can be transfered from singlet state of PTAA to the singlet state of Ir(piq)3.
Moreover, incorporation of iridium into amyloid fibrils decreases the importance of energy transfer by the Dexter
mechanism. Finally we propose a geometry of interaction to explain the obtained results.
Nyckelord
Key words
Singlet Triplet Förster Dexter Phosphorescent Amyloid PTAA Iridium
Acknowledgments
For first place, I would like to thank Prof. Olle Inganäs, for supporting me to do my thesis work
in his group and giving me this interesting topic. Furthermore, I wish to thank my supervisors,
Niclas solin and Aurora Rizzo, for all discussions, ideas and comments. My special thank
goes to Nils-Ola Persson and Peder Bergman, for valuable help in time-resolved measurements.
Finally, I want to thank my parents for supporting me to study here and for their love.
iv
Abstract
Phosphorescent metal complexes can be incorporated into amyloid-like fibrils, and these fibrils
can be decorated with conjugated polyelectrolytes (CPEs). In this study, fac-tris[2-phenylpyridinatoC 2 ,N]irdium(III) complexes [Ir(piq)3 ] were used as the phosphorescence emitter and Sodiumpoly(3-thiophene acetic acid) (PTAA-Na) compounds were used as CPEs. Herein we study
the energy transfer processes between the iridium complexes and the CPEs. To investigate
these mechanisms, the analysis of the emission quenching and time-resolved measurements
were done. Our measurements show that energy can be transfered from singlet state of PTAA
to the singlet state of Ir(piq)3 . Moreover, incorporation of iridium into amyloid fibrils decreases
the importance of energy transfer by the Dexter mechanism. Finally we propose a geometry of
interaction to explain the obtained results.
v
Contents
Acknowledgments
iv
Abstract
v
1 Introduction
1
2 Background Theories
3
2.1
Conjugated polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3
2.2
Optical transition in molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4
2.3
Lifetime and Quantum yield . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5
2.4
Optical property of metal-ligand complexes (MLCs) . . . . . . . . . . . . . . . .
6
2.5
Amyloid-like fibril . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7
2.6
Amyloid fibril incorporating Ir(piq)3 . . . . . . . . . . . . . . . . . . . . . . . . .
8
2.7
Energy transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9
2.7.1
Förster resonance energy transfer (FRET) . . . . . . . . . . . . . . . . . 10
2.7.2
Dexter energy transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
3 Experimental Procedures
3.1
3.2
Sample preparations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
3.1.1
Preparation of unmodified amyloid fibrils . . . . . . . . . . . . . . . . . . 12
3.1.2
Preparation of amyloid fibrils decorated with CPEs . . . . . . . . . . . . 12
3.1.3
Preparation of amyloid fibrils with Ir(piq)3 . . . . . . . . . . . . . . . . . 12
3.1.4
Preparation of amyloid fibrils with Ir(piq)3 with PTAA . . . . . . . . . . 13
Experimental methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
4 Results and discussions
vi
12
14
4.1
Spectral overlap between donor and acceptor . . . . . . . . . . . . . . . . . . . . 14
4.2
FRET . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
4.3
Dexter energy transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
4.4
Dependence of absorption and emission spectra on PTAA concentration . . . . . 16
CONTENTS
4.5
Time-resolved measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
4.6
Quantum yield of donor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
4.7
Distance between donor and acceptor . . . . . . . . . . . . . . . . . . . . . . . . 19
4.8
Geometry of interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
5 Conclusion
22
A Appendix A: Beer-Lambert Law
23
B Appendix B: Calibration Curve for amyloid fibril and Ir(piq)3 concentration 24
C Appendix C: Sample List
26
Bibliography
30
vii
Chapter 1
Introduction
Amyloid fibrils are structures made up of self-assembled proteins rich in β sheet content. Normally, these aggregated fibrils are present in several human disorders e.g. Alzheimer’s disease.
Several proteins and short peptides have been shown to undergo self-assembly into amyloid-like
fibrils. For example, by heating insulin in aqueous acid, amyloid fibrils are readily formed.
Typically the fibrils have a diameter of approximately 100 Å and lengths in the micrometer
range [1], [2].
Conjugated polyelectrolytes (CPEs) have been widely used in electronic, optoelectronic
and bimolecular applications such as printable logics, photovoltaics, light emitting diodes and
biosensors [3]. The interactions between CPEs and amyloid fibrils, by self-assembly in solution,
result in high signal enhancement and a color shift, which can be detected by the changes in
the absorption and emission spectra [4].
1.0
1.0
PTAA
Normalized absorbance
PTAA
Ir(piq)3
0.6
0.8
0.6
0.4
0.4
Ir(piq)3
0.2
0.2
0.0
350
400
450
500
550
600
650
700
PTAA
Normalized photoluminescence
0.8
0.0
Wavelength (nm)
Ir(piq)3
Figure 1.1: Absorption and emission spectra of PTAA and Ir(piq)3 . All
samples were excited at 380 nm.
It was recently reported that the amyloid fibrils incorporating phosphorescent metal complexes (metal-ligand complexes (MLCs)) can be prepared [5]. The MLCs consist of a heavy
metal surrounded by organic π-conjugated ligands, which lead to strong mixing of the singlet
1
Chapter 1. Introduction
and triplet states by spin-orbit coupling (SOC). This leads to high efficiency of the triplet
emission (phosphorescence). Because of phosphorescent emission, these materials are called
metalorganic phosphorescent complexes.
E(eV)
2.1
Donor: PTAA
S1
För
ster
E
Acceptor: Ir(piq)3
T
ISC
2.0
MLCT1
MLCT3
ET
1.9
1.8
Fluorescence
Phosphorescence
Non-radiative process
T1
ISC
De
xt
er
2.2
Absorption
Excitation energy
1.7
S0
PTAA
Ir(piq)3
Figure 1.2: The diagram of intra- and intermolecular energy levels of PTAA
and Ir(piq)3
The enhancement of phosphorescence, by incorporating Ir complexes into amyloid fibrils,
was reported recently in [6], suggesting that fibrils helps to block energy back transfer to the
lower dark triplet state of conjugated polymers. Therefore, it seems that the excitons are better
confined on the triplet emitter. In the present work, the energy transfer processes between CPEs
and Ir complexes confined in protein nanowires, are reported. The phosphorescent iridium complex, fac-tris[2-phenylpyridinato-C 2 ,N]irdium(III) [Ir(piq)3 ], was used as triplet emitter. Due to
the short excited state lifetime and high phosphorescent yield, Ir(piq)3 emits red light efficiently
from the metal-ligand charge transfer (MLCT) excited state [7]. As for CPEs, Sodium-poly(3thiophene acetic acid) (PTAA-Na) was used. The CPEs were decorated on insulin fibrils by
self-assembly in the solution.
The overlap spectra between the emission of PTAA and the absorption of Ir(piq)3 are shown
in figure 1.1. This indicates that, Ir complexes can absorb the energy from PTAA within this
overlapping region. The CPEs and triplet emitter incorporated into amyloid fibrils should
be located very close thereby making possible energy transfer between them. As described in
figure 1.2, the excited singlet state of PTAA has the higher energy level than the singlet state of
Ir(piq)3 . Therefore the exciton can transfer to the singlet state of Ir(piq)3 by Förster resonance
energy transfer (FRET). Then the energy can also transfer back by a Dexter process to the
dark triplet state of PTAA.
To investigate these mechanisms, the photophysical analysis of quenching emission were
done by varying the concentrations of acceptor. Moreover, the data from time-resolved measurements were used to determine whether the emission originates from fluorescence or phosphorescence. The approximated distance between donor and acceptor was calculated by Förster
energy transfer equations. From all results, the sketch of the interaction of PTAA and amyloid
fibril with Ir complexes is shown at the end of the report.
2
Chapter 2
Background Theories
2.1
Conjugated polymers
Polymers are made up of repeating structural units and are normally connected by covalent
bonds. Natural polymeric materials such as DNA, protein and rubber are well known. On
the other hand, synthetic polymers are also widely used in daily life; e.g. polyethylene and
polypropylene. The term conjugated refers to the alternating sequence of single and double
bond in the molecule, so that the carbon atom chain in conjugated polymers is built up with
an alternating bond pattern. One of the electronic configuration of the ground state of carbon
(C) is 1s2 2s2 2p2 . There are four electrons in the outer shell, two s electrons are paired and two
p electrons are unpaired. The mixing of one s- and three p-orbitals results in a set of four sp3
hybrid orbitals with four tetrahedral chemical bonds around the carbon atom such as methane
in figure 2.1(a).
(b)
1s
H
1s
sp3
1s
H
sp3
C
sp3
1s
1s
H
1s
π-bond
H 1s
sp3
H
(C)
sp2
Cσ-bondC sp
Energy
(a)
2
π-bond
H
1s
σ*-bond
π*-bond
Optical excitation
π-bond
σ-bond
Figure 2.1: sp3 and sp2 orbitals using the examples of methane (a) and
ethane (b). The energy descriptions of bonding and antibonding σ- and πorbitals are shown in (c).
Another possible configuration of carbon is the sp2 hybridization orbitals. In this case, one
s- and two p-orbitals form three degenerate orbitals, which are coplanar with an angle of 120◦
to each other. For example, the pz orbital remains unaltered, while s-orbital mixes with px and
py orbitals. The pz orbital is perpendicular to the plane of sp2 orbitals and overlaps with the
neighboring pz orbital, which results in a π-bond, such as in ethylene in figure 2.1(b).
For a π-conjugated molecule, the lowest excitation occurs between the π-orbital and π ∗ orbital as in figure 2.1(c). Conjugated polymers consist of many π-bonds connected with each
3
Chapter 2. Background Theories
other, resulting in a delocalization of the electrons over the chain of the polymer. The electronic
transitions from the π-bond to π ∗ -bond are referred as a π-π ∗ transition.
Some conjugated polymers are made soluble in polar solvents such as water by adding
permanent ionic charges on a side chain of each conjugated monomer. Such conjugated polymers
are called conjugated polyelectrolyte (CPEs). Conventionally CPEs are used as the detectors
of biospecific interaction and conformational changes of biomolecules [8].
2.2
Optical transition in molecules
Intersystem Crossing
Absorption
T1
Phosphorescence
Non-radiative process
Fluorescence
S1
Absorption
Energy
S2
Internal Conversion
Internal Conversion
The luminescence is the light emission from any substance, which can be divided into two
types, fluorescence and phosphorescence, depending on the excited states. If the spins of the
excited electron and ground state electron are antiparallel. The relaxation from the singlet
S0
Figure 2.2: Jablonski-diagram of typical organic molecule. All absorption,
emission and non-radiative processes are shown. (adapted from [9])
state to ground state conserve total spin, so this process is very fast and called fluorescence.
For phosphorescence, the light emission comes from the triplet excited state. The spins of the
electrons in the excited state and ground state are parallel, and as a result the transition is
forbidden and the rate of emission is very slow.
The processes of absorption and emission of light can be described by the Jablonski-diagram
in figure 2.2 [9],[10]. The diagram includes the ground state S0 , the first excited states for singlet
S1 and for triplet T1 , and the second excited singlet state S2 . Following the light absorption, an
electron is usually excited from ground state S0 to some higher singlet state Si and then rapidly
relaxes to the lowest vibration level of S1 by the internal conversion process within 10−12 s or
less. The excess energy is released by non-radiative process. The radiative transition from S1
to S0 is called fluorescence and T1 to S0 is called Phosphorescence.
The electron spin in S1 can convert to the first triplet state T1 by intersystem crossing
(ISC) process, but it is formally forbidden for the transition from T1 to S1 . However, the
spin-orbit coupling (SOC) increases the possibility of ISC and therefore the phosphorescence
4
2.3 Lifetime and Quantum yield
Table 2.1: A table of standard materials and their literature quantum yield
values
Compound
Sovent
Cresyl violet
Methanol
Quinine sulfate 0.1 M H2 SO4
Fluorescein
0.1 M NaOH
Harmane
0.1 M H2 SO4
Harmine
0.1 M H2 SO4
Anthracene
Ethanol
Quantum yield
0.54
0.54
0.90
0.83
0.45
0.27
Emission range
(nm)
600-650
400-600
500-600
400-550
400-550
360-480
Reference
[11]
[12]
[13]
[14]
[14]
[12]
is allowed. Since the atoms of high atomic number increase the spin-orbit interaction, the
forbidden transition is enhanced.
2.3
Lifetime and Quantum yield
The quantum yield and lifetime are important characteristics for photophysical analysis, such
as energy transfer, quenching rate constant, and radiative and non-radiative constant. The
lifetime determines the relaxation time of emission, and is easy to measure by the short pulsed
optical technique. A general expression of the fluorescence, τf , and phosphorescence, τp , lifetime
are given by
1
(2.1)
τf = S
S
kr + knr + kisc
1
τp = T
(2.2)
T
kr + knr
T
S
) is the
(knr
where krS (krT ) is the radiative rate constant for fluorescence (phosphorescence), knr
sum of all non-radiative transition from the first excited singlet (triplet) state and kisc is the
rate constant for ISC [8].
The quantum yield is the ratio of emitted photons to the number of absorbed photons, and
is used to describe the luminescence efficiency of substances. The quantum yield of fluorescence,
Φf , and phosphorescence, Φp , are given by
Φf =
krS
= krS τf
S +k
krS + knr
isc
Φisc krT
= Φisc krT τp
Φp = T
T
kr + knr
(2.3)
(2.4)
and Φisc is the quantum yield of ISC [8]. Because of the experimental difficulty to determine
the absolute quantum yield, the relative quantum yield is usually determined instead. The
quantum yield of unknown sample, ΦX , is related to a reference standard sample, ΦS , by the
equation
AS FX nX
ΦX =
ΦS
(2.5)
AX FS nS
where Φ is the quantum yield, A is the absorbance at the excitation wavelength, F is the
area under the corrected emission spectrum, n is the refractive of the used solvent, and the
subscripts, x and s, are referred to unknown and standard sample respectively [15]. Examples
of quantum yield standards are in table 2.1.
5
Chapter 2. Background Theories
To prevent the non-linear effect of the inner filter, the absorbance values from both unknown
and standard solution should be below 0.1 at the excitation wavelength when measured in a
10 mm path length cuvette. Moreover, the standard solution should have the absorption and
emission bands close to those of unknown sample.
2.4
Optical property of metal-ligand complexes (MLCs)
Metal-ligand complexes (MLCs) consist of organic π-conjugated ligands bonding to transition
metal complexes. The heavy central metal atom leads to a strong mixing of the excited singlet
and triplet states by spin-orbit coupling. This leads to triplet emission with high efficiency and
long-lived excited states.
The combination of a transition metal ion and organic ligands gives rise not only to phosphorescence but also to an optical transition with hybrid characteristics. For organic ligands,
the ligand-center (LC or π − π ∗ ) transitions are of high importance as discussed in section 2.1.
For the heavy metal, the d − d∗ transition is of high importance. However, the presence of
ligands splits the d orbital into three lower (t) and two higher (e) orbitals, and this leads to
that the d − d∗ transition is raised above the π − π ∗ transitions. Since both of t and e orbitals
have the same symmetry with respect to the metal center, transitions between those orbital
are forbidden (Laporte selection rules) resulting in the low emission in d − d∗ transition.
(b)
MLCT1
LC
Energy
Absorbance
ISC
S0
MLCT3
Phosphorescence
t
π
MLCT
π*
d-d*
Energy
e
Non-radiative process
(a)
Figure 2.3: (a) Orbital diagram for MLCs shows t, π-, e and π ∗ -bonding.
(b) Simplified diagram for absorption and emission processes of MLCs [10]
A new transition, due to the interaction between metal and ligands, is called metal to ligandcharge transfer (MLCT) transition as shown in figure 2.3(a). The electrons are moved from
the metal to the ligands upon the excitation. The emission from this transition is generally
phosphorescence. The MLCT has a large overlap with the metal atom giving a higher degree
of SOC, and therefore induces the strong mixing between singlet and triplet states. Because
of the fast rate of ISC of the MLCs, fluorescence is normally weak or unobservable. For many
MLCs, the triplet state of LC is the lowest state, but the SOC between MLCT1 to LC3 is
much smaller than MLCT1 to MLCT3 [16]. As discussed above, the most important optical
transition in MLCs is MLCT, and a simplified diagram is shown in figure 2.3(b).
6
2.5 Amyloid-like fibril
2.5
Amyloid-like fibril
Amyloids are made up of aggregated proteins, whose secondary structure is rich in β-sheet content. Amyloid deposits can be found in human’s diseases such as Parkinson’s and Alzheimer’s.
Proteins like insulin or lysozyme, have been shown to form amyloid fibrils [17],[18].
(a)
(d)
(b)
10 Å
4.8Å
(c)
10 nm
30 Å
Figure 2.4: (a) Insulin secondary structure (α-helix) shows the three native
disulfilde bonds. (b) After denaturation, the α-helix structure becomes βstrand. (c) Aggregation of β-strands becomes a protofilament. (d) Then
single amyloid fibril makes up with twisting protofilaments. The yellow dot
lines are disulfide bonds.
Amyloid fibrils are unbranched with a typical diameter of 10 nm and lengths up to 10
µm (figure 2.4)[19]. The morphology of fibrils can be studied by X-ray diffraction, which
presented cross-β structure with β-strands of the precursor protein arranged perpendicular to
fibril axis. The spacing of 4.8 Å between β-strand fibril assemblies was presented by cryoelectron microscopy [2].
During the heating process, the α-helical structure of native insulin unfolds to form the crossβ structure as in figure 2.5 (a) to (b). Then the 4.8 Å cross-β structure molecules aggregate
to be one protofilament with 20-35 Å in diameter and then a few protofilaments twist to each
other to form an amyloid fibril with approximately 100 Å wides [2].
At high pH value, above 12, amyloid fibrils are destroyed because the amino groups lose
their charge, while the carboxyl groups and some hydroxyl groups are ionized and become
charged. The high net charge results in the strongly repulsive forces which interrupt the inter
7
Chapter 2. Background Theories
(a)
Insulin
(b)
(d)
10 Å
4.8 Å
Secondary structure
Heating
65 °C
10 h
(c)
Aggregation
Aggregation
Dissolved in 2 M guanidine HCl
Dialyzed versus 25 mM HCl
Filtered through a 0.2 µm PVDF filter
100 Å
An amyloid fibril
30 Å
A protofilament
Figure 2.5: The amyloid fibril formation processes (a) native insulins with
α-helical in secondary structures, (b) misfold insulins with cross-β structures,
(c) a protofilament and (d) an amyloid fibril
(a)
(b)
(c)
Self-assembly
PTAA
100 Å
An amyloid fibril
An amyloid fibril
with PTAA
Figure 2.6: The combination of an amyloid fibril with PTAA by self-assembly
in the solution
fibril bonds until those fibrils disappear [4].
Amyloid fibrils are prepared under acidic conditions, meaning that the fibrils will acquire a
net positive charge. This means that negatively charged polyelectrolytes will interact strongly
with amyloid fibrils. Accordingly, upon mixing PTAA with the amyloid fibrils solution, the
PTAA will decorate amyloid fibrils as shown in figure 2.6.
2.6
Amyloid fibril incorporating Ir(piq)3
Ir(piq)3 complexes are insoluble in water or HCl solution. Therefore the grinding technique
was used in order to prepare soluble hybrid materials (figure 2.7) [5].
If the amyloid fibrils with Ir complexes are treated with PTAA solution, it can be expected
that PTAA, in the same way as described in section 2.5, will decorate this type of fibrils as well
(figure 2.8).
8
2.7 Energy transfer
(a)
Insulin
Ir(piq)3
(d)
(c)
Aggregation
10 Å
(b)
Grinding
Heating
65 °C
72 h
An amyloid fibril
with Ir(piq)3
Dissolved in 2 M guanidine HCl
Dialyzed versus 25 mM HCl
Filtered through a 0.2 µm PVDF filter
Figure 2.7: The combination of an amyloid fibrils with Ir(piq)3 , (a) native
insulin with α-helical in secondary structures and Ir(piq)3 , (b) after grinding,
hybrid materials of insulin and Ir(piq)3 , (c) misfolded insulin molecules with
cross-β structures around Ir(piq)3 molecule, (d) an amyloid fibril with Ir(piq)3
complexes
The goal of this study is to investigate the energy transfer between PTAA and the Ir
complexes incorporated into amyloid fibrils.
(b)
PTAA
(c)
(a)
PTAA
Self-assembly
Insulin
Ir(piq)3
10 Å
An amyloid fibril
with Ir(piq)3 and PTAA
Figure 2.8: The combination of an amyloid fibril with PTAA and Ir(piq)3
by self-assembly in the solution
2.7
Energy transfer
There are two mechanisms for triplet and singlet energy transfer from donor to acceptor
molecule. The first one is Förster resonance energy transfer (FRET), which is dipole-dipole
coupling process depending on the inverse sixth power of the distance between donor and acceptor. This energy transfer process can be effective only between singlet and singlet states.
Another is Dexter energy transfer which refers to non-radiative process of electrons exchange.
This energy transfer needs very short-range between donor and acceptor, and usually happens
9
Chapter 2. Background Theories
between triplet and triplet states.
2.7.1
Förster resonance energy transfer (FRET)
Today FRET is applied to plenty of works involving the energy transfer in light-emitting devices
[1], biological macromolecules[20], and biosensors [21]. The incoming radiation induces the
oscillating electric dipole moment of the donor molecule. After the donor absorbs the energy
at frequency ν, the oscillating dipole from the donor can affect electrons bound to a nearby
acceptor molecule by inducing an oscillating dipole moment at the same frequency ν, and then
the acceptor absorbs the energy from donor. Figure 2.9(a) shows the mechanism of FRET. This
energy transfer normally occurs between singlet and singlet states because only multiplicityconserving transitions have large transition dipoles. The rate of FRET (kF ) is given by
kF =
ΦD κ2 9000(ln 10)
(
)J
τD R6 128π 5 N n4
(2.6)
where ΦD is the quantum yield of the donor in the absence of acceptor, κ2 is orientation factor
which equals to 2/3 for free movement of donor and acceptor, n is the refraction index of
the medium (commonly uses 1.4 for the solution), N is Avogadro’s number, R is the distance
between donor and acceptor, τD is the lifetime of the donor in the absence of acceptor and J is
the overlap integral between donor and acceptor. J is defined as
Z ∞
FD (λ)A (λ)λ4 dλ
(2.7)
J=
0
where FD is the peak-normalized fluorescence spectrum of donor, A is the molar absorption
coefficient of the acceptor when is in unit of M −1 cm−1 and wavelength λ in unit of nm [1].
To get high rate of FRET, the donor should have high quantum yield and the spectral overlap
between donor and acceptor should be considerable.
Another important parameter is R0 which refers to the Förster or critical transfer distance
in Å when the energy transfer is equal to the decay rate, so that
R06 = 8.79 × 1023 (κ2 n−4 ΦD J)
(2.8)
Generally, the transfer efficiency (E ) is the ratio between fluorescence intensity of the donor
in presence (F ) and absence (F0 ) of acceptor, which is defined by
E =1−(
F
)
F0
(2.9)
On the other hand, the transfer efficiency (E) is related to the FRET by
R06
E= 6
R0 + R6
(2.10)
From this relationship, if R has the same value as R0 , the transfer efficiency will be equal to 50
percent.
2.7.2
Dexter energy transfer
Dexter energy transfer is a short-range process involving the overlap between two molecular
orbitals. The process is based on quantum mechanics, and more information can be found
10
2.7 Energy transfer
(a)
Förster resonance energy transfer
S1
Di
p
int ole
er
ac dipo
tio
n le
S0
D
S1
S1
S1
S0
S0
S0
D
A
A
(b)
Dexter energy transfer
T1
T1
T1
T1
S0
S0
S0
Exchange electrons
S0
D
D
A
A
Figure 2.9: (a) The FRET mechanism normally happens between singletsinglet states. (b) Dexter energy transfer mechanism, the combined spin of
the participating molecules has to be conserved.
in [22] The Dexter mechanism is shown in figure 2.9(b). The donor and acceptor exchange
their electrons, and also preserve the combined spins during the energy transfer. This process
normally happens in triplet-triplet states of molecules [23],[24],[25]. The simple rate constant
of Dexter energy transfer (KD ) is given by
KD = KJexp(
−2R
)
L
(2.11)
where K is the specific orbital interactions, J is the normalized spectral overlapping integral,
R is the distance between donor and acceptor, and L is the sum of Van der Waals radii [24].
Obviously, the rate constant of this process depends on the R and decays steeply because of
the exponential relation.
11
Chapter 3
Experimental Procedures
3.1
Sample preparations
In this section, the preparations of four amyloid fibril solutions are described. There are preparations of amyloid fibrils, amyloid fibrils with CPEs, amyloid fibrils with Ir complexes, and
amyloid fibrils with CPEs and Ir(piq)3 . The synthesis of PTAA-Na and Ir(piq)3 were reported
elsewhere [7],[26].
3.1.1
Preparation of unmodified amyloid fibrils
80 mg of insulin was dissolved in 5 ml of 2 M guanidine hydrocloride, and was then dialyzed
versus three rounds of 25 mM HCl at 4◦ C over night. The solution was filtered through a 0.2
µm PVDF filter and was diluted to 10 ml. The sample was then heated at 65◦ C in 25 mM HCl
for 10 hours.
The heating resulted in the formation of amyloid fibrils. The amyloid fibril concentration
was checked by measuring the absorbance at 280 nm and using 280 = 5840 M−1 cm−1 as
extinction coefficient and calculated following Beer-Lambert law.
3.1.2
Preparation of amyloid fibrils decorated with CPEs
PTAA-Na was dissolved in deionized water. The solution of PTAA-Na was added to the stock
solution of amyloid fibrils, which resulted in the formation of amyloid fibrils decorated with
PTAA. The amount of PTAA and amyloid fibrils are shown in table C.1 in Appendix C.
3.1.3
Preparation of amyloid fibrils with Ir(piq)3
About 2 mg of Ir complex and 50 mg of insulin were put in a mortar. After grinding for 10
minutes, the mixed materials were obtained as in figure 2.7(b), and were dissolved in 5 ml of 2
M guanidine hydrochloride. This solution was further dialyzed versus three rounds of 25 mM
HCl at 4◦ C for 24 hours. This resulted in a solution of the hybrid material in 25 mM HCl.
Then this solution was filtered through a 0.2 µm PVDF filter and diluted to a volume of 10
ml. The solution was then heated at 65◦ C in 25 mM HCl for 72 hours.
The heating process resulted in a 25 mM HCl solution containing Ir complexes confined
12
3.2 Experimental methods
in amyloid fibrils. The amyloid fibril and Ir(piq)3 concentration were calculated following the
method in Appendix B. The simple methods of this combination are shown in figure 2.7.
3.1.4
Preparation of amyloid fibrils with Ir(piq)3 with PTAA
The solution of amyloid fibrils with Ir(piq)3 was prepared as described in section 3.1.3, then
a PTAA solution was added to this solution. This resulted in amyloid fibrils with Ir(piq)3
decorated with PTAA as in the figure 2.8. The relative concentrations of the different materials
in the prepared solutions were listed in table C.1 in Appendix C.
3.2
Experimental methods
The performed experiments are described in this section. The used samples in each experiment
are listed in the table C.1 in Appendix C.
Absorbance measurements were done by using UV-vis spectrophotometer, UV-2450 Shimadzu, slit width of 0.5 nm.
The emission data of quantum yield experiments and overlapping graph were obtained with
Fluormax-4 spectroflurometer, Horiba Jobin Yvon, slit width 3 nm.
The emission for FRET and Dexter energy transfer experiments were done by using Tecan
Safire2 microplate reader in Top fluorescence sensitive mode.
Fluorescence lifetime measurements were done with FL900 Fluorescence Lifetime Spectrometer, Edinburgh, using PLD 800 picosecond diode laser as excitation source at 380 nm. The
setup of this instrument was suited to measure lifetime of sample in solution with very short
lifetime within ns unit. The data were collected by time-corrected single photon counting as
wavelength intensity versus time plot. All data were fitted to a stretch exponential in lifetime
analysis software program.
The phosphorescence lifetime measurements were done with time-corrected single photon
counting (in-house construction) by the excitation wavelength at 355 nm. This time-resolved
measurements were done using a frequency tripled diode-pumped YAG laser as excitations
source. This produces 40 ns pulses with 5kHz repetition rate. The detection wavelength was
selected using interference filters at 650 nm, each with bandwidth of 20 nm. The time decay
of the emission was recorded using a picosecond time analyzer, which used for time-correlated
photon counting. The time resolution of the system was limited by the length of the laser
pulses of 40 ns. The data were collected as number of counting photon versus time and then
fitted to exponential curve by Matlab.
13
Chapter 4
Results and discussions
Herein two mechanisms for energy transfer are considered. Förster resonance energy transfer
(FRET) is the dipole-induced interaction. This mechanism requires the spectra overlap between
the absorption of acceptor and the emission of donor. Dexter energy transfer is the exchange
electron mechanism which needs the orbital overlap of neighbor molecules, therefore this process
is observable only at very short distance between donor and acceptor. Dexter transfer typically
occurs between triplet-triplet states because it preserves the symmetry of the donor and acceptor
pair. But between singlet-singlet states, the dipole-induced interaction is dominating so that
FRET always happens between these states.
In figure 1.1, the decorated PTAA on amyloid fibrils has maximum emission at 579 nm (2.14
eV) which refers to a localized-singlet exciton from direct absorption at sites on the thiophene
backbone. The triplet state of PTAA chain emits at 701 nm (1.77 eV) [26]. For the hybrid
materials of Ir(piq)3 complexes and insulin fibrils, the emission originates from the lowest energy
MLCT3 transition with the maximum wavelength of approximately 625 nm (1.98 eV). However,
the energy between MLCT1 and MLCT3 of Ir(piq)3 is very small (1370-1520 cm−1 (0.027-0.030
eV)) [7].
As discussed in the introduction, excitation energy can be transfered between PTAA and
Ir(piq)3 incorporated into amyloid fibrils. This transfer might occur by FRET and Dexter
energy transfer processes. FRET was investigated by donor-acceptor quenching experiment.
Then the Dexter mechanism was investigated by the donor-acceptor quenching as well as timeresolved measurements of high PTAA concentration solutions. With these data, the approximate distance between PTAA and Ir(piq)3 molecules was calculated. Finally, a proposal for
the interaction between amyloid fibril, PTAA, and Ir(piq)3 is shown. The constitutions of all
samples in this study are listed in table C.1 of Appendix C.
4.1
Spectral overlap between donor and acceptor
The absorption and emission spectra (excited at 380 nm) were acquired with samples A and
B, and the spectra are shown in figure 1.1. This indicates that, Ir complexes can absorb the
energy from PTAA within this overlapping region. Herein two mechanisms for energy transfer
are considered such as Förster resonance energy transfer (FRET) and Dexter energy transfer.
Na-phosphate pH 7 solution was used as the buffer because of two reasons. First, at low pH
solution, the quantum yield of PTAA is very low [27], which results low FRET efficiency [10].
Second, when pH is higher than 10, the amyloid fibrils can be destroyed by the actions of alkali
[28],[29]. Therefore pH 7 seems to be an optimum pH for both PTAA and amyloid fibrils.
14
4.2 FRET
4.2
FRET
To investigate FRET, the donor was choosen to be PTAA and the acceptor to be Ir(piq)3 . The
measurements were done by varying the concentrations of Ir(piq)3 . The samples were prepared
in cuvettes in the same way as the sample C with different Ir(piq)3 concentrations. Then each
sample was split up into five parts, that was transfered into five wells of a microplate, and then
the emission was measured. All samples were excited at 380 nm because PTAA absorbs energy
more than Ir(piq)3 at this wavelength (see figure 4.1(a)). In figure 4.1(b), the solutions with
increasing Ir(piq)3 concentrations showed obvious quenching of PTAA emission spectra. These
results indicated that some amounts of energy transfered by FRET from PTAA to Ir complexes
which were confined in amyloid fibrils.
(a)
355
1.0
380
PTAA
Normalized absorbance
0.8
0.6
0.4
Ir(piq)3
0.2
0.0
350
450
400
500
600
550
(b)
3
Ratio PTAA:Ir(piq)3
5x10
1:0
1.0.75
1:1.65
1:2.26
1:3.02
1:3.77
3
Photoluminescence
650
Wavelength (nm)
4x10
3
3x10
3
2x10
3
1x10
0
500
550
600
650
700
750
Wavelength (nm)
(c)
4
1.0x10
Ratio Ir(piq)3:PTAA
1:0
1:0.70
1:4.68
1:7.00
Photoluminescence
3
8.0x10
3
6.0x10
3
4.0x10
3
2.0x10
0.0
600
700
Wavelength (nm)
Figure 4.1: The optical data of the interaction between amyloid fibril, PTAA
and Ir(piq)3 , (a) the absorption spectra of PTAA and Ir(piq)3 in amyloid solution, (b) the emission quenching of 0.35 mM PTAA by increasing concentrations of Ir(piq)3 at 380 excitation, (c) the slightly quenching of 66 µM Ir(piq)3
by increasing concentration of PTAA at 355 nm excitation
15
Chapter 4. Results and discussions
4.3
Dexter energy transfer
Since Ir(piq)3 has a higher triplet state energy level than PTAA, the Ir complexes were chosen as
donor for the Dexter energy transfer measurement. A high concentration of PTAA is required
in order to decrease the distance between donor and acceptor. Solutions were prepared in
the same way as sample D but with different PTAA concentrations. Then each solution was
split into two parts and transferred to two wells of a microplate. As shown in figure 4.1(a),
Ir complexes absorb the energy more than PTAA at 355 nm so this wavelength was used as
excitation wavelength for every sample. These measurements were performed in 25 mM HCl
(in place of pH 7 buffer) in order to minimize FRET by reducing the quantum yield of PTAA.
The figure 4.1(c) shows that if the amounts of PTAA were increased, the spectra in Ir(piq)3
region slightly decreased. This suggests some degree of energy back transfer, but the data are
not of sufficient quality to conclude how important the dexter mechanism is in these hybrid
materials.
4.4
Dependence of absorption and emission spectra on
PTAA concentration
For more information of the interactions, the sample G, H, I and J, were prepared (see figure
4.2(c)). All solutions, which had Ir complexes confined in amyloid wires, came from the same
preparation. In sample I, PTAA solution was added before fibril formation. In this case,
many PTAA molecules might be located inside the amyloid fibrils, and thus be located close to
Ir(piq)3 molecules. In sample J, PTAA solution was added after fibril formation, and we can
thus expect most PTAA molecules to be located on the outside of the fibril.
The absorption spectra of sample G, H, I, and J, were measured. Then all samples were
split into five part and transferred to five wells of a microplate. The emission was measured
with the excitation wavelength at 355 nm.
(a)
(b)
(c)
1.0
Color Sample
G
H
I
J
0.6
4
5x10
Photoluminescence
0.8
Absorbance
4
6x10
0.4
0.2
0.0
300
4
4x10
4
3x10
4
2x10
4
1x10
350
400
450
500
Wavelength (nm)
550
600
650
0
500
550
600
650
700
750
Wavelength (nm)
Figure 4.2: The data of photochemical analysis of high PTAA concentration
experiment, (a) the absorption spectra, (b) the emission spectra with 355 nm
excitation, and (c) the sample list
16
4.5 Time-resolved measurement
The absorption spectra in figure 4.2(a) shows the absorption spectra of sample J, which
mixed with PTAA after the fibril formation process, should come from both of PTAA and
Ir(piq)3 in that solution. While the absorbance from the sample I which mixed with PTAA
before the fibril process, is similar to the other Ir complex solutions. In figure 4.2(b), sample
J gave rise to the strongest emission. On the other hand, sample I, presented the similar
emission to the spectrum of the sample H which had no PTAA. Since it was found that most
of PTAA in the sample I precipitated to the bottom of the container after the heating process
resulting in lower PTAA emission. However, by this experiment we could not conclude if
the high emission of sample J is the result of fluorescence or phosphorescence. Therefore the
time-resolved measurement was also carried out.
4.5
Time-resolved measurement
The equation 2.3 and 2.4 indicate that quantum yield is proportional to lifetime. Besides,
quantum yield is the number of absorbed photons divided by the number of emitted photon.
All solutions which had Ir complexes in experiment 4.4 had similar absorption spectra. Therefore if any solution gave rise to high phosphorescence, that solution should also present high
phosphorescence lifetime.
(a)
(b)
Fluorescence lifetime of PTAA
1.0
Phosphorescence Lifetime of Ir(piq)3
1.0
Sample
0.8
Normalized photon counting
Normalized Photon counting
0.8
Sample G
0.6
0.4
0.2
0.0
0
1
2
3
4
5
H
I
J
0.6
0.4
0.2
0
2
4
6
8
10
Time (ns)
Figure 4.3: The time-resolved measurement data, (a) the fluorescence lifetime decay of sample G with 380 nm excitation, (b) the phosphorescence
lifetime decay of sample H, I, and J, with 355 nm excitation and 650 nm
detection
The fluorescence lifetime of sample G was measured with the excitation wavelength at 380
nm. The phosphorescence lifetime of samples H, I, and J, were measure with the excitation
at 355 and the detection at 650 nm. All of the four samples from section 4.4 were analyzed
by time-resolved measurements at room temperature. The lifetime of PTAA is very short
(ns) when compared with the phosphorescence lifetime of Ir(piq)3 (µs). The time-resolved
measurements were thus performed on different instruments with suitable lifetime range. Figure
4.3(a) shows that the fluorescence lifetime of PTAA is about 0.48 ns from software analysis.
The phosphorescence samples, in figure 4.3(b), had the counting photon intensities fitted to
17
Chapter 4. Results and discussions
Table 4.1: The summary of phosphorescence lifetime data with excitation at
355 nm and detection at 650 nm
Sample
H
I
τ1
(µs)
2.03
2.06
τ2
(µs)
8.89
9.70
J
1.32
5.39
Comment
Added PTAA before
fibril formation process
Added PTAA after
fibril formation process
biexponential decay equation following
I(t) = I1 exp(t/τ1 ) + I2 exp(t/τ2 ) + c
(4.1)
where I is the Intensity of photon counting, τ1 is the average phosphorescence lifetime of Ir
complexes in that solution, τ2 is the effect of noise from the environment, and c is the value of
baseline. The phosphorescence lifetime data are summarized in figure 4.3(b) and table 4.1.
The sample J, which gave rise the highest emission in figure 4.2(b), showed the shortest
phosphorescence lifetime. This indicates that most of the emission come from fluorescence of
PTAA, and thus some energy of Ir(piq)3 was transfered to the triplet state of PTAA by the
Dexter mechanism. Since this solution had the molar ratio of Ir(piq)3 to PTAA as 1:3, so that
many short polymer chains of PTAA, which were not coupled with Ir(piq)3 , did not transfer
energy, and thus emitted their energy as fluorescence (figure 4.2(b)).
Since Ir(piq)3 molecules are located randomly in the amyloid fibrils, some of them might be
deposited at the edge of the fibrils and might thus be located very close to PTAA molecules.
Therefore the dexter energy transfer might be allowed. However, the value of the lowest phosphorescence lifetime showed an insignificant drop when compared with the value from the solution without PTAA. This means that the amyloid fibrils prevented the dexter energy transfer
by increasing the distance between Ir(piq)3 and PTAA molecules.
4.6
Quantum yield of donor
The distance between donor and acceptor can be obtained by using equation 2.7, 2.8, 2.9, and
2.10. All of these equations are related to FRET measurement. Besides, the quantum yield of
donor is required to complete the distance calculation. Nevertheless, it is difficult to get real
quantum yield value from direct measurement of the quantum yield. Therefore the method of
comparing the unknown sample with a reference quantum yield sample is a convenient choice
in order to obtain an approximate quantum yield value of the sample.
A solution of fluorescein in 0.1 M NaOH having a quantum yield of 0.90 was used as
the reference quantum yield standard [13]. To obtain reasonable data, the concentration of
fluorescein should never exceed 1 µM. Moreover, this solution should be used within 12 hours
and be excited within the 400-500 nm region. For this study, the solutions of 1 µM fluorescein
were prepared and then the concentrations of the test solutions were increased by adding small
amount of the initial solution. Equation 2.5 is an important relation between referent standard
and unknown sample. In this work, The absorbance and integrated fluorescence intensity data
of Fluorescein is presented in figure 4.4(a).
PTAA was the donor for FRET, and therefore three kinds of PTAA solutions were prepared
18
4.7 Distance between donor and acceptor
(b)
(a)
Sample
8
9
1.0x10
2.5x10
Fluorescein
(K )
y = 1.425E9x + 2.151E5, χ2 = 1.00
y = 5.623E10x + 2.904E7, χ2 = 0.99
(L )
8
2.0x10
y = 5.218E9x + 1.002E8, χ2 = 1.00
8.0x10
Intergrated fluorescence
Integrated fluorascence
8
8
6.0x10
8
4.0x10
8
2.0x10
(M )
y = 5.503E9x + 2.067E6, χ2 = 1.00
8
1.5x10
8
1.0x10
7
5.0x10
0.0
0.0
0.000 0.002 0.004 0.006 0.008 0.010 0.012 0.014 0.016 0.018
-0.005 0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.040
Absorbance
Figure 4.4: The plots between absorbance and integrated fluorescence of
Fluorescien and PTAA solutions with excitation at 380 nm
Table 4.2: The summary of the quantum yields of donor solution
Sample
Φ
Fluorescein
K
0.90
0.023
L
0.084
M
0.088
in the same way as samples K, L, and M. First, 1 mM PTAA solution was prepared, and then the
small amount of this solution were added to several test samples. The plots of the absorbance
and integrated fluorescence intensity of three PTAA solutions are shown in figure 4.4(b). The
slope of the fitted linear equation of each solution was compared to the data from the fluorescien
solution by equation (2.5). By assuming equal refractive indexes of both types of solutions, the
relative quantum yields in table 4.2 were calculated.
An earlier study [27] suggested that the quantum yields of PTAA in pH 2, pH 5.9 and
pH 10, are 0.0014, 0.020, and 0.027 respectively. Therefore PTAA in buffer pH 7 solution (as
sample K ) with quantum yield of 0.023 is reasonable. The same procedure was performed on
the other solutions, and it can therefore be assumed that their relative quantum yields are
acceptable. Obviously, having amyloid fibrils in the solution enhanced the quantum yield of
PTAA [3]. By comparing the quantum yields for sample L and M , it can be concluded that
increasing amyloid concentration leads to a slight increase in quantum yield of PTAA.
4.7
Distance between donor and acceptor
The distance between donor and acceptor can be determined by FRET equations. But these
equations are suitable for the molecules with confined excited states. This is not true for PTAA,
and therefore following calculations might contain large errors. However, we still performed
them in order to get a rough estimate of donor-acceptor distances.
For FRET calculation, PTAA was chosen as donor and Ir(piq)3 was acceptor. A solution
with low PTAA concentration was added to sample F, while a solution with high PTAA concentration was added to sample J. All samples were excited at 380 nm. The emission data
comparing these solutions (F and J ) with the solutions without Ir(piq)3 (E and G) are shown
19
Chapter 4. Results and discussions
(a)
(b)
Low PTAA concentration
Sample
Sample
E
F
3
5x10
High PTAA concentration
Sample
Sample
4
7x10
G
J
4
3
6x10
548
4
Photoluminescence
Photoluminescenc
4x10
3
3x10
3
2x10
5x10
Sample
548
4
4x10
4
3x10
4
2x10
3
1x10
4
1x10
0
500
550
600
650
700
750
0
500
550
Wavelength (nm)
600
650
700
750
Wavelength (nm)
Figure 4.5: The emission spectra from sample E, F, G, and J with 380 nm.
excitation
Table 4.3: The summary of the quantum yield of donor (ΦD ), overlab integral
(J ), Förster distance (R0 ), transfer efficiency (E ) and distance between donor
and acceptor (R) in different concentrations of PTAA, Ir(piq)3 and amyloid
in the solution
Sample
F
J
ΦD
J
−1
(%) (M cm−1 )
0.084 2.49 × 1016
0.088 2.36 × 1016
R0
(Å)
53
52
E
(at 548 nm)
0.38
0.30
R
(Å)
57
60
in figure 4.5(a) and (b). To obtain the transfer efficiency from equation 2.9, the ratio between
fluorescence intensity of the donor in presence to absence of acceptor was taken at 548 nm.
The important values and the approximated distances between donor and acceptor are
shown in table 4.3. The results of the calculated distance from the solutions with low and high
PTAA concentrations were similar. This suggests that the amyloid fibrils separated the PTAA
and Ir(piq)3 molecules, resulting in a distance around 57 Å to 60 Å. These long ranges are
not suitable for dexter energy transfer, so that there was phosphorescent emission of Ir(piq)3
complexes from sample J.
4.8
Geometry of interaction
In figure 4.6 is shown a schematic drawing of a proposed structure for the hybrid material formed
from CPEs, triplet emitter, and protein nanowire. The molar ratio of materials in this image
was taken from sample B (Ir(piq)3 : amyloid : PTAA = 3 : 5 : 9). Sample J showed highest
emission in figure 4.2 and most of the emission originates from short chains of PTAA which
had no Ir(piq)3 couple for FRET process. At the same time, the result of the time-resolved
measurement of sample J suggested that Ir(piq)3 molecules still emitted phosphorescence. All
of these results agrees with the calculated approximate distances between donor and acceptor,
which are caused by the amyloid fibril structure. Most of the Ir(piq)3 molecules were confined
20
4.8 Geometry of interaction
inside the amyloid fibrils but some of them were located at the edge of fibril, and which might
allow some energy back transfer.
Molar ratio Ir(piq)3:Amyloid:PTAA = 3:5:9
<60 Å
100 Å
>60 Å
10 Å
PTAA
Ir(piq)3
Figure 4.6: The simple conclusion drawing of the interaction between PTAA
chains, Ir complexes and amyloid fibril. The molar ratio was calculated from
the solution J. Assuming that one fibril consisted of four protofilaments as in
left hand side image and the cross section of this fibril is shown in right side
view.
21
Chapter 5
Conclusion
The quenching of PTAA spectra in FRET experiments confirmed FRET from PTAA singlet
state to Ir(piq)3 singlet state. On the other hand, the slightly quenched Ir(piq)3 spectra for
Dexter process implied poor energy back transfer. It thus seemed that amyloid fibrils acted as
a wall which prevented the energy from transferring back to the triplet state of PTAA.
However, the data from all experiments were difficult to analyze because of the absorption
regions overlap between PTAA and Ir(piq)3 . Therefore, any excitation wavelength could absorb
by molecules from both of CPEs and triplet emitter. Moreover, PTAA and Ir(piq)3 molecules
were randomly located in amyloid fibrils, leading to a range of possible donor-acceptor distances.
22
Appendix A
Appendix A: Beer-Lambert Law
The absorbance (A) or optical density is usually defined as
A = − log(I/I0 )
(A.1)
where I is the passed light intensity of the sample and I0 is the initial light intensity, which
I0
Sample solu
of concentrattion
ion c
I
d
Figure A.1: A thin slab of solution and light absorption parameters
are described in figure A.1. If the absorbance is measured, the unknown concentration can be
determined by Beer-Lambert law following
A = cd
(A.2)
where is the molar extinction coefficient with unit M −1 cm−1 ; M is moles/liter, c is the
concentration of solution in M and d is the path length of the sample in cm. The BeerLambert law is limited by chemical and instrument factor, which cause of nonlinearity, e.g.
high concentration solution, scattering of light and non-monochromatic radiation.
23
Appendix B
Appendix B: Calibration Curve for
amyloid fibril and Ir(piq)3
concentration
To calculate the concentration of hybrid materials between amyloid and Ir(piq)3 in solution, the
calibration curve of hybrid materials were prepared by keeping the concentration of insulin and
varying the concentration of Iridium complexes following table B.1. After the mixing process,
sample c, d, and e, were diluted by 25 mM HCl into 10 ml. The absorbance data at 280 nm
and 430 nm of all samples, were linear with respect to concentration as in figure B.1. These
linear relations used as the calibration curves for calculating the concentration of insulin fibrils
and Ir(piq)3 .
For example, if one solution consists of Ir(piq)3 complexes confined in insulin fibrils presents
the absorbances of 1.387 and 0.271 at 280 nm and 430 nm respectively. The concentration of
both compounds can be determined following:
- Substitute 0.271 for y of the equation for 430 nm, the value x will become the concentration
of Ir(piq)3 complexes (xred )in mg/ml unit.
y = 19x + (9 × 10−17 )
y − (9 × 10−17 )
0.271 − (9 × 10−17 )
xred =
=
19
19
xred = 0.014
- Substitute xred in x of the equation for 280 nm, y will become the total absorbance (Atotal )
which come from both of two materials.
Table B.1: The samples constitution for calibration curve are listed as follow:
sample a, 50.51 mg of bovine insulin was grinded for 10 minutes with 1.02 mg
of Ir(piq)3 , then dissolved in 10 ml 25 mM HCl; sample b, 50.50 mg insulin
was dissolved in 10 ml 25 mM HCl.
Sample
c
d
e
a
(ml)
3
2
0
b
(ml)
0
1
3
24
(e )
(d )
(c )
(c )
(d )
(e )
Figure B.1: Calibration curve for Ir(piq)3 complexes and insulin fibrils: the
latter c, d, and e, are referred to the sample in table B.1.
y = 46.5x + 1.335 = 1.999
Atotal = 1.999
- Find absorbance of Ir(piq)3 (Ared280 ) at 280 nm by abstracting 1.335 from Atotal , because
this value belongs to the solution without Ir(piq)3 complexes.
Ared280 = 1.999 − 1.335 = 0.664
- Calculate the absorbance of insulin in the hybrid material solution at 280 nm Ains280 by
abstracting Ared280 from the detected absorbance at 280 nm.
Ains280 = 1.387 − 0.664 = 0.664
From this calculation, the concentration of insulin can be obtained by Beer Lamber law.
25
Appendix C
Appendix C: Sample List
The samples that were used in this diploma thesis work are described in table C.1 with the
samples constitution. Moreover, the tested samples in each experiment are listed in table C.2.
26
Table C.1: The list of samples constitution: amyloid fibrils and Ir(piq)3
complexes were dissolved in 200 µl of 25 mM HCl, PTAA were dissolved in 10
µl of deionized water, and 1000 µl of Na-phosphate pH 7 solution was used as
the buffer in every sample but D.
Sample
A
B
C
D
E
F
G
H
I
Amyloid Ir(piq)3
(µM)
(µM)
400
400
66
400
Variable
400
66
400
400
66
200
200
120
200
120
PTAA
Comment
(µM)
6.6
0.35
Variable
No buffer
0.35
0.35
6.6
6.6
PTAA was added before
J
200
120
6.6
K
L
M
200
400
-
6.6
Variable
Variable
fibril formation process
PTAA was added after
fibril formation process
Table C.2: The list of experiments and the used samples
Experiment
Sample
Spectra overlap
A, B
Absorption spectra
A, B
FRET
C
Dexter energy transfer
D
High PTAA concentration
G, H, I, J
Time-resolved measurement
G, H, I, J
Quantum yield of donor
K, L, M
Distance between donor and acceptor E, F, G, J
Geometry of interaction
J
27
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