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FUNCTIONALIZED QUANTUM DOTS AND ITS
APPLICATIONS
XU JIA
(B. Sc, Sichuan University)
A THESIS SUBMITTED
FOR THE DEGREE OF MASTER OF SCIENCE
FOOD SCIENCE AND TECHNOLOGY PROGRAMME
DEPARTMENT OF CHEMISTRY
NATIONAL UNIVERSITY OF SINGAPORE
2008
Table of Contents
Acknowledgement
IV
Summary
V
List of Tables
Ⅶ
List of Figures
Ⅷ
Chapter 1. General Introduction
1.1 Quantum Dots and Its Properties
1
1
1.1.1
Introduction to quantum dots
1
1.1.2
Quantum dots optical properties
2
1.2 Quantum Dots in Optical Sensing Applications
5
1.2.1
Anion and small molecules sensing
1.2.2
Hydrosulfide biology and its sensing
11
1.2.3
Aim and objectives
12
References
Chapter 2. Thiolated Caffeic Acid Functionalized Qua ntum do ts
8
13
15
2.1 Introduction
15
2.2 Experimental Section
19
2.2.1
Materials and instruments
19
2.2.2
Synthesis of caffeic acid derivative
20
2.2.3
Quantum dots ligand exchange with thio-caffeic acid
25
-I-
2.3 Results and Discussion
26
2.4 Conclusion
33
References
35
C h a p t e r 3 . Tr o l o x F u n c t i o n a l i z e d Q u a n t u m d o t s
3.1 Introduction
35
35
3.1.1
Quantum dots and its synthesis
35
3.1.2
Quantum dots ligand exchange strategies
36
3.1.3
Electron transfer based sensor
40
3.2 Experimental Section
41
3.2.1
Materials and instruments
41
3.2.2
Experimental procedures
43
3.3 Results and Discussion
52
3.3.1
Synthesis and structural characterization of Trolox derivatives
52
3.3.2
Functionalization of quantum dots with Trolox derivative
56
3.3.3
Trolox-QDs sensing of peroxyl radicals
62
3.4 Conclusion
References
C h a p t e r 4 . Tr i s ( 2 - a m i n o e t h y l ) a m i n e ( Tre n ) F u n c t i o n a l i ze d Q D s
4.1 Introduction
68
70
71
71
- II -
4.2 Experimental Section
78
4.2.1
Materials and instruments
78
4.2.2
Sensing of metal ions
80
4.2.3
The effect of carbon disulfide on QDs-metal ions complex
81
4.2.4
Tren-CS2-metal complex synthesis and its reaction with
sodium hydrosulfide
82
4.2.5
Tren-QDs-CS 2 -Fe(II) complex synthesis
82
4.2.6
Tren-QDs-CS2-Fe(II) complex sensing hydrosulfide anion
and nitric oxide
83
4.3 Results and Discussion
84
4.3.1
Tren-QDs system
84
4.3.2
Optical characteristics of the Tren-QDs
86
4.3.3
Sensing of transition metals
88
4.3.4
Tren-QDs-CS 2 -Fe(II) complex system
95
4.3.5
Tren-QDs-CS2-Fe(II) complex synthesis and its application
4.4 Conclusion
References
101
109
112
- III -
Acknowledgement
This research is financially supported by A*STAR’s Science and Engineering
Research Council (SERC). The guidance and the fundamental works from Dr. Huang
Dejian and Dr. Wang Suhua are acknowledged with gratitude. Miss Karen Hay and
Miss Lim Pui Yee were also acknowledged for technical supports.
- IV -
Summary
In this research the ability of several ligands bounding to the surface of ZnS capped
CdSe quantum dots (QDs) was studied. Different ligands for quantum dots
functionalization was synthesized and characterized by ESI-MS, NMR and FT-IR
techniques. The ligand exchange was carried out between synthesized ligands and
trioctylphosphine oxide (TOPO) functionalized CdSe/ZnS quantum dots under
different conditions. The functionalized CdSe/ZnS quantum dots was purified and
characterized to ensure the successful bounding. Preliminary sensing studies were
carried out using the functionalized quantum dots sensing peroxyl radicals and
hydrosulfide anion.
The possibility for thiolated caffeic acid (3,4-dihydroxycinnamic acid) to serve as a
quantum dot ligand was explored although the vulnerability of this ligand to air
made ligand exchange difficult. The α-tocopherol (α-TOH) analogue Trolox
(5,7,8-tetramethylchroman-2-carboxylic Acid) was modified to its diamine
derivative. Using carbon disulfide as a bridging unit, Trolox diamine was capped
onto CdSe quantum dots surface by ligand exchange process. The significant
changes of functionalized QDs in solubility and optical properties,
31
P-NMR
spectrum and FT-IR spectrum was consistent with literature reports which provide
evidences for successful ligand exchange. The sensitivity of Trolox-QDs to peroxyl
radicals and analysis to the reaction products showed us the promising future of this
electron transfer based sensing Trolox-QDs system and the in depth studies are still
-V-
on going. The tris(2-aminoethyl)amine (Tren)-QDs system was developed in the
same way as Trolox system but with a stronger bounding ligand. After ligand
exchange, Tren-QDs can be well dissolved in water, PBS buffer and other polar
solvents. It has been found the fluorescence of Tren-QDs has selective sensitivity to
Co(III) and Cu(II). Different mechanisms for this effective quenching was proposed
and the verification is still on the way. Moreover, Tren-QDs was used as the basis of
development of new fluorescent probes. The Tren-QDs-CS2-Fe(II) complex
material was synthesized and utilized for biologically important hydrosulfide anion
(HS-) sensing. It was our speculation that a sulfide containing complex, which has
strong absorbance at Tren-QDs excitation and emission wavelength was form from
the reaction between the Tren-QDs-CS2-Fe(II) complex and HS- therefore the inner
filter effect of this new complex quenched emission of Tren-QDs in the system.
- VI -
LIST OF TABLES
Table 1.1 QD-based fluorescent probes for sensing of small molecules and
ions
Table 2.1 Scavenging properties of caffeic acid
9
17
Table 3.1 The rate constants of α-tocopheryl radical and the biological
concentrations of the substrates
41
- VII -
LIST OF FIGURES
Figure 1.1
Orbital Energy levels in semiconductor QDs.
Figure 1.2
Tuning the QD emission wavelength by changing the nanoparticle
size or composition.
1
2
Figure 2.1
ESI-MS of thio-caffeic acid reaction mixture
22
Figure 2.2
Separation and purification of thio-caffeic acid
23
Figure 2.3
ESI-MS (negative) of thio-caffeic acid extraction
24
Figure 2.4
ESI-MS (negative) of purified thio-caffeic acid
24
Figure 2.5
ESI-MS result of DTT protective effect on thio-caffeic acid
31
Figure 3.1
ESI-MS (negative mode) spectrum of Trolox methyl ester
44
Figure 3.2
ESI-MS (positive mode) spectrum of Trolox methyl ester
44
Figure 3.3
1
45
Figure 3.4
13
45
Figure 3.5
ESI-MS spectrum of Trolox diamine
47
Figure 3.6
1
48
Figure 3.7
13
48
Figure 3.8
FT-IR spectra of Trolox methyl ester and Trolox diamine
53
Figure 3.9
Trolox methyl ester standard curve in methanol.
54
H-NMR spectrum of Trolox methyl ester
C-NMR spectrum of Trolox methyl ester
H-NMR spectrum of Trolox diamine
C-NMR spectrum of Trolox diamine
Figure 3.10 The reaction kinetic of AMVN(3.65 mM) and AAPH (3.35 mM) on
Trolox methanol solution (4.52 mM) absorbance
55
Figure 3.11 ESI-MS (negative) spectrum of reaction mixture of Trolox methyl
ester and AAPH
Figure 3.12 Proposed Structure of Trolox functionalized quantum dots
56
57
Figure 3.13 Absorption spectrum and fluorescence emission spectra of TOPO
Figure 3.14
QDs and Trolox functionalized QDs
58
31
60
P NMR spectra of TOPO-QDs and Trolox functionalized QDs
Figure 3.15 FT-IR spectrum of Trolox functionalized QDs and TOPO QDs
61
Figure 3.16 Effect of AAPH and AMVN solution on the fluorescence of Trolox-QDs
63
Figure 3.17 Effect of AAPH solution on the fluorescence of Tren-QDs and Trolox-QDs
64
- VIII -
Figure 3.18 FT-IR spectrum of Trolox-QDs and oxidized Trolox-QDs
Figure 4.1
Ten distinguishable emission colors of ZnS-capped CdSe QDs
excited with a near UV lamp with sizes of QDs increase from left to right
Figure 4.2
66
73
Proposed surface structure of CdSe QDs capped by Tren ligands
and their coordination complexes
with transition metal ion.
85
Figure 4.3
Absorption spectrum and fluorescence emission spectra of Tren-QDs
86
Figure 4.4
The concentration-dependent of fluorescence property of QDs.
87
Figure 4.5
Effect of metal ions on the fluorescence of Tren-QDs.
88
Figure 4.6
Electronic spectra of Co(III) complexes of various concentrations
in aqueous solution.
Figure 4.7
90
Electronic spectra of Cu(II) complexes of various concentrations
in aqueous solution.
91
Figure 4.8
Energy scheme of the Tren-QDs in the presence of Cu(II).
92
Figure 4.9
Effect of Cu(II) and Co(III) concentration on the fluorescence
intensity of Tren-QDs.
93
Figure 4.10 Stern-Volmer plot of Cu(II) and Co(III) concentration dependence
of the fluorescence intensity of Tren-QDs.
Figure 4.11 Effect of metal ions and CS2 on the fluorescence of Tren-QDs.
95
97
Figure 4.12 UV-Vis spectrum of Tren-CS2-Fe(II) complex before and after NaSH
addition
99
Figure 4.13 Effect of hydrosulfide anion on the UV-Vis absorbance of
Tren-QDs-CS2-metal complex at 550nm.
100
Figure 4.14 Proposed surface structure of CdSe QDs capped by Tren ligands
and their coordination complexes with Iron (II) ions.
Figure 4.15 Spectra of Tren-QDs-CS2-Fe(II) complex quenched by NaSH solution
102
103
Figure 4.16 Effect of HS - concentration on the fluorescence intensity of
Tren-QDs-CS2-Fe(II) complex
104
Figure 4.17 Effect of hydrosulfide anion concentration on the fluorescence
intensity of Tren-QDs-CS2-Fe(II) complex
105
Figure 4.18 UV-VIS spectrum of Tren-QDs-CS2-Fe(II) complex before and after
- IX -
NaSH addition
106
Figure 4.19 Quenching Tren-QDs-CS2-Fe(II) complex by NaSH at different
excitation wavelength
107
Figure 4.20 S p e c t r a o f Tr e n - Q D s - C S 2 - F e ( I I ) c o mp l e x q u e n c h e d b y
nitric oxide solution
108
-X-
Chapter 1
General Introduction
1.1 Quantum Dots and Its Properties
1.1.1 Introduction to quantum dots Quantum dots (QDs) are nanostructured
semiconductor
materials[1].
These
colloidal
nanocrystalline
semiconductors
comprising elements from the periodic groups II-VI, III-V or IV-VI, are featured
with roughly spherical and with typical sizes (diameter) in the range 1-12 nanometer
(nm). At such reduced sizes that close or smaller than dimensions of the exciton
Bohr radius within the corresponding bulk material, these nanoparticles behave
differently from bulk solids due to quantum confinement effects
[2,3]
. The result of
quantum confinement are that the electron and hole energy states within the
nanocrystals are discrete, but the electron and hole energy levels and therefore the
band-gap is a function of the QDs diameter as well as composition[5]. The band-gap
of semiconductor nanocrystals increase as their size decreases, resulting in shorter
emission wavelength[6,7]. Hence, the quantum confinement effects are responsible for
unique optoelectronic properties exhibited by QDs which includes high emission
quantum yields, size-tunable emission profiles and narrow spectral bands[3,4].
Especially, their size-dependent properties result in a tunable emission that allows
one to choose an emission wavelength that is well suited to a particular experiment
and to synthesize the QD-based probe by using an appropriate semi-conductor
material and nanocrystal size.
-1-
Semiconductor QDs are characterized by a band-gap between their valence and
conduction electron bands (Figure 1). When a photon having an excitation energy
exceeding the semiconductor band-gap is absorbed by a QD, electrons are promoted
from the valence band to the high-energy conduction band. The excited electron may
then relax to its ground state by the emission of another photon with energy equal to
the band-gap[4].
Conduction
Band
e
Band gap
Hole
Electron
e
+
e
e
e
e
Valence
Band
Figure 1.1: Orbital Energy levels in semiconductor QDs.
1.1.2 Quantum dots optical properties In recent years there has been intense
research in the fundamental study of the synthesis and photophysical properties of
QDs[8-11]. Researches on II-VI semiconductor QDs, such as CdSe or CdS
nanocrystals have been studied in order to characterize the relationship between size,
shape and electronic properties[2-4]. And the initial applications of QDs were heavily
focused on their use in microelectronics and opto-electrochemistry (e.g.,
light-emitting diodes, solar energy conversion et al.)[3,12,13].
-2-
But soon the unique sized-dependent emission attracted enough attention from
researchers and became probably the most intriguing and the most studied optical
property of QDs. As the emission properties of semiconductor nanocrystals depend
strongly upon the energy and the density of the electron state, they can be altered by
engineering the size and the shape of their structure. As Figure 1.2 shows, different
size CdSe nanoparticles can be tuned in the 500-700nm range. Moreover, by altering
the chemical composition of QDs, fluorescence emission may be tuned from the
near-infrared spectrum, spanning a broad wavelength range of 400-2000nm[14].
Figure 1.2 Tuning the QD emission wavelength by changing the nanoparticle size or composition.
(A) The emission of a CdSe QD may be adjusted to anywhere within the visible spectrum
(450-650 nm) by selecting a nanoparticle diameter between 2 and 7.5 nm (B) While keeping the
nanoparticle size constant (5 nm diameter) and varying the composition of the ternary alloy
CdSexTe1-x, the emission maximum may be tuned to any wavelength between 610 and 800
nm.(figure adapted from refs.16)
Another advantage of quantum dots fluorescence emission is its typically narrow
emission profile compared with traditional organic dyes with full width at
half-maximum (FWHM) around 15-40 nm[1]. This property is due to QDs’ discrete,
atom-like electronic structure. Since the emission lines are comparatively narrow,
-3-
detection of the QDs suffers much less from cross-talk that might result from the
emission of a different fluorophore bleeding into the detection channel of analyte.
On the other hand, QDs typically exhibit higher fluorescence quantum yields than
conventional organic dyes which give it greater analytical sensitivity. The quantum
yield of a fluorophore is a function of the relative influences of radiative
recombination (producing light) and non-radiative recombination mechanisms.
Non-radiative recombination, which largely occurs at the nanocrystal surface, is a
faster mechanism than radiative recombination an is greatly influenced by the
surface chemistry. By capping the nanocrystal with a shell of an inorganic wide-band
semiconductor, such as ZnS, reduces such non-radiative deactivation and results in
brighter emission[14].
The suitably surface protected QDs also have superior photoluminescent stability as
compared to typical fluorescent organic dyes. Several studies have demonstrated that
the photo luminescence properties of CdSe nanocrystals did not show any detectable
change upon aging in air for several months [15] and were observed to be 100 times
more stable than conventional organic fluorophores against photobleaching[12]. The
long fluorescence lifetimes of QDs, on the order 10-50 ns, are advantageous for
distinguishing QD signals from background fluorescence and for achieving
high-sensitivity detection[17].
-4-
1.2 Quantum Dots In Optical Sensing Applications
The application of luminescent QDs as biological labels was first reported in 1998 in
two breakthrough papers published by A. P. Alivisatos at UC-Berkeley and S. M. Nie
at Indiana University-Bloomington respectively[17,18]. Their research simultaneously
demonstrated that semiconductors QDs could be made water soluble and could be
conjugated with biological molecules by surface modification and bioconjugation
methods. It was their pioneer work paved the way for QDs application as highly
sensitive fluorescent bio-marker and biochemical probes. Developments in recent
years have more focused on the importance of adequate surface modifications in
developing luminescent QDs for labeling in bioanalysis and sensing. The nature of
the ligands being coordinated to the QDs surface and the particular type of bonds
which it forms with the nanocrystal surface atoms are of great importance to
quantum dots researchers. By ligand designing and fabrication, several important
properties of quantum dots can be tuned for specific purpose: processibility,
reactivity and stability. All of these have direct consequences on quantum dots’
spectroscopic properties[19]. Three principal functions of the surface ligands can be
described by Querner et al. as follows: (1) They prevent individual colloidal
nanocrystals from aggregation. (2)They facilitate nanocrystals’ dispersion in a large
variety of solvents. In the presence of surface ligands, the ability to disperse
nanocrystals is governed by the difference between the ligand and the solvent
solubility parameters, which can be precisely tuned. (3) Ligands containing
appropriate functional groups may serve as bridging units for the coupling of
-5-
molecules or macromolecules to nanocrystals or their grafting on substrates[19].
As the luminescence of QDs is very sensitive to the surface states of the QDs, it is
reasonable to expect that the chemical or physical interaction between a given
chemical species and the surface of the nanoparticles would also result in changes in
the efficiency of the core electron-hole recombination[20]. This has been the basis of
the increase in research activity on the development of novel optical sensors based
on QD probes.
Following this approach, Cd-based QDs have been widely reported for optical
sensing of small molecules and ions. In some pioneering works, the enhancement of
fluorescence were reported by L. Spanhel et al. when Cd ions was added to a basic
aqueous solution containing unpassivated CdS nanoparticles without detectable
changes in particle sizes[10]. Similar phenomenon was observed when Zn and Mn
ions was introduced to colloidal solutions of CdS or ZnS QDs[20,21]. These
photoluminescence-activation effect could be attributed to passivation of surface trap
sites that either being “filled” or energetically moved closer to the band edges.
Besides the activation effect, QD-based optical sensing quenching strategies has also
been proposed. The mechanism of the quenching by the analyte that affects the
luminescence emission of the nanoparticle was summarized as: (1) inner filter effects;
(2) non-radiative recombination pathways; (3) electron-transfer processes and (4)
-6-
ion-binding interactions. These above four quenching mechanisms have been
proposed and intensely studied in recent years to elucidate QDs based sensing.
Electron transfer between semiconductor nanoparticles and organic molecules bound
to their surface is a fundamental process that has been studied extensively in the
recent years, especially for the creation of solar cells and optoelectronic devices[22].
When electron transfer occurs, the nanoparticle and its attached molecule exist in
highly reactive charged forms long enough to interact with the surrounding
environment[22]. The redox potential of the organic ligand can be chosen or modified
to maximized the efficiency of charge transfer or to yield a radical of the desired
reactivity able to oxidize the target molecule. Currently most work in this area was
performed with TiO2 nanocrystallites[23,24]. But research in recent years has also
established the same system with CdSe and CdSe/ZnS quantum dots: D. S. Ginger et
al reported photoinduced electron transfer from conjugated polymers to CdSe
nanocrystals[25]; C. Landes et al used n-butylamine as an acceptor to occupies hole
sites, thus blocking the recombination process, which results in decreasing the
density of luminescent centers[26]. J. A. Kloepfer and coworkers observed CdSe
solubilized with mercaptoacetic acid emission quenched by a hole acceptor
adenine[27]; in a most recent publication S. J. Clarke et al reported electron transfer
between neurotransmitter dopamine and CdSe/ZnS QDs[22]; Maurel et al. reported a
non-linear quenching effects of fluorescent quantum dots by nitroxyl free radicals
TEMPO (2,2,6,6-tetramethylpiperidine-N-oxide free radical) and suggested the
-7-
mechanism would involve electron transfer from the conduction band to the
nitroxide (a mild acceptor), and back electron transfer from the nitroxide to the
valence band, effectively leading to quenching using the nitroxide SOMO as a
shuttle for electron and hole[28]. Later, in another publication them utilized
4-amino-TEMPO (4-AT) bounding on QDs surface to create a as highly selective
prefluorescent sensors for the detection of carbon-centered free radicals[29].
The potential for the use of QD-electron-donor systems as biosensors is nonetheless
great, as electron transfer eliminates (“on-and-off” system )or activate (“off-and-on”
system ) fluorescence from the particle, thus providing a visible signal of its
occurrence.
In this work, we used derivatives of antioxidant caffeic acid and Trolox to study the
electron transfer between small molecule ligands and CdSe/ZnS quantum dots.
1.2.1 Ions and small molecules sensing Methods based on chemical or physical
interactions between target chemical species and the surface of the nanoparticles are
very simple. But those methods appear to be restricted to sensing just a few reactive
small molecules or ions (Table 1.1).
-8-
Table 1.1 QD-based fluorescent probes for sensing small molecules and ions (refs. 1)
Chen et al. explained the quenching of L-cysteine capped QDs by Fe(III) is
attributed to an inner filter effect as a result of the strong absorption by Fe(III) at the
excitation wavelength used. This interference caused by Fe(III) can be eliminated by
adding fluoride ions to form a colorless complex FeF63-, which will also dissociate
from the surface of the QDs due to same charge repulsions. Also, the quenching of
thioglycerol capped CdS QDs by Cu(II) is through an electron transfer from
thioglycerol to Cu(II). The reduction of Cu(II) to Cu(I) by thioglycerol, formed
CdS+-Cu+ on the surface, which has a lower energy level than pure CdS QDs,
therefore causing a red-shift of fluorescence. Moreover, Cu(I) quenches by
facilitating non-radiative recombination of excited electrons in the conduction band
and holes in the valence band[30].
On the other hand, fluorescence enhancement of QDs has been reported by Moore et
al. in 2001[20]. The reversible fluorescence activation process caused by Zn(II) and
-9-
Cd(II) adsorption on the surface of CdS was studied. These ions enhanced the
fluorescence intensity of QDs, was attributed to some form of passivation of surface
trap states. In 2002, Chen et al. worked on Zn(II) determination and found the
formation of a Zn-cysteine complex on the surface of cysteine capping QDs which is
believed responsible for the activation of the surface states and therefore exhibiting
the fluorescence enhancement[30].
Apart from intensive studies for potential use of QDs in sensing cations,
functionalized QDs were also used for detection of inorganic anions although they
are still in embryonic stages. In 2002, Watanabe et al. reported that a gold
nanoparticle capped with amide ligands showed enhanced optical sensing of anions.
The presence of anions would cause a marked decrease in extinction as a result of
anion-induced aggregation of amide-functionalized gold nanoparticle via the
formation of hydrogen bonding between the anions and the interparticle amide
ligands. However, there was no selectivity and the binding affinity of anions was
low[31]. In 2005, Jin et al. developed water soluble fluorescent CdSe quantum dots
which are capped with 2-mercaptoethane sulfonate (MES) for the selective detection
of free cyanide. Consequently, a slight blue-shift fluorescence quenching with
detection limit of 1.1 x 10-6 M was observed. The blue-shift implies the changes in
size or surface properties of MES-CdSe QDs which brings about the decrease in
fluorescence[32]. However, the mechanism of fluorescence quenching was not
discussed.
- 10 -
In the present paper, transition metal ions sensing are performed by
tris(2-aminoethyl)amine (Tren) capped QDs. However, only the quenching pathways
of selective determination of Co(III) and Cu(II) by using CdSe QDs which is capped
by Tren ligands are thoroughly investigated. Furthermore, the Tren-QDs and
transition metal Fe(II)complex hybrid materials are also utilized for sensing
physiologically important hydrosulfide anion.
1.2.2 Hydrosulfide biology and its sensing Hydrogen sulfide is the most recent
small endogenously generated species touted as a biological signal species[33]. Like
CO, H2S is not a radical but has the apparent ability to interact with and
disrupt/modulate the actions of other radicals. In various papers, hydrogen sulfide
was reported to cause vasorelaxation in the vascular system[34] and enhance the
vasorlaxant effect of
·
NO[35]. In the brain, H2S can have numerous effects, one of
them is the ability to act as a neuromodulator enhancing N-methyl-D-aspartate
(NMDA) receptor responses[36], which was reported to be related to cAMP
production[37]. H2S also have antioxidant properties, protecting neurons from
oxidative stress[38] which was due to its ability to raise the intrcellular glutathione
(GSH) concentration by as much as twofold without increasing oxidized GSH levels,
as well as increasing the levels of the GSH biosynthetic enzyme γ-glutamylcystein
synthase. It has also been demonstrated that H2S can increase the ability for the
antioxidant enzyme superoxide dismutase (SOD) to scavenge superoxide[39].
Hydrogen sulfide has a pKa of 6.8, making the anionic species the predominant form
- 11 -
under most physiological conditions[33]. Hence the detection of HS- anion is of great
importance to biological and medical sciences. The highly sensitive detection of HS(as low as 125 ± 9.8 nM ) can be achieved by anion chromatography with ultraviolet
detection (IC/UV) method[40,41], but a easy, simple and inexpensive quantitative
detection method has not yet been reported and is still required development.
1.2.3 Aim and Objectives
In this paper, electron transfer meditated QDs quenching were studied with
thio-caffeic acid functionalized QDs system and Trolox diamine functionalized QDs.
Tris(2-aminoethyl)amine (Tren) functionalized QDs system was also established for
transition metal ions sensing. Selective determination of Co(III) and Cu (II) by using
CdSe QDs which is capped by Tren ligands are thoroughly investigated. Furthermore,
the Tren-QDs and transition metal Fe (II) complex hybrid materials are also utilized
for sensing hydrosulfide anion (HS-).
- 12 -
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9996–10003.
- 13 -
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Chemical Communications, 2002, 2866-2867 .
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- 14 -
Chapter 2
Thiolated Caffeic Acid Functionalized Quantum dots
2.1 Introduction
Semiconductor nanocrystals (or quantum dots, QDs) have size-dependent fluorescent
emissions wavelengths with high quantum yields and superior photostability
comparing to organic dyes[1]. Thus QDs have great potential in various biological
imaging applications, including labeling cells and other biological components.
However, the prerequisite for the development of QD-based bioimaging systems is
to gain access to photostable, and biocompatible nanocrystals. To achieve that,
quantum dots synthesis and coating usually followed by surface modification as a
preferred route for obtaining highly fluorescent water-soluble QDs[1]. Among the
various techniques for surface modification, two general methods are most widely
used: (1) Ligand exchange of hydrophobic surfactant molecules for bifunctional
linker molecules (2) phase-transfer methods using amphiphilic molecules that act as
detergents for solubilizing the QDs coated with hydrophobic groups[1].
Electron transfer between semiconductor nanocrystal and organic molecules bond to
their surface has been studied extensively in recent years[2]. This kind of charge
transfer has already been established with CdSe and ZnS coated CdSe quantum dots
(CdSe/ZnS QDs) to create a sensor of changes in redox potential[2-5]. A suitable
- 15 -
ligand is the key to fabrication of such a sensor.
Caffeic acid (3,4-dihydroxycinnamic acid) is a natural phenolic compound, which is
reported to exist widely in vegetable and coffee products, and presents as a
degradation product of chlorogenic acid
[6-8]
. Some pharmacological properties of
this substance have been described previously. For example, it has a strong and
specific inhibitory activity towards 5-1ipoxygenase, and can inhibit platelet
aggregation and thromboxane biosynthesis [9-12].
Among the hydroxycinnamates, caffeic acid retains the structural features that
maximize radical-scavenging activity in flavonoids, which is predictive of higher
rate constants toward several types of oxidant species, as shown in Table 2.1[14]. It
was argued that caffeic acid is the “active site ” of flavonoids for radical scavenging
in particular flavones and flavonols. The o-dihydroxy group is typically the radical
target site, which after one-electron oxidation produces a phenoxyl radical
(o-semiquinone). Accordingly, the caffeic acid-derived o-semiquinone radical has
been observed by electron paramagnetic resonance (EPR) after reaction of the
phenolic acid with several oxidants, including peroxynitrite and ferrylmyogloben [15].
Even so, a recent study on the scavenging activity of caffeic acid derivatives against
1,1-diphenyl-2-picrylhydrazyl (DPPH) radical indicated that a saturated lateral group
in the aromatic ring had a slightly higher inhibitory activity when compared with an
unsaturated group [16].
- 16 -
Oxidant
Product/rate constant
ROO.
O2.OH.
1.5×107 M-1s-1
0.96×106 M-1s-1
3.24×109 M-1s-1
ONOO-
Quinone
a
b
b
5
-1 -1
(for chlorogenic1.6×10 M s )
.
NO2
MbFeIV=O
8.6×10 M s
b
b
MbFeIII
Semiquinone radical
c
8
9
-1 -1
-1 -1
N2O3
HOCl
o-Quinone of chlorogenic
1
5.1×106 M-1s-1
O2
8.6×10 M s
d
e
Table 2.1 Scavenging properties of caffeic acid (or Its Ester Derivative, Chlorogenic Acid)
(refs.14) a Kinetic chemiluminescence in chlorobenzene at 50℃ and peroxyl radical derived from
diphenyl methane, b Pulse radiolysis, c MbFeIV=O, ferrylmyoglobin; MbFeIII, metmyoglobin,
d
Competition spectrophotometric assay, e Time-resolved infrared phosphorescence in acetonitrile.
It was claimed that the higher radical scavenging activities of caffeic acid can be
ascribed to oxidative dimerization or even higher degrees of polymerization through
which oxidizable -OH moieties are reproduced in the “oxidation” products after
electrochemical studies[17 –19]. And it was also assumed that the oxidation of caffeic
acid involves formation of dimer by a coupling reaction of the semiquinone radical
as an intermediate of one-electron oxidation. Although various dimers of caffeic acid
are reportedly formed by chemical autooxidations [8, 9], the electrochemical oxidation
products could not be isolated despite considerable effort [17, 18], because the dimer(s)
was susceptible to further polymerization.
Caffeic acid is water soluble and vulnerable to free radicals, which indicate
- 17 -
possibility for this compound to serve as a quantum dot ligand. But the bounding
between carboxyl group of caffeic acid and quantum dot surface was proved to be
difficult by our preliminary ligand exchange experiments. Thiol compounds were
reported to have the strongest affinity towards CdSe nanoparticles[19] although their
has a major drawback: instability towards oxidation. The surface thiol ligands were
found to undergo a photocatalytic oxidation using CdSe nanocrystals as
photocatalysts and form the disulfides during the process. But severity of this
oxidation varies with different thiol ligands, and some of them can sustain long
enough in solution for practical applications[19]. Another disadvantage of Thiol
ligands is that it can cause severe fluorescence quenching of quantum dots after
ligand exchange process, but ZnS coating of quantum dots provides significant
protection to QDs fluorescence against this quenching[20]. Except few reports on
synthesis of thiol ester, so far has no literature report on thiolation of caffeic acid[21].
If we can synthesize thio-caffeic acid, it might be a good ligand with strong ability
bounding to CdSe/ZnS surface, but whether CdSe/ZnS QDs can be stabilized well
by thio-caffeic acid is yet to be found out.
In addition, through ligand exchange process, we hope to establish a donor-receptor
system between CdSe/ZnS QDs and thiolated caffeic acid. The quantum dots capped
with thio-caffeic acid should be soluble in water or other polar solvent and still
possess acceptable level of fluorescence. As electron transfer quenches or enhances
fluorescence of quantum dot by interfering the efficiency of the CdSe core
- 18 -
electron-hole recombination, this “donor-receptor” caffeic acid-QDs system can
provide a visible signal when it reacts with free radicals or other species and serves
as a fluorescent probe. Our goal is to synthesize and purify thio-caffeic acid for
quantum dot ligand exchange and try to replace the original hydrophobic
trioctylphosphine oxide (TOPO) ligand of quantum dots with thio-caffeic acid.
2.2 Experimental Section
2.2.1 Materials and instruments All solvents used were of reagent grade unless
otherwise specified. The tetrahydrofuran (THF) used in synthesis was dried over Na
and distilled under N2. Caffeic acid, 4-dimercapto-2,3-butanediol (DTT) and
dicyclohexylcarbodiimide (DCC) were purchased from Sigma-Aldrich Chemical
Company (St. Louis, MO, USA).
1
H and
13
C{1H} NMR spectra were recorded in deuterated methanol with a Bruker
AC300 spectrometer (Karlsruhe, Germany) at 300 and 75 MHz, respectively. The
electrospray ionization mass spectra were obtained from a Finnigan / MAT LCQ ion
trap mass spectrometer (San Jose, CA, USA) equipped with an electrospray
ionization (ESI) source. The heated capillary and voltage were maintained at 250 ℃
and 4.5 kV, respectively. The full-scan mass spectra from m/z 100 to 1000 were
recorded. The samples were dissolved in methanol and the solution was introduced
into the ion spray source with a syringe (100 μL).
- 19 -
2.2.2
Synthesis of caffeic acid derivatives
OH
OH
OH
OH
OH
DCC
OH
2
O
O
OH
Caffeic Acid
Fw: 180.16
O
O
Caffeic Acid Anhydride
Fw: 342.30
Scheme 2.1 Synthetic route of caffeic acid anhydride
Caffeic acid (1.000g, 5.5mmol) and dicyclohexylcarbodiimide (DCC) (0.453g, 2.2
mmol) were added into two schlenk flasks which were connected to a schlenk line,
respectively. Freshly distilled tetrahydrofuran (THF) (20mL) was added into the two
flasks to dissolve caffeic acid and DCC, respectively. The DCC solution was added
dropwise into caffeic acid solution under vigorous stirring. The reaction mixture was
kept stirring for 12 hours. The product mixture was later separated by filtration. The
caffeic acid anhydride in filtrate was kept for further use (Scheme 2.1). The
anhydride was directly used in next step of synthesis without characterization, but in
unpurified reaction mixture it could be identified as a peak at 341 in ESI-MS
negative model (Figure 2.2).
The 4-dimethylaminopridine hydrosulfide salt precursor was prepared according
literature reports. Freshly distilled THF (30 ml) was added into the a three-neck flask
to dissolve 4-dimethylaminopridine (DMAP) 0.46 g (3.8 mmol). And the DMAP
- 20 -
solution was cooled in an ice bath. H2S gas was produced by dropping phosphoric
acid on solid NaHS and dried over P2O5. The dried H2S gas was then bubbled
through the cooled solution for 2 hours. The reaction was stopped when a slightly
yellow color appeared in the reaction mixture (Scheme 2.2).
N
N
N
N
H+ HS-
H2S +
Scheme 2.2 Preparation route of 4-dimethylaminopridine hydrosulfide salt
The caffeic acid anhydride THF solution was added into the ice-cooled sulfur
precursor solution and stirred for 3 hours. The temperature of cooled mixture was
naturally increased to room temperature. Finally, a yellow precipitate appeared in
reaction mixture, which can be separated by filtration. The precipitate is kept for
characterization and further purification (Scheme 2.3).
OH
OH
OH
OH
OH
OH
OH
OH
+
M HS
O
O
O
C affeic Acid A nhydride
O
SH
Thio-caffeic A cid
O
OH
C affeic Acid
Scheme 2.3 Synthetic route of thio-caffeic acid salt
- 21 -
8
2.5x10
8
2.0x10
8
1.5x10
PPT_Positive
123
HN
N
Relative Abundance
8
1.0x10
7
5.0x10
0.0
8
1.0x10 100
150
200
8.0x10
7
6.0x10
135 161
150
350
400
195
341 359
200
250
300
Filtrate_Negative
7
1.2x10
6
9.0x10
350
400
385
O
N
OH
C O
6
6.0x10
6
3.0x10
0.0
100
300
179
7
4.0x10
7
2.0x10
0.0
7
1.5x10 100
250
PPT_Negative
7
OH
NH
N-caffeic acid acyl DCC urea
Fw: 386.48
150
200
250
300
350
400
m/z
Figure 2.1 ESI-MS of thio-caffeic acid reaction mixture
In ESI-MS spectra of this precipitate, thio-caffeic acid was corresponded by peak
195 in negative mode and coexist with caffeic acid peak 179 (Figure 2.1).
- 22 -
OH
OH
OH
OH
N
S
N
H+
+
Dissolved in H2O
un-identified
impurities
+
N
600 ml
N
H+
O
pH = 6.1
measured
O
O
Filtration
Precipitated products
OH
OH
N
+
+
+
HCl solution Transparent
yellow solution
pH = 3.2
pH = 6.5
OH
OH
N
Cl H+
O
SH
OH
Caffeic Acid
Fw: 180.16
O
Thio-caffeic Acid
Fw: 196.22
Extraction with Diethyl ether
OH
OH
OH
O
OH
HO
+
+
OH
OH
OH
OH
S
SH
O
O
S
Fw: 390.43
O
Figure 2.2 Separation and purification of thio-caffeic acid
As illustrated in Figure 2.2, the precipitate obtained from last step was added into
600mL deionized water. Sodium hydroxide solution (0.1M) was added into the
mixture to adjust its pH to 6.5. Then the mixture was filtrated to afford a transparent
yellow solution. Hydrochloric acid (0.1 M) was added to adjust solution pH to 3.2.
The solution was then extracted with 500mL diethyl ether. The organic phase turned
yellow color after extraction and it was washed 5 times with deionized water. After
that the organic phase was then collected and rotary evaporated, the yellow colored
solid residue was kept under nitrogen. The extraction process was repeated 3 times.
It was later confirmed that water phase mainly contained caffeic acid and organic
phase contains thio-caffeic acid and impurities (Figure 2.3). And further purification
was able to increase the thio-caffeic acid ratio in the mixture (Figure 2.4).
- 23 -
6
2.0x10
179
6
Water phase
1.6x10
6
1.2x10
5
8.0x10
201
5
4.0x10
0.0
7100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400
2.0x10
Organic phase
7
1.6x10
179
7
1.2x10
161
6
8.0x10
195
135
6
4.0x10
389
341
0.0
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400
m/z
Figure 2.3 ESI-MS (negative ) of thio-caffeic acid extraction
195
7
1.0x10
6
8.0x10
Relative Abundance
375
6
6.0x10
179
6
359
4.0x10
391
161
6
2.0x10
0.0
100
200
300
400
m/z
Figure 2.4 ESI-MS (negative) of purified thio-caffeic acid
- 24 -
2.2.3 Quantum dots ligand exchange with thio-caffeic acid The 100 nM quantum
dots solution in 2mL chloroform and then added into 1mL 100 times equivalent
amount of thio-caffeic acid diethyl ether solution dropwise. This mixture was
vigorously stirred under nitrogen for 12 hours at room temperature in darkness.
When the reaction was stopped, it was found the quantum dots precipitated out from
solution. The quantum dots solid was centrifuged and collected. It was found this
solid immiscible in any polar or non-polar solvent.
2.3 Result and Discussion
The reaction between caffeic acid and dicyclohexylcarbodiimide (DCC) would form
an intermediate after addition of reagents(Scheme 2.4). The caffeic acid-DCC
intermediate is crucial to the following steps of synthesis. This intermediate would
swiftly converted to desired product caffeic acid anhydride as well as another
by-product N-acyl urea (Scheme 2.5). The two products were separated by filtration
and the solid was conformed to be by-product. The anhydride formed from the
intermediate stayed in solution (Scheme 2.6) and would be used in the next step of
synthesis.
- 25 -
OH
OH
OH
OH
N
+
C
N
Fast
O
O
OH
O
H
N C N
Intermediate
Scheme 2.4 Formation of intermediate
OH
OH
O
N
By-product reaction
O
THF
O
OH
C O
OH
NH
H
N C N
N-acyl urea
Fw: 386.48
Intermediate
Scheme 2.5 Formation route of by-product N-acyl urea
OH
OH
OH
OH
OH
OH
+
Desired reaction
C O
NH
+ Caffeic acid
O
NH
O
O
H
N C N
O
O
Caffeic acid anhydride
Intermediate
DCC-urea; precipitate
Fw: 342.30
Fw: 224.34
Scheme 2.6 Synthetic route of caffeic acid anhydride
The instability of caffeic acid was a big problem we encountered during synthesis.
- 26 -
Caffeic acid has a pKa1 = 4.43±0.02 and pKa2 = 8.69±0.03[22]. It was found that
caffeic acid tends to oxidize by oxygen in the atmosphere if environmental pH is
above 7.0. As Figure 2.7 shows, the o-dihydroxy group would be easily oxidized to
o-quinone. Not to mention the caffeic acid anhydride is extremely sensitive to water.
For this reason, the synthetic process of caffeic acid anhydride was carried out under
N2 and freshly distilled and dried THF was chosen as solvent.
HO
O
O
O
pH > 7.0
HO
O
O2
O
O
Scheme 2.7 Oxidation of caffeic acid
The synthesis was started with the formation of hydrosulfide anion donor. The
commercially available sodium sulfide and sodium hydrosulfide were tried out, but
their poor solubility in anhydrous THF was a serious problem in quantitative
synthesis. This problem caused extremely slow reaction rate and low yield of
thio-caffeic acid and also made experiment results not reproducible. Hence,
4-dimethylaminopridine (DMAP) hydrosulfide salt was prepared to serve as an
alternative hydrosulfide anion donor (Scheme 2.2).
The caffeic acid anhydride THF solution was added into the ice-cooled sulfur
precursor solution and the temperature of cooled mixture was slowly increased to
room temperature. A yellow precipitate appeared in reaction mixture and it was
separated by filtration. The filtrate, which contained mainly by-product was
discarded. From the ESI-MS result of the precipitates, 3 main components were
- 27 -
identified: caffeic acid (M.W.=180), thio-caffeic acid (M.W.=196) and unreacted
caffeic acid anhydride (M.W.=342). The precipitate is kept for further purification
(Figure 2.2).
As Scheme 2.4 illustrated, the precipitates obtained from above steps were first
dissolved in water and the pH was adjusted to 6.5 (below caffeic acid pKa2 and
above pKa1). The mixture was filtered and the solid was discarded. Hydrochloric
acid was added into the filtrate to adjust the pH to 3.2. Then the solution was
extracted with diethyl ether. It was later confirmed that water phase mainly contained
caffeic acid and unidentified impurities (Figure 2.3).
The organic phase turned yellow color after extraction and it was washed 5 times
with water. After that the organic phase was then collected and rotary evaporated, the
yellow colored solid residue was kept under nitrogen. The extraction process was
repeated 3 times to afford even purer thio-caffeic acid (before server oxidation took
place), which is analyzed by ESI-MS and results are demonstrated in Figure 2.4.
Caffeic acid and thio-caffeic acid are similar in chemical structure. Hence their
solubility and polarity are also quite similar, which makes separation of pure
thio-caffeic acid a tricky task. Furthermore, the amount of caffeic acid in the mixture
was in largely excess comparing to the amount of thio-caffeic acid which makes
purification even more difficult. The silica gel column chromatography separation
method was taken into consideration, but later it was found that Rf value of the two
- 28 -
compound was too close for this kind of separation. And the vulnerability of
thio-caffeic acid to oxygen also made this method impossible.
Finally, we took advantage of difference in pKa of these two compounds. Caffeic
acid has a pKa1 at 4.35 and its thiolated derivative has a pKa around 3.3. Although
the two values are close, a progressive separation was proved possible. When we
dissolved the mixture containing caffeic acid and thio-caffeic acid into water and
adjusted its pH to 3.2, most of caffeic acids were not able to ionize and stay in
solution in acid form. Thio-caffeic acid at this point was partially ionized and was
safe from oxidation at this pH value. The mixture was first filtered and the filtrate
was extracted with diethyl ether. The organic phase was separated and washed with
water. The caffeic acid, which is more soluble in water (both ionized and unionized
forms), was washed off by water repeatedly to afford a product with higher
thio-caffeic acid ratio.
Initial quantum dots ligand exchange was carried out with the thio-caffeic acid
mixture. The quantum dots were dissolved in chloroform and then added into
thio-caffeic acid diethyl ether solution (100 times equivalent amount) dropwise. This
mixture was vigorously stirred under nitrogen for 12 hours at room temperature.
When the reaction was stopped, it was found the quantum dots precipitated out from
solution and was not soluble in any polar or non-polar solvent.
- 29 -
The ligand exchange step of thio-caffeic acid was not as successful as we expected.
The possible reasons are: The ligand was oxidized or photooxidazied during the
exchange process and formed disulfide dimmer. According to literatures, the
disulfides would form micelle-like structure around the particles and kept them
soluble in the solution at beginning. As the oxidation products diffused out of the
ligand shell instead of surrounding the CdSe core, the quantum dots would gradually
became smaller. At a critical point, the small CdSe inorganic core could not be
supported by micelle-like ligand anymore and the whole system would become
unstable and begin to precipitate out[19].
The thio-caffeic acid was more unstable than the caffeic acid because thio-caffeic
acid has a lower pKa than caffeic acid which is around 3.3. If the environmental pH
value is above 3.3 thio-caffeic acid tends to form disulfide dimmer even without
photooxidation (Scheme 2.8). This dimmer formation could be protected by addition
of 4-dimercapto-2,3-butanediol (DTT), which confirmed its nature of oxidation
(Figure 2.5). With the addition of quantum dots solution, it was highly possible the
oxidation process was photocatalized. And free thio-caffeic acid was converted to
disulfide dimmer instead of bounding to CdSe/ZnS quantum dots surface.
OH
OH
OH
O2
2
OH
OH
HO
pH = 5.5
O
O
SH
O
S S
Fw: 390.43
- 30 -
Scheme 2.8 Oxidation of thio-caffeic acid
6
3.5x10
6
3.0x10
6
2.5x10
OH
OH
OH
HO
6
2.0x10
6
1.5x10
O
O
161
389
S S
Fw : 390.43
6
1.0x10
5
179
5.0x10
0.0
6100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400
3.5x10
6
3.0x10
153
DTT
189
6
2.5x10
OH
OH
6
195Thio-caffeic Acid
2.0x10
6
1.5x10
6
1.0x10
161
O
S
O
O
193
O
5
5.0x10
S
0.0
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400
m/z
Figure 2.5 ESI-MS result of DTT protective effect on thio-caffeic acid; Above: without addition
of DTT; Below: with addition of DTT
The quantum dots ligand exchange with thio-caffeic acid needs further quantitative
studies. It was reported by Peng et al.[19] that the amount of free thiol ligands is an
important factor to the success of thiol ligand exchange. In this report, authors
proposed that excessive thiol ligand could protect QDs by replacing the oxidized
ligands so that they can keep quantum dot soluble in the solution for a longer period
of time. And if there was insufficient free thiols in solution, quantum dot may not
remain soluble through the exchange process. It is possible the amount of
thio-caffeic acid we used in the preliminary studies was not sufficient for
nanocrystals to survive from oxidation. Since precise control of amount of ligand is
the key to make exchange process reproducible, the amount of thio-caffeic acid
- 31 -
requires further study and determination before anymore QDs exchange is conducted.
Another factor that affects oxidation of quantum dot is reaction time. Many thiol
ligand functionalized quantum dot has limited photochemical stability. From the
literature report, the optical density of 3-mercaptopropionic acid functionalized
quantum dots could start to change within 120 minutes[20]. For this reason, the
experimental condition of thio-caffeic acid ligand exchange could be optimized and
deduct the 12 hours reaction time to an acceptable level before serious oxidation of
QDs or ligand occurs.
It was also reported that the diffusion of oxygen was the rate-determining step for
the photooxidation of quantum dots. Because the thiol ligand monolayer could serve
as a barrier for the oxygen diffusion process[20]. Hence the quantum dots stability
was closely related to packing of respective ligand. According to Peng et al.,
aromatic thiols can not stabilize quantum dots as good as aliphatic thiols with a
similar length dimension because they can not pack as densely as the latter and the
diffusion of oxygen is much easier through the loosely packed ligand shell. In this
aspect, thio-caffeic acid may not be a good ligand to stabilize quantum dots in
aqueous or polar solution[19].
However, other studies suggested carbodithiolic acid can be used to functionalized
quantum dots regardless aromatic part in vicinity[20]. If we can modify caffeic acid
with this carbodithiolic structure, maybe caffeic acid derivative could still be used as
- 32 -
a ligand to functionalize quantum dots or extend its use in other fields.
2.4 Conclusion
By bounding antioxidant caffeic acid sulfide analogue to CdSe/ZnS quantum dots
surface, we hope to create hydrophilic quantum dots which are also sensitive to
oxidants like free radicals. The thiolization of caffeic acid, which has never been
reported in literatures, was explored. The synthetic process, including formation of
the caffeic acid-DCC intermediate and caffeic acid anhydrate was studied to provide
information for further separation. The synthesis method was then optimized, which
includes preparation of a new hydrosulfide anion donor. The pH-dependent
thio-caffeic acid oxidation was also investigated by ESI-MS. The vulnerability of
thio-caffeic acid to oxygen made it difficult for separation and further application.
Hence the purification of thio-caffeic acid with water was stopped before serious
oxidation occurred. Preliminary CdSe/ZnS quantum dots ligand exchange with
thio-caffeic acid in chloroform was also carried out. The negative result of ligand
exchanged step suggests the oxidation of thio-caffeic acid must be reduced before
successful ligand exchange with CdSe/ZnS quantum dots. Possible reasons for this
unsuccessful result was proposed and further studies on quantum dot ligand
exchange using thio-caffeic acid is still underway.
- 33 -
References
[1] R. E.Bailey, A. M.Smith, S. Nie, Physica E, 2004, 25, 1-12
[2] S. J. Clarke, C. A. Hollmann, Z. Zhang, D. Suffern, S. E. Bradforth, N. M. Dimitrijevic, W. G.
Minarik, J. L. Nadeau, Nature materials, 2006, 5, 409-417
[3] D. S. Ginger; N. C. Greenham, Physical Review B, 1999, 59, 10622-10629
[4] A. J. Nozik, Physica E, 2002, 14, 115-120
[5] C. Landes, C. Burda, M. Braun, and M. A. El-Sayed, Journal of Physical Chemistry B, 2001,
105, 1981-1986
[6] T. P. Cameron, T. J. Hughes, P. E. Kirby, K. A. Palmer, V. A. Fung, V. C. Dunkel, Mutation
Research/Genetic Toxicology, 1985, 155, 17-25.
[7] H. Hsu, Y. Chen and M. Hong, The Chemical Constituents of Oriental Herbs, 1982, 1, 264
[8] A. Nahrstedt, M. Albrecht, V. Wray, H. G. Gumbinger, M. John, H. Winterhoff, F. H. Kemper,
Planta Medica, 1990, 56,395.
[9] M. Sugiura; et al, Chemical and Pharmaceutical Bulletin, 1989, 37, 1039.
[10] H. Cho, M. Ueda, M. Tamaoka, M. Hamaguchi, K. Aisaka, Y. Kiso, T. Inoue, R. Ogino, T.
Tatsuoka, and et al., Journal of Medicinal Chemistry, 1991, 34, 1503.
[11]T. Okuyama, K. Fujita, S. Shibata, M. Hoson, T. Kawada, M. Masaki, N. Yamate, Planta
Medica, 1986, 52, 171
[12] W. C. Chang and F. L. Hsu, Prostaglandins Leukotrienes and Essential Fatty Acids, 1992, 45,
307.
[13] Y. Koshihara, et al, FEBS Letter, 1982, 143, 13.
[14] L. Packer, E. Cadenas; Handbook of Antioxidants, second edition, Marcel Dekker, New York,
2002
[15]J. Laranjinha; E. Cadenas; IUBMB life, 1999, 48, 1-9
[16]CP. Chen,; et al, Experimental and Toxicologic Pathology, 1999,51,59-63
[17]Y. S. Uang; et al., J. Chromatogr. B ,1995, 673, 43-49
[18] R. Arakawa, M. Yamaguchi, H. Hotta, T. Osakai, T. Kimoto, Journal of the American Society
for Mass Spectrometry, 2004, 15, 1228–1236
[19] J. Aldana, Y. A. Wang, and X. Peng, Journal of the American Society,2001,123,8844
[20] J. A. Kloepfer, S. E. Bradforth, and J. L. Nadeau, Journal of Physical Chemistry B, 2005,109,
9996-10003
[21] A. R. Kroon, W.D. Hoff, H. P.M. Fennema, J. Gijzen, G. Koomen, J. W. Verhoeven, W.
Crielaard, and K. J. Hellingwerf, The Journal of Biological Chemistry, 1996, 271,
31949-31956
[22] IUPAC, Ionization Constants of Organic Acids in Aqueous Solutions, Pergamon Press,
Oxford, 1979.
- 34 -
Chapter 3
Trolox Functionalized Quantum Dots
3.1 Introduction
3.1.1 Quantum dots and its synthesis Quantum dots (QDs) are spherical
semiconductor nanoparticles that composed of atoms from groups II-VI or III-V
elements in the periodic table, such as CdSe, ZnTe, GaAs and InSb. With quantum
confinement effect, QDs present considerable advantages over traditional organic
dyes. Their unique optical and electronic properties, including broad excitation
spectra with high molar absorptivities, size-tunable emission properties, narrow and
symmetric emission and comparatively bright and photostable properties[1].
For routine preparation, the CdSe core is usually capped with an organic layer such
as trioctylphosphine/trioctylphosphine oxide (TOP/TOPO). This layer coordinates to
Cd sites and stabilizes QD’s surface, preventing aggregation of the nanocrystals[2].
Since the ligands are hydrophobic, they make the QDs incompatible with aqueous
solution and did not appear to have an immediate application for analytical system.
Two breakthrough papers by Nie’s and Alivisatos’ groups in 1998 demonstrated the
fluorescent QDs can be made water-soluble and biocompatible by surface
modification and bioconjugation while maintaining their large fluorescent quantum
yield[3,4]. Hence, by tailoring QDs with different capping ligands, they can serve for
- 35 -
different purposes. In the following years, there are two approaches to surface
modification to obtain highly fluorescent and water-soluble QDs. Firstly, the
formation of a layer of another semiconductor of higher band gap on the QDs
surface such as ZnS, giving rise to a bi- or three-layered structure QD resulting in
highly fluorescent QDs[5]. Another approach is to use competing capping agent such
as mercaptosulphonic acid or dihydrolipoic acid to displace TOP/TOPO from the
QDs surface and endow it solubility in polar solvents[6, 7].
3.1.2 Quantum dots ligand exchange strategies The nature of the ligands being
coordinated to the QDs surface and the particular type of bonds which it forms with
the nanocrystal surface atoms are of great importance to quantum dots researchers.
By ligand designing and fabrication, several important properties of quantum dots
can be tuned for specific purpose: processibility, reactivity and stability. All of these
have direct consequences on quantum dots’ spectroscopic properties[8]. Three
principal functions of the surface ligands can be described by Querner et al. as
follows: (1) They prevent individual colloidal nanocrystals from aggregation.
(2)They facilitate nanocrystals’ dispersion in a large variety of solvents. In the
presence of surface ligands, the ability to disperse nanocrystals is governed by the
difference between the ligand and the solvent solubility parameters, which can be
precisely tuned. (3) Ligands containing appropriate functional groups may serve as
bridging units for the coupling of molecules or macromolecules to nanocrystals or
their grafting on substrates[8].
- 36 -
So far we have reported quantum dot ligand exchange with thio-caffeic acid. The
ligand exchange step of thio-caffeic acid was not as successful as we expected
because the thiol ligand was oxidized or photooxidazied during the exchange process
and formed disulfide dimmer. According to Peng et al., the disulfides would form
micelle-like structure around the particles and kept them soluble in the solution at
beginning. As the oxidation products diffused out of the ligand shell instead of
surrounding the CdSe core, the quantum dots would gradually became smaller. At a
critical point, the small CdSe inorganic core could not be supported by micelle-like
ligand anymore and the whole system would become unstable and begin to
precipitate out[9].
Since thiols suffer from their major drawback, i.e. instability towards oxidation.
Their alternatives was soon synthesized and applied to quantum dots ligand
exchange. Alkyl or aryl derivatives of carbodithiolic acids (R-C(S)SH) were reported
by Querner et al. in their publication. This family of ligands for CdSe nanoparticle
surface functionalization has two main advantages comparing to thiols: they nearly
quantitatively exchange the initial surface ligands (TOPO) in very mild conditions;
they significantly improve the resistance of nanocrystals against photooxidation
because of their ability of strong chelate-type binding to metal atoms[8]. However,
one problem of this chelating-type ligands exchange method remains: a specific and
adapted synthesis is required each time new ligands are needed.
An improved method was proposed by Zhao et al.[10], by using dithiocarbamate
- 37 -
compounds they successfully assembled secondary amine containing ligands on Au
surface. Recently this method was adapted by Dubois et al.[11], and it was used on
CdSe/ZnS quantum dots for ligand exchange. Depending on the intrinsic nature of
substituents borne by the amino group, different amine ligands were chelated to QDs
surface and different properties were conferred to nanocrystals, such as solubility in
different solvents. With these recent developments of quantum dots surface
modification techniques, it would be possible for rational design and tailoring
quantum dots’ ligand for specific purpose and application, such as biological sample
labeling and radical sensing.
The α-tocopherol (α-TOH), as a principal scavenger of peroxyl radicals in biological
membranes, is probably the most important inhibitor of the free-radical chain
reaction of lipid peroxidation in animal. Antioxidant properties of α-TOH appear to
play a critical role in preventing oxidative injury by toxic and carcinogenic
chemicals[12]. Research shows the lipid antioxidant activity of α-TOH comes from its
preferential localization within the lipid, its high chemical reactivity toward radicals
and the stability of the phenoxyl radical intermediate (α-TO.) generated[13].
Tocoherols inhibit lipid peroxidation largely because they scavenge lipid peroxyl
(LO·2) radicals much faster than these radicals can react with adjacent fatty acid
side-chains or with membrane proteins. Rate constants for the following reaction
α-TOH + LO·
α-TO· + LO2H
are about 106M-1s-1, which is four orders of magnitude faster than those for reaction
- 38 -
of LO·2 radicals with lipids(~102M-1s-1)
[13]
. In addition, tocopherols both quench
and react with singlet O2 and might protect membranes against this species.
-
α-tocopherol reacts slowly with O·2 and, like most other biological molecules, at an
almost diffusion-controlled rate with OH·. The α-TO· radical might also react with a
further peroxyl radical to give non-radical products [14].
α-TO· + LO2
·
α-TOOOL
That means theoretically one molecule of α-tocopherol is capable of terminating two
peroxidation chains (Scheme 3.1). Products of the above reaction include eight
α-substituted tocopherones and epoxy (hydroperoxy) tocopherones: the former
would readily hydrolyze to tocopherylquinone and the latter to epoxyquinones[14].
Scheme 3.1 Peroxyl radical scavenging by α-TOH and α-TO· (refs 15)
It has been shown that α-TO. radical reacts with peroxyl radical to give a peroxo
adduct at the para 8a-position. It may react with another α-TO. radical to give a
dimer. This α-TO. radical may be reduced by a reductant (e.g. ascorbic acid, uric acid,
ubiquinol and polyphenolic compounds) to regenerate α-TOH. In some cases, this
radical may react with the substrate and/or hydroperoxide to give active radicals,
- 39 -
(which may start a chain reaction) and acts as a chain-transfer agent[12].
The rate constants of α-TO. were obtained by E. Niki et al by a stopped-flow ESR
method [12] (together with the biological concentrations of the substrates) are showed
blow:
Table 3.1 The rate constants of α-tocopheryl radical (obtained by a stopped-flow ESR method)
and the biological concentrations of the substrates(refs 12)
From Table 3.1, it is clear that α-TO. is sensitive to several biologically important
·
species but reacts most efficiently with LOO radical.
3.1.3 Electron transfer based sensor By bonding α-tocopherol analogue to
quantum dots surface as ligand, we have reasons to expect a photoinduced electron
transfer (PET) between this ligand and quantum dots excited state, which cause
intramolecular emission quenching. This kind of quenching have already been
observed by Oleynik et al. when they coupled a α-tocopherol analogue--Trolox
(5,7,8-tetramethylchroman-2-carboxylic Acid) to a dipyrrometheneboron difluoride
(BODIPY) fluorescent probe[15]. And trough radical-mediated oxidation of the
- 40 -
chromanol head (Trolox part), the Trolox-BODIPY complex significantly restored
fluorescent emission by10-fold. The same kind of “off-and-on” phenomenon was
also observed by Maurel et al.
[16]
. Their research showed the QDs capped by
4-amino-TEMPO (QD-4AT) which was initially quenched by the nitroxide in a
QD-4AT complex and the fluorescent emission was readily restored when nitroxide
moiety react with carbon-centered free radicals to form alkoxyamines.
By synthesizing Trolox-QDs complex, which has never been reported before, we
hope to establish a “off-and-on” system for sensitive peroxyl radical sensing. But a
remaining problem is that Trolox’s carboxyl group can not directly bond to
CdSe/ZnS quantum dots surface as it coupled to BODIPY. By modifying this
carboxyl group to a amide or amino group, we can easily adopt the method
developed by Dubois and coworkers, and take advantage of strong chelating ability
of carbodithioate group after CS2 addition[11]. And finally make a strong bonding of
Trolox on QDs’ surface. The ligand exchanged quantum dots would be applied for
peroxyl radical sensing.
3. 2 Experimental Section
3.2.1 Materials All solvent used were of reagent grade unless otherwise specified.
TOPO CdSe/ZnS quantum dots and Tren-CdSe/ZnS QDs were synthesized by Dr.
Wang
Shuhua
in
our
research
((±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic
group.
acid)
Trolox
and
- 41 -
2,2′-Azobis(2-methylpropionamidine) dihydrochloride (AAPH) were purchased
from
Sigma-Aldrich
Chemical
2,2'-azobis(2,4-dimethylvaleronitrile)
Company
(St.
Louis,
MO,
USA).
(AMVN) was purchased from cayman
Cayman Chemical Company(Ann Arbor, MI, USA). Hydrochloric acid and sodium
bicarbonate anhydrous were purchased from Fisher Scientific (Fair lawn, NJ, USA).
Ethylenediamine were purchased from J.T. Baker (Phillipsburg, NJ, USA). Carbon
Disulfide were purchase from BDH limited (Poole England).
1
H and
13
C{1H} NMR spectra were recorded in deuterated methanol with a Bruker
AC300 spectrometer (Karlsruhe, Germany) at 300 and 75 MHz, respectively.
The
electrospray ionization mass spectra were obtained from a Finnigan / MAT LCQ ion
trap mass spectrometer (San Jose, CA, USA) equipped with an electrospray ionization
(ESI) source. The heated capillary and voltage were maintained at 250 ℃ and 4.5 kV,
respectively. The full-scan mass spectra from m/z 50 to 1000 were recorded. The
samples were dissolved in methanol and the solution was introduced into the ion
spray source with a syringe (100 μL). UV-Vis spectra were recorded using a
Shimadzu UK1601 spectrophotometer fitted with a quartz cell and Bio-TEK Synergy
HT Plate Reader (Winnoski, Vermont, USA). Fluorescence measurements were
performed using a Perkin–Elmer LS55 luminescence spectrometer with an excitation
wavelength of 400 nm. FT-IR spectroscopy was taken from Perkin–Elmer Spectrum
one-B spectrometer.
- 42 -
3.2.2 Experimental Procedures
Synthesis of 6-hydroxy-2,5,7,8-tetramathyl-chroman-2-carboxylic acid methyl
ester (Trolox methyl ester)
HO
O
MeOH
OH
O
HO
O
Room Temp.
OCH3
O
Scheme 3.2 Synthetic route of trolox methyl ester
Trolox (250mg) was weighed out and dissolved in MeOH (5mL). Five microliters of
3.3% (v/v) HCl/MeOH solution was added into this solution and stirred at room
temperature for 12 hours (Scheme 3.2). When the reaction was stopped, solvent was
removed by rotary evaporation to give white colored solids[17]. After separation by
silica gel column with chloroform as mobile phase, 0.2351g white colored solid was
obtained with a yield of 89.05%, rf value 0.35, and M.W.=264 (Figure3.1, Figure
3.2)
The 1H-NMR results are: (δ)1.6 (s, 3H); 1.82–1.89 (m, 2H); 2.06, 2.15, 2.19 (3s,
9H); 2.39–2.61 (m, 2H); 3.67 (s, 3H). As Figure 3.7 shows, These result are
consistent with literature report by Palozza et al[17].
The 13C-NMR was also carried
out in CDCl3 and the results are: (δ)174.5, 145.5, 145.3, 122.5, 121.3, 118.4, 116.8,
52.3, 30.6, 25.3, 20.9, 12.1, 11.8, 11.2 (Figure 3.3, Figure 3.4).
- 43 -
troloxester #81 RT: 1.66 AV: 1 SB: 7 1.03-1.14 NL: 1.05E6
T: - c ESI Full ms [50.00-1000.00]
263.2
100
95
90
85
80
75
70
Relative Abundance
65
60
55
50
45
40
35
30
163.4
25
20
15
264.2
10
5
97.0
159.3
143.4
187.2
261.8
297.1
318.9 361.9
424.9
491.8 515.9
645.9 680.1
582.8
710.5
960.4
851.9 878.4 938.1
990.3
784.9
0
100
200
300
400
500
600
700
800
900
1000
m/z
Figure 3.1 ESI-MS (negative mode) spectrum of Trolox methyl ester
troloxester #91 RT: 1.88 AV: 1 SB: 16 1.35-1.66 NL: 4.53E7
T: + c ESI Full ms [50.00-1000.00]
282.0
100
95
90
85
80
75
265.0
70
Relative Abundance
65
60
55
50
45
40
35
30
205.2
550.9
25
20
287.1
15
10
5
92.9
196.1 206.1
165.2
545.7 551.9
315.1
364.6
416.0
429.4 481.7 526.1
588.1 630.9
687.2
742.3
809.1 852.8 906.0 934.2
992.6
0
100
200
300
400
500
600
700
800
900
1000
m/z
Figure 3.2 ESI-MS (positive mode) spectrum of Trolox methyl ester
- 44 -
7.8
7.6
7.4
7.2
7.0
6.8
6.6
6.4 6.2
6.0
5.8
5.6
5.4
5.2
5.0
4.8
4.6
4.4
4.2
4.0
3.8
3.6 3.4
3.2
3.0
2.8
2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6 0.4
0.2
(ppm)
Figure 3.3 1H-NMR spectrum of Trolox methyl ester (CDCl3 )
195 190 185 180 175 170 165 160 155 150 145 140 135 130 125 120 115 110 105 100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
(ppm)
Figure 3.4 13C-NMR spectrum of Trolox methyl ester (CDCl3)
- 45 -
Synthesis of N-(3,4-Dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-carbonyl) ethylenediamine (Trolox diamine)
HO
O
NH2CH2CH2NH2
OCH3
O
HO
O
110oC, 4Hours
NHCH2CH2NH2
O
Scheme 3.3 Synthesis of Trolox ethylenediamine
Trolox methyl ester (500 mg, 1.9 mmol) and 5 mL of the ethylenediamine was
heated at 110 °C and vigorously stirred for 4 h (Scheme 3.3). The reaction mixture
was diluted with CHCl3 (5mL) and washed with 10 mL of saturated aqueous
NaHCO3. The organic layer was dried with Na2SO4, and the solvent was evaporated
in vacuo
[18]
. The remaining yellow colored solids was purified by silica gel
chromatography using chloroform: MeOH (v/v=10:1) as mobile phase,
and white
colored solids of Trolox diamine was obtained with a yield of 76%, rf value 0.5, and
M. W. 292 (Figure 3.5).
- 46 -
troloxamine #26 RT: 0.42 AV: 1 SB: 5 0.02-0.09 NL: 5.15E8
T: + c ESI Full ms [50.00-1000.00]
293.12
100
90
Relative Abundance
80
70
60
50
40
30
584.94
20
10
102.85 137.89
0
50
100
150
191.98 239.65
200
276.19
250
331.18 359.29
300
350
471.00
411.64
400
450
556.87
606.96
662.93 704.87
499.00
500
550
600
650
700
778.02 817.02 838.76 876.36
750
800
850
900
936.84 978.36
950
1000
m/z
troloxamine #55 RT: 0.99 AV: 1 SB: 5 0.56-0.64 NL: 2.23E6
T: - c ESI Full ms [50.00-1000.00]
291.4
100
90
Relative Abundance
80
70
60
50
40
30
292.4
20
163.4
201.5 227.0
95.1 129.1 162.7
10
0
50
100
150
200
290.7
250
337.2 369.1 389.2 435.2
300
350
400
450
491.2
583.1
523.3 581.4 584.1
603.2
538.4
500
550
600
671.3 693.3
650
700
827.3 872.4 894.0
733.6 751.9
750
800
850
900
956.4
950
997.3
1000
m/z
Figure 3.5 ESI-MS spectrum of Trolox diamine (up: positive mode; down: negative mode)
The 1H-NMR results are: (δ)1.52 (s, 3H, CH3-C2), 1.91-1.82 (m, 1H, ArCH2CH2),
2.07, 2.16, 2.18(3s, 9H), 2.40-2.32 (m, 1H), 2.74-2.55 (m,4H), 3.37-3.13 (m, 2H),
6.77 (bs, 1H). Most apparently, the disappeared peak at 3.67 corresponding to methyl
ester clearly indicates the success of conversion of methyl ester group to amine
group(Figure 3.6). The
13
C-NMR results are: (δ)174.72, 145.72, 144.31, 122.04,
121.81, 119.57, 117.90, 41.82, 41.35, 29.60, 24.53, 20.56, 12.32, 11.92,
11.38( Figure 3.7). These results are consistent with literature report by Koufaki et
al[18].
- 47 -
7.8
7.6
7.4
7.2
7.0
6.8
6.6
6.4
6.2
6.0
5.8
5.6
5.4
5.2
5.0
4.8
4.6
4.4
4.2
4.0
3.8
3.6
3.4
3.2
3.0
2.8
2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
(ppm)
Figure 3.6 H-NMR spectrum of Trolox diamine (CDCl3)
195 190 185 180 175 170 165 160 155 150 145 140 135 130 125 120 115 110 105 100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
(ppm)
Figure 3.7 13C-NMR spectrum of Trolox diamine (CDCl3)
- 48 -
Trolox methyl ester react with AMVN and AAPH Trolox methyl ester was
dissolved in methanol to afford a sample solution (4.55×10-3M) for kinetic reaction.
-3
2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN) methanol solution (40.25×10 M)
and 2,2′-Azobis(2-methylpropionamidine) dihydrochloride (AAPH) methanol
solution (36.87×10-3M) was prepared as free radical initiator.
Synergy HT Plate Reader was to monitor absorbance change of sample solution.
Each 300μL sample solution was transferred into 96 well plate followed by addition
of 20μL free radical initiator solution. In each case, 300uL methanol was used as
blank solution to deduct absorbance of the solvent. The machine internal temperature
was adjusted to 37℃. A programmed shaking was used before each measurement.
The data points were collected each 5 minutes and the entire kinetic test lasted 120
minutes.
Quantum dot ligand exchange with Trolox diamine CdSe/ZnS quantum dots
(10mL) was precipitated by adding 2mL of CHCl3 and 10mL of MeOH. The
precipitates were reconstituted in 2 mL of toluene for the step of synthesis. The
concentration of the solution was determined by Peng’s method [19].
The QDs in toluene solution (54.62×10-6 M, 2 mL , 109.23×10-9 moles) were
precipitated out by adding MeOH (5 mL) and centrifugation at 4000rpm for 10
minutes. The supernatant was discarded and the QDs are redissolved in CHCl3 (1
- 49 -
mL). Trolox diamine in largely excess (100 mg, 0.4 mmol) was added, followed by
an equal molar quantity of carbon disulfide chloroform solution (1:4 v/v, 0.1mL).
The solution was stirred for 12 hours at room temperature in darkness under nitrogen
(Scheme 3.4).
HO
O
HO
NHCH2CH2NH2
O
+
O
S
CS2
R.T, 12 hours
NHCH2CH2NH
O
S
Scheme 3.4 Quantum dots ligand exchange with Trolox diamine
Five milliliter of methanol was added into 1mL of reaction mixture and it was
thoroughly mixed by vortexing, followed by centrifugation at 4000 rpm for ten
minutes in order to precipitate out ligand exchanged quantum dots. The precipitates
were reconstituted in 3mL of acetone. The reconstituted mixture was centrifuged at
3000 rpm for 5 minutes. The precipitates of this step were discarded and only the
homogeneous supernatant was collected or diluted for further studies. The absorption
and emission spectra of this ligand exchanged quantum dots were recorded. The size
and extinction coefficient (ε) of QDs were determined by using equation [1] and [2]
respectively[19].
D = (1.6122 x 10-9 )λ 4 -(2.6575 x 10-6 )λ 3 +(1.6242 x 10-3 )λ 2 -(0.4277)λ+41.57 [1]
where D (nm) is the size of CdSe QDs sample and λ (nm) is the wavelength of the
- 50 -
first excitonic absorption peak.
ε = 5857(D) 2.65
[2]
The concentration of the QDs sample was then calculated using Lambert-Beer’s Law
[3]:
A=εCL
[3]
where A is the absorbance at the peak position of the first excitonic absorption peak
of QDs sample, C is the molar concentration (M) of QDs sample and L is the path
length (cm) of the radiation beam used for the recording of the absorption spectrum,
and path length was set at 1 cm in this study. The sizes were determined to be 3.19
nm after the ligand exchange.
Trolox functionalized quantum dots Sensing of Peroxyl Radicals The stock
solution of AMVN and AAPH were prepared in Methanol. The concentration of
these two free radical initiators were 40.15×10-6 M and 36.15×10-6 M respectively.
The concentration of the ligand exchanged quantum dots was determined by
equation [1], [2] and [3] and was diluted to be 235×10-9 M.
Synergy HT plate reader was used in sensing peroxyl radicals with excitation
wavelength at 360±40nm and emission wavelength at 540±25nm. Each type of
- 51 -
working solutions was injected into 3 plate wells repetitively for accuracy purposes.
Besides, the total amount of working solutions in each plate well is kept the same to
avoid any deviation came from the volume differences. The general procedure was:
for each well, 200μL Trolox-QDs were added followed by 20μL of free radical
initiator solutions. In each case 200μL methanol was sued as blank solution and 200
μL Trolox-QDs with 20μL methanol was used as control.
3.3 Results and Discussion
3.3.1 Synthesis and characterization of Trolox derivatives The white solid
obtained from Trolox esterification has a 89% yield. The product obtained from
purification was sent for ESI-MS, 1H-NMR and
13
C-NMR essay to confirm its
structure. The molecular weight of the compound is determined by ESI-MS
spectroscopy as 263 from the anionic mode MS (Figure 3.1) and 265 from the
cationic mode (Figure 3.2). The major peak in Figure 3.6 is at m/z 265
corresponding to the unionized molecule (C15H20O4, requires 264.3).
The yellow color Trolox diamine solid obtained from synthetic reaction has a 76%
yield. The white solid obtained from purification was dried in vaccum and sent for
ESI-MS, 1H-NMR and
13
C-NMR study to confirm its structure. As Figure 3.5
demonstrates, the molecular weight of the compound is determined from ESI-MS
spectroscopy as 291 from the anionic mode MS, corresponding to the loss of one
proton; and 263 from the cationic mode, corresponding to the obtaining of one
proton (C16H24N2O3, requires 292.18).
UV-Visible spectrum is a crucial character of Trolox diamine-QDs, therefore the
UV-Vis and fluorescence spectra of the Trolox ligand were studied. The major
- 52 -
absorbance peaks of both Trolox methyl ester and diamine are at 290nm whereas the
emission maximum is located at 400 nm (data now shown). Since the CdSe/ZnS
quantum dots are normally exited at 400nm and emission signal collected at around
550nm, there would be no interference when we study the Trolox diamine-QDs in
the same way.
The Fourier Transform Infrared spectroscopy (FT-IR) was used to extract more
information of the quantum dot ligand exchange reaction. The FT-IR spectrum of
Trolox methyl ester and Trolox diamine was obtained before further functionalized
Normalized Transmittance
quantum dots characterization.
Trolox methyl ester
Trolox diamine
4000
3500
3000
2500
2000
1500
1000
500
-1
cm
Figure 3.8 FT-IR spectra of Trolox methyl ester and Trolox diamine
- 53 -
In above Figure 3.8, the primary amino Trolox diamine has two absorption bands
situated at 3418 cm-1 and 3341 cm-1 because of the symmetrical and asymmetrical
stretching vibration of NH2, which clearly indicates the formation of amino group.
Moreover, The C=O stretching band at 1737 cm-1 of Trolox methyl ester (Figure 3.8
redline above) was shifted to 1667cm-1 in the spectrum of diamine (Figure 3.8 black
line below), and this could be the proof of formation of amide functional group.
Figure 3.9 shows a plot of the absorbance versus concentration of Trolox methyl
ester. By establishing quantitative relation between Trolox ester concentration in
methanol and its UV-Vis absorbance, we hope to understand the kinetic effect of
peroxyl radical on Trolox analogue concentration.
1
y = 0.1283x - 0.0448
2
R = 0.9944
A b s or b an c e
0.8
0.6
0.4
0.2
0
0
1
2
3
4
5
-0.2
Trolox methyl ester conc.(mM)
Figure 3.9 Trolox methyl ester standard curve in methanol. Absorbance was recorded at 290nm
2,2′-Azobis(2-methylpropionamidine)
dihydrochloride
(AMVN)
and
- 54 -
2,2′-Azobis(2-methylpropionamidine) dihydrochloride (AAPH) was chosen to be the
initiator of peroxyl radicals. The kinetic UV-Vis absorbance of mixture containing
-3
-3
-3
4.52×10 M Trolox methyl ester and 3.65×10 M AMVN or 3.35×10 M AAPH
was measured at 290nm for 120 minutes(reaction temperature
37℃). Figure 3.10
shows that the drop of absorbance occurred in both solutions containing AMVN and
AAPH. This result demonstrates that AMVN reacts much faster with Trolox methyl
ester than AAPH, and it made mixture absorbance decreased 13.9% whereas the one
containing AAPH decreased only 6.4%. This result also indicates that both AMVN
and AAPH could be used as effective and efficient free radical initiator to oxidize
Trolox analogue within 120 minutes. And in future studies they would be used to
challenge Trolox functionalized quantum dots.
1
0.98
Normalized Absorbance
0.96
0.94
0.92
0.9
0.88
0.86
0.84
0
20
40
60
80
100
120
140
Time (mins)
Figure 3.10 The reaction kinetic of AMVN(3.65 mM) and AAPH (3.35 mM) on Trolox methanol
solution (4.52 mM) absorbance in 120 minutes at room temperature.
- 55 -
To determine the nature of the reaction between Trolox methyl ester and peroxyl
radical initiators, the mixture of Trolox methyl ester and AAPH was diluted 10 times
and transferred into a round bottom flask. The mixture was stirred vigorously for 24
hours and sent for ESI-MS examination. According to MS spectrum result (Figure
3.11), the oxidation reaction completed in 24 hours and Trolox methyl ester was
converted to more complicated oxidized products which we have not yet indentified.
XJ1 #40-61 RT: 1.15-1.76 AV: 22 SB: 6 0.89-1.03 NL: 1.85E6
T: - c ESI Full ms [50.00-2000.00]
783.5
100
747.5
95
90
85
80
75
70
Relative Abundance
65
60
55
784.5
50
472.5
45
40
35
30
819.2
25
20
508.4
829.3
15
10
376.5
163.3
427.5
738.6
570.3 702.6
534.4
244.7
5
1085.3
831.3
847.2
955.3
152.2
1101.7
1330.6
0
200
400
600
800
1000
m/z
1200
1400
1512.8
1647.9 1718.2 1838.9 1935.0
1600
1800
2000
Figure 3.11 ESI-MS (negative) spectrum of reaction mixture of Trolox methyl ester and AAPH
3.3.2 Functionalization of quantum dots with Trolox derivatives As Dubois et al
reported[11], dithiocarbamate moiety formed by amine and CS2 can provide strong
coordination to CdSe/ZnS surface. The bidentate chelating of amine-CS2 ligand
- 56 -
displayed high affinity for metal atoms, hence they can readily increase stability of
nanocrystals. And at the same time, this ligand exchange process can confer
properties of the ligands to the nanoparticle, such as solubility in different solvents
or affinity to other specific compounds. The hydrophilic Trolox diamine ligand was
proved to be capable of significant changing QDs solubility. Depending on
ligand/QDs ratio and the size of original TOPO quantum dots, functionalized QDs
could be precipitated out from reaction mixture by diethyl ether and reconstituted in
various solvent like acetone, chloroform, methanol and PBS buffer (Figure 3.12).
HO
OH
O
O
O
HN
HN
O
NH
-
S
S
NH
-
S
S
CdSe
S
HN
NH
O
S
ZnS
S
-
-
S
N
H
H
N
O
O
OH
O
HO
Figure 3.12 Proposed structure of Trolox functionalized Quantum Dots
- 57 -
The fluorescence emission spectrum and absorption spectrum of Trolox
functionalized QDs dissolved in acetone are shown in Figure 3.13. From this Figure,
the absorption maximum is located at 557 nm whereas the emission maximum is
located at 596.5nm. Both absorption and emission maximum has a about 3nm red
shift comparing to TOPO QDs before ligand exchange process.
Figure 3.13 Absorption spectrum and fluorescence emission spectra of TOPO QDs (black line)
and Trolox functionalized QDs (red line)
On the other hand, the concentration of Trolox-QDs was calculated by using
equations [1], [2] and [3] following Peng’s method[19]. From the absorption spectrum
(Figure 3.17), the absorbance value at first excitonic absorption peak (λ = 557nm) is
0.989. Therefore
- 58 -
D=(1.6122×10-9)λ4-(2.6575×10-9) λ3+(1.6242×10-3) λ2-(0.4277λ)+41.57
D=3.19
Extinction Coefficient, ε=5857(D)2.65
= 126683.64
By Lambert Beer’s law, A=εCL
C=7.81×10-6 M
The similar calculation steps were performed for obtaining the concentration of other
solution of TOPO QDs or Trolox-QDs.
To determine the fate of the original phosphine ligands upon exposure to
dithiocarbamate formed by Trolox diamine and carbon disulfide, we carried out 31P
NMR measurements and the results shown in Figure 3.14.
- 59 -
1.0000
Integral
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
-5
25
20
15
10
5
0
-5
(ppm)
85
80
75
70
65
60
55
50
0.5887
1.4876
1.0088
1.0000
Integral
TOPO
45
40
35
30
(ppm)
Figure 3.14 31P NMR spectra of TOPO-QDs (above) and Trolox functionalized QDs (below)
The starting TOPO QDs exhibit only one peak at 55 ppm. After 12 hours of ligand
exchange process, new peaks emerge in the reaction mixture although no new
phosphine compound was introduced into this reaction. The appearance of the new
peak situated at 48.7 ppm corresponds to free TOPO that released from the QDs
surface. This phenomenon is in agreement with quantum dots ligand exchange study
reported by Dubois [11]. Furthermore, other two new peaks at 54.2 ppm and 39.9ppm
respectively suggest there might have chemical reaction between TOPO and Trolox
- 60 -
Normalized Transmittance
diamine or CS2 and formed unidentified phosphine product in the system.
Trolox-QDs
1514 cm-1
1651 cm-1
TOPO QDs
4000
3500
3000
2500
2000
1500
1000
500
-1
cm
Figure 3.15 FT-IR spectrum of Trolox functionalized QDs (Red line above) and TOPO QDs
(Black line below)
The FT-IR spectrum of Trolox functionalized quantum dots is significantly different
from the spectrum of ligand unchanged TOPO quantum dots(Figure 3.15). The peaks
at 2928 cm-1 and 2860 cm-1, which originate from TOPO ligand’s C-H stretching
vibration, are the most characteristic feature of TOPO quantum dots. But these two
peak drastically diminished in Trolox-QDs’ spectrum. Instead, the two strong peaks
featuring C=O stretching vibration of amide group of Trolox diamine ligand appear
at 1651 cm-1 and 1514 cm-1 which proved the successful bonding of Trolox diamine
- 61 -
ligand and replacing of the original TOPO ligand.
3.3.3 Trolox-QDs sensing of peroxyl radicals The preliminary study of peroxyl
radicals’ effect on Trolox functionalized quantum dots was performed using AMVN
and AAPH as free radical initiators. Fluorescence response of
0.235×10-6 M
Trolox-QDs acetone solution to peroxyl radical at concentration of 3.65×10-6 M
(AMVN) and 3.35×10-6 M (AAPH) respectively are shown in Figure 3.16. It shows
that the fluorescence intensity of Trolox-QDs was significantly quenched by peroxyl
radical generated by AAPH at 37℃ in 120 minutes ( i.e. 66.7%) whereas the
fluorescence was quenched moderately by AMVN ( i.e. 48.2%). Therefore, the
sensing result demonstrates that Trolox-QDs system has highly sensitivity towards
peroxyl radical.
- 62 -
1.200
Normalized Fluorescence Intensity
1.000
0.800
0.600
0.400
0.200
0.000
Control
AAPH
AMVN
Figure 3.16 Effect of AAPH and AMVN solution on the fluorescence of Trolox-QDs.
Concentration of AAPH is 3.35×10-6 M and concentration of AMVN is 3.65×10-6 M. (The
excitation and emission wavelength are 360 nm and 540 nm respectively.)
Tris(2-aminoethyl)amine, which do not possess antioxidant ability, functionalized
QD (Tren-QDs) was used for comparison. The Tren-QDs powder was dissolved in
water to afford a 0.161×10-6 M Tren-QD water solution followed by addition of
3.35×10-6 M AAPH. After 120 minutes reaction at 37 ℃ , the fluorescence intensity
of the mixture was measured by HT platereader. As Figure 3.17 shows, peroxyl
radical generated by AAPH did not have profound effect on the fluorescence of
Tren-QDs (i.e.11.2%) whereas it significantly quenched antioxidant functionalized
Trolox-QDs (i.e. 66.7%) as we previously showed. Therefore, the sensing result
demonstrates that Trolox-QDs system has highly sensitivity towards peroxyl radical.
- 63 -
1.200
Normalized Fluorescence Intensity
1.000
0.800
0.600
0.400
0.200
0.000
Control
Trolox QD
Tren QD
Figure 3.17 Effect of AAPH solution on the fluorescence of Tren-QDs and Trolox-QDs.
Concentration of AAPH is 3.35×10-6 M. (The excitation and emission wavelength are 360 nm
and 540 nm respectively.)
We speculate this sensitivity to peroxyl radical related intramolecular emission
quenching could be attributed to photoinduced electron transfer (PET) from quantum
dots to the chromanol moiety of Trolox ligand. It is possible that peroxyl radicals
oxidized Trolox ligand on QDs surface and produce Trolox phenoxyl radical
intermediate (α-TO.), electron transfer from the conduction band of quantum dots to
the α-TO., which is also a mild electron acceptor, and transfer back to QDs’ valence
band to interfere the fluorescence excitation-relaxation process of QDs. In another
words, the process of peroxyl radicals quenching Trolox-QDs fluorescence could be
simply interpreted as the electron use the Trolox radical intermediate SOMO as a
- 64 -
shuttle and hole, therefore blocked the pathway of fluorescence. This result resemble
the report of M. Laferriere et al. when they observed non-linear quenching of CdSe
quantum dots by nitroxyl free radicals[20].
This preliminary study show Trolox-QDs system is promising for peroxyl radical
sensing, but several problems remains. The photoluminescence profile of the ligand
exchanged quantum dots depended greatly on the condition of TOPO coated
quantum dots. The stability of Trolox-QDs vary from batch to batch which can make
the ligand exchange results not repeatable. Generally ligand exchanged QDs can
sustain their fluorescence for 4-8 hours in room temperature exposed to air and could
be stable under nitrogen protection for nearly a week. But we also observed ligand
exchanged QDs lost their fluorescence overnight after the functionalization and
turned into a much darker color. These oxidized Trolox-QDs were precipitated out
several times by diethyl ether and methanol and sent for FT-IR examination.
- 65 -
Normalized Transimittance
1633 cm-1
801 cm-1
Oxidized Trolox-QDs
Trolox-QDs
1514 cm-1
-1
1651cm
4000
3500
3000
2500
2000
1500
1000
500
-1
cm
Figure 3.18 FT-IR spectrum of Trolox-QDs (Red line below) and oxidized Trolox-QDs (Blue
line above)
As Figure 3.18 shows, the spectrum of Trolox-QDs and oxidized Trolox-QDs are
compared to illustrate the nature of reaction. The peaks at 1651cm-1 and 1514 cm-1
which originate from C=O stretching vibration of Trolox diamine ligand
significantly diminished. The former peak disappeared while the latter became a
shoulder. This result is in accordance with that of the oxidation products of Trolox
functionalized gold nano-particle reported by Z. Nie et al. [21]. Two new peaks of the
oxidized Trolox-QDs appear at 1633cm-1 and 801cm-1. This two peaks’ wavenumber
are similar to the oxidized Trolox product reported by Nie et al.
[21]
. From the
spectrum of oxidized form of Trolox-QDs we speculate there might be two products
of Trolox diamine ligand: one has a tetraalkylbenzoquinonoid group (IR 1633 cm-1),
- 66 -
one has a cyclohexanone structure conjugated with adiepoxide (IR 801 cm-1)
Possible mechanism for this rapid oxidation remain unclear. Since severe oxidation
was not observed when Trolox analogues were bounded to other compounds or
chemicals [15], we speculate photooxidation catalyzed by quantum dots emission and
traps on the quantum dot surface could be the culprits for oxidation and lose of
fluorescence. Also according to Z. Nie et al., Trolox functionalized gold particle
appeared to enhanced the activity of the Trolox derived ligand and increase the
reaction rate to 8 times compared with Trolox monomer[21]. Their explanation to this
phenomenon is that by self-assembling on gold nanoparticle surface, both reactants
in close proximity create a highly effective concentration which would lead to
enhanced reaction rate. If this speculation also applies to our case, Trolox
functionalized CdSe QDs should be oxidized faster than the ligand--Trolox diamine
alone and functionalized quantum dots would be more sensitive to oxidants or free
radicals.
Dubois et al. suggested
[11]
that the thickness and the type of the shell grown on the
CdSe core of quantum dots are essential for the preservation of the QD optical
properties. In the same paper, they also suggested multilayer CdSe/CdS/CdZnS/ZnS
QDs should have greater resistance to the lose of fluorescence. Our future work is to
carry out in depth study on oxidation of Trolox-QDs. The relationship between
functionalized QDs’ solubility and “TOPO-QD: CS2: Ligand ratio” also requires
investigation. Since Trolox-QDs tend to be oxidized in reaction mixture, reaction
- 67 -
time of ligand exchange step also needs further optimization to achieve balance
between ligand exchange ratio and photoluminescence of product. Ideally, enough
amount of Trolox diamine should be able to almost completely replace original
TOPO ligand from QDs, and make functionalized product water soluble.
Hydrophilic Trolox-QDs should have wide application in bio-compatible
environment. Finally, multilayer CdSe/CdS/CdZnS/ZnS QDs could be developed to
optimize Trolox-QDs system and provide us more stable quantum dots with a higher
quantum yield.
3.4 Conclusion
By quantum dots ligand exchange using Dubois’ method with this Trolox diamine,
we successfully developed Trolox-QDs system. Characterization with FT-IR shows
ligand exchange QDs obtained new peaks from Trolox ligand. And
31
P-NMR also
illustrates release of TOPO ligand during ligand exchange process. Both of these,
along with the change in solubility and optical properties all indicates modification
of quantum dots surface and bounding between ligand and QDs surface. Preliminary
peroxyl radicals sensing study was carried out. Trolox-QDs has significant response,
which represented by quenching of fluorescence intensity, to radicals generated by
AMVN and AAPH in 120 minutes. The oxidized Trolox-QDs was studied with
FT-IR, and its spectroscopic changes were in accordance with literature report by Z.
Nie et al. In future work, the sensitivity of Trolox system can still be optimized (by
washing steps or dialysis ); Photostability can also be improved by developing more
- 68 -
robust multilayer quantum dots; the Trolox-QDs quenching mechanism requires
more evidence to confirm the occurrences of electron transfer between ligand and
quantum dots.
- 69 -
References
[1] R. E. Bailey, A. M. Smith, S. Nie, Physica E, 2004, 25, 1-12.
[2] R. Eran, The Journal of Chemical Physics, 2001, 115, 1493-1497.
[3] W. C. Chan, nbsp, W, and S. Nie, Science, 1998, 281, 2016-2018.
[4] M. Bruchez, M. Moronne, P. Gin, S. Weiss, and A. P. Alivisatos, Science, 1998, 281,
2013-2016.
[5] S. J. Rosenthal, I. Tomlinson, E. M. Adkins, S. Schroeter, S. Adams, L. Swafford, J. McBride,
Y. Wang, L. J. DeFelice, and R. D. Blakely, Journal of the American Chemical Society, 2002,
124, 4586-4594.
[6] W. J. Jin, M. T. Fernandez-Arguelles, J. M. Costa-Fernandez, R. Pereiro, and A. Sanz-Medel,
Chemical Communications, 2005, 883-885.
[7] A. J. Sutherland, Current Opinion in Solid State and Materials Science, 2002, 6, 365-370.
[8] C. Querner, P. Reiss, J. Bleuse, and A. Pron, Journal of the American Chemical Society, 2004,
126 , 11574-11582.
[9] J. Aldana, Y. A. Wang, and Xiaogang Peng, Journal of the American Chemical Society, 2001,
123, 8844-8850.
[10] Y. Zhao, W. P. Segarra, Q. Shi, and A. Wei, Journal of the American Chemical Society, 2005,
127, 7328-7329.
[11] F. Dubois, B. Mahler, B. Dubertrer, E. Doris, and C. Mioskowski, Journal of the American
Chemical Society, 2007, 129, 482-483.
[12] E. Niki, and N. Nouguchi, Accounts of Chemical Research, 2004, 37, 45-51.
[13] D. C. Liebler, J. A. Burr, S. Matsumoto, and M. Matsuo, Chemical Research in Toxicology,
1993, 6, 351-355.
[14] B. Halliwell and J. M. C. Gutteridge, Free Radicals in Biology and Medicine, Third Edition,
Oxford University Press, New York, 1999.
[15] P. Oleynik, Y. Ishihara, and Gonzalo Cosa, Journal of American the Chemical Society,
(Communication), 2007, 129, 1842-1843.
[16] V. Maurel, M. Laferriere, P. Billone, R. Godin, and J. C. Scaiano, J. Phys. Cem. B, 2006,
110, 16353-16358.
[17] P. Palozza, E. Piccioni, L. Avanzi, S. Vertuani, G. Calviello, and S. Manferdini, Free Radical
Biology and Medicine, 2002, 33, 1724-1735.
[18] M. Koufaki, T. Calogeropoulou, A. Detsi, A. Roditis, A. P. Kourounakis, P. Papazafiri, K.
Tsiakitzis, C. Gaitanaki, I. Beis, and P. N. Kourounakis, Journal of Medicinal Chemistry,
2001, 44, 4301.
[19] W. W. Yu, L. Qu, W. Guo, and Xiaogang Peng, Chemistry of Mateials, 2003, 15, 2855.
[20] M. Laferriere, R. E. Galian, V. Maurel and J. C. Scaiano, Chemical Communications, 2006,
257-259.
[21] Z. Nie, K. J. Liu, C. J. Zhong, L. F. Wang, Y. Yang, Free Radical Biology & Medicine, 2007,
43, 1243-1254.
- 70 -
Chapter 4
Tris (2-aminoethyl) amine (Tren) Functionalized QDs
4.1 Introduction
The demand for chemosensors that are selective for specific target ionic species and
radicals is continuously increasing. Most importantly in this aspect is the sensors that
monitor transition metal ions, reactive oxygen species and other species which have
played important roles on the human health and environment even at very low
concentration level[1,2]. Currently, many fluorescent small molecules (organic dyes)
have been used to detect transition metal ions[3,4]. However, most of them tend to
have narrow excitation spectra, and often exhibit broad and asymmetric emission
bands with red tailing, which makes simultaneous quantitative evaluation of relative
amounts of different probes present in the same sample difficult due to spectral
overlap[5]. Besides, organic dyes are also subject to photobleaching and instability.
Quantum dots (QDs) are semiconductor nanoparticles that are composed of atoms
from groups II-VI or III-V elements in the periodic table, with all three dimensions
confined to the 1 to 10 nm length scale, such as those made of CdSe, ZnTe, GaAs
and InSb. QDs present considerable advantages over organic dyes because they have
unique optical and electronic properties, including broad excitation spectra with high
molar absorptivities and tunable emission[6-8]. The former property is due to the
relatively large densities of states and overlapping band structures. QDs are able to
- 71 -
absorb photons even when the excitation energy exceeds the band gap due to the
presence of multiple electronic states in QDs[9]. Therefore it allows the ease of light
sources usage from various wavelengths and also simultaneous excitation of QDs
with a single light source. The latter property exhibits an attractive way of using QDs
as fluorescent sensors for various ionic species and radicals since the wavelength of
fluorescence emission of QDs can be tuned by altering the QDs size and its chemical
composition. Besides, present methods of QDs synthesis are able to manufacture
QDs with excellent control over the QDs size and its distribution, yielding QDs with
narrow and symmetric emission peaks[6,10].
Those properties mentioned are as a result of quantum confinement effect[9] which
occurs when the size of semiconductor crystal becomes small enough that it
approaches the size of its Exciton Bohr radius, which is the physical separation in a
crystal between an electron in the conduction band and the hole it leaves behind in
the valence band. Under these conditions, the electron energy levels are treated as
discrete and the semiconductor material ceases to resemble bulk and can be called a
quantum dot.
According to Planck’s law, E=hc/λ where E is the energy of light, h is Plank constant,
c is speed of light and λ is the wavelength of light. The wider the QDs’ band gap (i.e.
larger energy) which separates the conduction band and valence band, the bluer the
fluorescence (i.e. shorter wavelength) and vice versa. Figure 4.1 shows the colors of
- 72 -
ZnS-capped CdSe QDs produced are size-dependent. On the contrary with organic
dyes, QDs also has easily accessible synthetic routes, large fluorescence quantum
yields,
high
extinction
coefficients,
low
photobleaching
and
excellent
photostability[6].
Figure 4.1: Ten distinguishable emission colors of ZnS-capped CdSe QDs
excited with a near –UV lamp with sizes of QDs increase from left to right.
(refs. 6)
For routine preparation, the CdSe core is usually capped with an organic layer such
as trioctylphosphine/trioctylphosphine oxide (TOP/TOPO). This layer coordinates to
Cd sites and stabilizes QD’s surface, preventing an aggregation of the
nanocrystals[11]. Since the ligands are hydrophobic, they make the QDs incompatible
with aqueous solution and did not appear to have an immediate application for
analytical system. Two breakthrough papers by Nie’s and Alivisatos’ groups in 1998
demonstrated the fluorescent QDs can be made water-soluble and biocompatible by
surface modification and bioconjugation while maintaining their large fluorescent
quantum yield[8,12]. Hence, by tailoring QDs with different capping ligands, they can
serve for different purposes. In the following years, there are two approaches to
- 73 -
surface modification to obtain highly fluorescent and water-soluble QDs. Firstly, the
formation of a layer of another semiconductor of higher band gap on the QDs
surface such as ZnS, giving rise to a bi- or three-layered structure QD[13] resulting in
highly fluorescent QDs[14]. An alternative approach is to use competing capping
agent such as mercaptosulphonic acid[15] or dihydrolipoic acid[16] to displace
TOP/TOPO from the QDs surface.
Surface modification is important in designing QDs for different analytical
applications. The conjugation of suitable recognition groups will target specific ionic
species or radicals, creating a hybrid QDs-receptor sensing system. This is because
the fluorescence of QDs arises from the recombination of the exciton, it is expected
that the changes of surface components or charges of QDs would affect the
efficiency of core exciton recombination and consequently the fluorescence of QDs.
Therefore QDs in which activated by different ligands can act as novel fluorescence
selective ion probes[10,15,17-23]. The key successful implementation of the uses relies
on the ability to add functionality to QDs and to stabilize their emission.
The earlier work performed on QDs focused mainly on the physical and
photochemical properties. To date, QDs have been developed for their applications
in sensing analytes. Until now, either fluorescence quenching or enhancement of
functionalized QDs has been observed dependent on the nature and environment of
the ionic species or radicals. There are three quenching mechanisms reported: inner
- 74 -
filter effects, electron transfer processes, and non-radiative recombination pathways.
Chen et al. explained the quenching of cysteine capped QDs by Fe(III) is attributed
to an inner filter effect as a result of the strong absorption by Fe(III) at the excitation
wavelength used. This interference caused by Fe(III) can be eliminated by adding
fluoride ions to form a colorless complex FeF63-, which will also dissociate from the
surface of the QDs due to same charge repulsions Also, the quenching of
thioglycerol capped CdS QDs by Cu(II) is through an electron transfer from
thioglycerol to Cu(II). The reduction of Cu(II) to Cu(I) by thioglycerol, formed
CdS+-Cu+ on the surface, which has a lower energy level than pure CdS QDs,
therefore causing a red-shift of fluorescence. Moreover, Cu(I) quenches by
facilitating non-radiative recombination of excited electrons in the conduction band
and holes in the valence band[10].
On the other hand, fluorescence enhancement of QDs has been reported by Moore et
al. in 2001. The reversible fluorescence activation process caused by Zn(II) and
Cd(II) adsorption on the surface of CdS was studied. These ions enhanced the
fluorescence intensity of QDs, was attributed to some form of passivation of surface
trap states. In 2002, Chen et al. worked on Zn(II) determination and found the
formation of a Zn-cysteine complex on the surface of cysteine capping QDs which is
believed responsible for the activation of the surface states and therefore exhibiting
the fluorescence enhancement[10].
- 75 -
Apart from intensive studies for potential use of QDs in sensing cations,
functionalized QDs were also used for detection of inorganic anions although they
are still in embryonic stages. In 2002, Watanabe et al. reported that a gold
nanoparticle capped with amide ligands showed enhanced optical sensing of anions.
The presence of anions would cause a marked decrease in extinction as a result of
anion-induced aggregation of amide-functionalized gold nanoparticle via the
formation of hydrogen bonding between the anions and the interparticle amide
ligands. However, there was no selectivity and the binding affinity of anions was
low[24]. In 2005, Jin et al. developed water soluble fluorescent CdSe quantum dots
which are capped with 2-mercaptoethane sulfonate (MES) for the selective detection
of free cyanide. Consequently, a slight blue-shift fluorescence quenching with
detection limit of 1.1 x 10-6 M was observed. The blue-shift implies the changes in
size or surface properties of MES-CdSe QDs which brings about the decrease in
fluorescence[15]. However, the mechanism of fluorescence quenching was not
discussed.
Currently, researchers also raise considerable attention on sensing the paramagnetic
radicals such as reactive oxygen species and reactive nitrogen species by using QDs
as fluorescence probes. This is due to the importance of radicals in maintenance of
vascular homeostasis and injury in biological system. However, when these reactive
species are overproduced (due to exogenous stimulation), they will become highly
harmful, leading to cellular damage. Unfortunately, reactive species present some
- 76 -
characteristics that make functionalized QDs difficult to detect in biological system
such as their very short lifetime and variety of antioxidants existing in vivo which
capable of capturing these reactive species. Maurel et al. showed the QDs capped by
4-amino-TEMPO (QD-4AT) which was initially quenched by the nitroxide in a
QD-4AT complex, was readily restored when carbon-centered free radicals were
trapped by the nitroxide moiety to form alkoxyamines. The initial quenching was
due to the electron exchange or assisted intersystem crossing[25] upon the binding of
4AT to QDs’ surface without displacing TOPO while the subsequent restoration was
due to radical reaction with ligand and blocked this quenching effect[26]. In the other
work, Neuman et al. described a fluorescence quenching pathway of CdSe/ZnS
core/shell QDs, leading by NO production from trans-Cr(cyclam)(ONO)2+. These
observations are interpreted in terms of Förster resonance energy transfer (FRET)
mechanism and the presence of a number of Cr(III) cations within the QDs hydration
sphere. FRET occurs via space in which the dipole oscillation in QDs is induced by
the dipole oscillation in trans-Cr(cyclam)(ONO)2+. In the other words, FRET
requires that the absorption spectrum of trans-Cr(cyclam)(ONO)2+ overlap the
fluorescence emission spectrum of the QDs[27].
In the present paper, transition metal ions sensing are performed by
tris(2-aminoethyl)amine (Tren)-QDs. However, only the quenching pathways of
selective determination of Co(III) and Cu(II) by using CdSe QDs which is capped by
Tren ligands are thoroughly investigated. Furthermore, the Tren-QDs and transition
- 77 -
metal complex hybrid materials are also utilized for preliminary sensing study of
hydrosulfide anion and nitric oxide.
4.2 Experimental Section
4.2.1 Materials and Instruments Tren-CdSe/ZnS QDs were synthesized by Dr.
Wang Suhua. Iron(II) sulfate heptahydrate, magnesium chloride hexahydrate,
cadmium
nitrate
tetrahydrate,
manganese(II)-chloride-2-hydrate,
magnesium
chloride hexahydrate, cadmium nitrate tetrahydrate and hydrogen peroxide were
purchased from Merck (Germany). Chromium(II) fluoride, manganese(III) acetate
dihydrate, vanadyl sulfate hydrate, ruthenium(II) nitrosyl chloride hydrate, Cobalt(II)
tetrafluoroborate
hexahydrate,
manganese(III)
acetate
dehydrate,
sodium
hydrosulfide, L-Ascorbic Acid (Vitamin C) and dialysis tubing cellulose membrane
were purchased from Sigma Aldrich (USA). Tris(2-aminoethyl)amine (Tren) and
palladium(II) acetate were purchased from Fluka. Nickel(II) acetate tetrahydrate and
pentaaminechloruthenium (III) dichloride were purchased from Alfa Aesar. Iron(III)
chloride anhydrous was purchased from Comak. Copper(II) sulfate pentahydrate was
from J.T. Baker (USA). Cuprous chloride was from British Drug House. Zinc
chloride was from Riedel-de Haën. Carbon Disulfide were purchase from BDH
limited (Poole England). All solvents used were analytical grade and used without
any purification.
Ultraviolet-visible (UV-Vis) absorption spectra were obtained using UV-Visible
- 78 -
Spectrophotometer UV-1601 (Shimadzu (Asia Pacific) Pte. Ltd, Singapore). The
absorbance readings were obtained from 900 nm to 400 nm. The fluorescence was
monitored by using Bio-tek Synergy HT (Winnoski, Vermont, USA) plate reader.
Excitation and emission wavelengths were set at 360±40 nm and 540±25 nm
respectively. The working solutions were injected into 384 Well HiBase polystyrene
microplates and the fluorescence was measured with prior shaking for ten seconds
with intensity of one for homogeneity. Temperature control was not necessary while
reading fluorescence values. On the other hand, the fluorescence spectra were
obtained on a Perkin Elmer LS55 Luminescence Spectrometer. The fluorescence
spectra were obtained from 700 nm to 500 nm. The excitation slit width and the
emission slit width were set at 10 nm and 20 nm respectively. The scan speed was set
at 500 nm/min. PTFE vials (4mL, screw-top 15x45 vial with cap and bonded
PTFE/silicone septa) from Waters® (USA) were utilized. All optical measurements
were performed at room temperature under ambient conditions. After fluorescence
measurement, the vials were further wrapped with aluminum foil to minimize the
exposure to light. All pH measurements were made with Thermo Orion pH meter
(Model 410). FT-IR spectroscopy was performed on Perkin–Elmer Spectrum one-B
spectrometer.
Choosing suitable pH of Phosphate Buffer Saline (PBS)
for Tren-QDs system
Different pH of PBS buffer were prepared by using commercial 10X
Phosphate
Buffer Saline which contains 137 mM NaCl, 2.7 mM KCl and 10 mM Phosphate
Buffer. By adjusting pH of PBS buffer with NaOH and HCl and measuring the pH
- 79 -
value of PBS with pH meter, PBS 4.0, PBS 5.0, PBS 6.0, PBS 6.5, PBS 7.0, PBS 7.5,
PBS 8.0, PBS 9.0 were prepared. The same amounts (1.2 mg) of Tren-QDs were
then dissolved in 3.0 mL of different pH of PBS buffer respectively. After vortex
each solution to make the solution homogenous, the fluorescence was measured for
every five minutes (0-60 minutes). The stock solution of Tren-QDs was prepared by
dissolving the powder form of Tren-QDs in PBS buffer, followed by centrifugation
(1000 rpm) for three minutes in order to obtain saturated Tren-QDs in which they
were well dispersed in PBS buffer. The precipitates were removed by centrifugation.
4.2.2 Sensing of metal ions The stock solutions with 1 mM concentration of
Na(I), K(I), Mg(II), Ca(II), VO2+, Cr(II), Cr(III), Mn(II), Mn(III), Fe(II), Fe(III),
Co(II), Ni(II), Cu(II), Zn(II), Ru(II), Ru(III), Pd(II), Ag(I) and Cd(II) were prepared
by dissolving suitable amount of NaCl, KBr, MgCl2.6H2O, CaSO4.2H2O,
VOSO4.xH2O, CrF2, CrCl3.6H2O, MnCl2.2H2O, (C2H3O2)3Mn.2H2O, FeSO4.7H2O,
FeCl3,
Co(BF4)2.6H2O,
(CH3COO)Ni2.4H2O,
CuSO4.5H2O,
ZnCl2,
Ru(NO)Cl3.xH2O, [RuCl(NH3)5]Cl2, Pd(OAc)2, AgNO3 and Cd(NO3)2.4H2O in 10
mL deionized water respectively and were further diluted when necessary. The stock
solutions were prepared in 10 mL PTFE vials with further sealed with parafilm and
kept under dark. However, freshly-prepared Fe(II) solution was needed due to the
easy-oxidized property of Fe(II). The white precipitates adhere on the wall of vial
indicated the oxidized Fe(II) solution.
- 80 -
The general detection procedures for metal ions were as follows: 3.0 mL of
Tren-QDs in PBS buffer (stock solution) was introduced into a PTFE vials, followed
by adding 20 μL of 1.0 mM standard solution of metal ions every time (0 -80
microliters). The fluorescence intensity was measured immediately after the working
solution mixed thoroughly by vortex.
4.2.3 The effect of carbon disulfide on QDs-metal ions complex The stock
solutions with 1 mM concentration
of
Na(I), Mn(III), Fe(II), Fe(III), Co(II),
Ni(II), Cu(II), Zn(II), Ag(I) and Cd(II) were prepared by dissolving suitable amount
of
NaCl,
(C2H3O2)3Mn.2H2O,
FeSO4.7H2O,
FeCl3,
Co(BF4)2.6H2O,
(CH3COO)Ni2.4H2O, CuSO4.5H2O, ZnCl2, AgNO3 and Cd(NO3)2.4H2O in 10 mL
deionized water respectively and were further diluted when necessary. The stock
solutions were prepared in 10 mL PTFE vials with further sealed with cap and kept
in darkness. However, freshly-prepared Fe(II) solution was needed due to the
easy-oxidized property of Fe(II). The white precipitates adhere on the wall of vial
indicated the oxidized Fe(II) solution.
The general detection procedures for metal ions were as follows: Trens-QDs
methanol/H2O 1:1(v/v) solution (30nM) 10mL was mixed with 1mL CS2 methanol
solution (30μM) and vigorously vortexed. This mixture was left in darkness for 30
minutes for stabilization and then transferred into NUNCTM 96 well micro plate
followed by adding 20μL of various transition metal water solution (50μM) was
- 81 -
added for fluorescence reading. Each type of working solutions was injected into 3
plate wells repetitively for accuracy purposes. Besides, the total amount of working
solutions in each plate well is kept the same to avoid deviation came from the
volume differences. The fluorescence intensity was measured immediately after
addition.
4.2.4 Tren-CS2-metal complex synthesis and reaction with sodium hydrosulfide
Tren solution and CS2 solution was prepared by diluting these liquid into suitable
amount of methanol. Tren solution (10mL, 2.67mM) and CS2 (10mL, 8.32mM)
solution was mixed and vigorously stirred overnight. Each 5 mM of this reaction
mixture was mixed with 1mL of copper(II) acetate methanol solution(6mM), Iron(II)
acetate methanol solution (6mM), Iron(III) chloride methanol solution (6mM) and
1mL of methanol (as control) respectively. Above four mixtures were all fully
vortexed following by adding of 20uL NaSH (200mM) water solution. Hence the
finally concentration of each component was Tren: CS2: M+: HS-=0.56mM: 1.73mM:
0.5mM: 1.98mM. One milliliter of this mixture was withdrew and 2 times diluted to
afford a 2 mL methanol solution for UV-Vis spectrum scan.
4.2.5 Tren-QDs-CS2-Fe(II) Complex synthesis Tren-QDs water solution(43nM,
10mL) was prepared by dissolving Tren-QDs powder into deionized water. The
solution was fully vortexed and centrifuged at 1000rpm for 5minutes in order to
obtain saturated Tren-QDs water solution. After that, the precipitates were removed
- 82 -
and the supernatant was transferred out for further applications. CS2 methanol
solution (52μM, 2mL) was added into the saturated Tren-QDs water solution and
fully vortexed for 30 seconds, followed by adding 1mL of Fe(OAc)2 water solution
(135μM). The reaction mixture was vigorously stirred overnight in darkness at room
temperature.
When the reaction stopped, the mixture was withdrew from reaction vessel and
purified by dialysis using Sigma-Aldrich dialysis tubing cellulose membrane. By one
hour of dialysis in deionized water, the remaining solution of Tren-QDs-CS2- Fe(II)
complex was withdrew from the membrane and stored in sealed PTEE vials in
darkness and was ready for sensing or other applications.
4.2.6 Tren-QDs-CS2-Fe(II) complex sensing hydrosulfide anion and nitric oxide
Hydrosulfide anion was provided by dissolving 265mg NaSH solid into 25mL
deionized water(189× 10-3M). NO was generated by mixing NaNO2 and ascorbic
acid water solution dropwise at room temperature. By passing NO gas trough
deionized water, saturated NO water solution ( 2× 10-6M) was prepared. The general
detection procedures for HS- and NO were as follow: 3.0 mL of Tren-QDs-CS2-Fe(II)
complex in deionized water was introduced into a PTFE vials, followed by adding
20μL of NaSH (0-140μL)or 100μL NO water solution every times (0-500μL). The
fluorescence intensity was measured on Perkin Elmer LS55 Luminescence
Spectrometer five minutes after the working solution mixed thoroughly by vortex.
- 83 -
4.3 Results and Discussion
4.3.1 Tren-QDs system The surface structure of QDs capped with Tren ligands and
their interaction with transition metal ion are shown in Figure 4.2. Due to the
presence of lone pairs on nitrogen atoms of amines groups in Tren ligands, they have
high degree of basicity and are completely miscibility with water. Therefore, by
capping QDs with Tren ligands, the functionalized QDs become water-soluble. Tren
ligands are attached to the surface of QDs with the assistance of carbon disulfide
which is bidentate chelating moieties with high affinity for metal atoms[28] and
therefore leading to a secure coating of the QDs. The sulfur atoms of carbon
disulfide are bound to the zinc atom of zinc disulfide via electrostatic interaction.
One molecule of Tren consists of four amine groups. From Figure 4.2, N1 is tertiary
amine and N2, N3 and N4 are primary amines. N2, N3 and N4 are chemically
equivalent since they are in the same chemical environment. Therefore, either of
them has the same reactivity and readily to react with carbon disulfide. This is
because primary amines are more reactive than tertiary amines. Consequently, the
remaining amines are believed corresponding to the coordination with transition
metal ions.
- 84 -
NH2
4
M (Ln)
N
1
NH2
3
NH2
(Ln) M
HN
3
2
-
S
H2N
S
4
N
1
S
S
CdSe
-
NH
2
-
NH
2
S
S
N
ZnS
S
H2N
NH
4
N
2
1
S
M
NH2
3
(Ln)
H2N
4
1
(Ln)M
NH2
3
Figure 4.2: Proposed surface structure of CdSe QDs capped by Tren ligands and their
coordination complexes with transition metal ion. M and Ln represent transition metal and other
ligands.
4.3.2 Optical characteristics of the Tren-QDs The fluorescence emission spectrum
and absorption spectrum of Tren-QDs are shown in Figure 4.3. The fluorescence
spectrum shows a nonzero tail toward longer wavelength suggesting the presence of
surface traps. This fluorescence is attributed to the recombination of the charge
carriers immobilized in traps of different energies[29]. General fluorescence
measurements were significantly simplified due to long wavelengths emission and a
high separation between the excitation and emission wavelengths.
- 85 -
600
559
0.10
Emission
0.09
Absorbance
0.08
500
400
361
0.07
300
0.06
200
0.05
100
0.04
300
350
400
450
500
550
600
650
0
700
F lu o rescen ce In ten sity
A b so rb an ce
0.11
Wavelength (nm)
Figure 4.3: Absorption spectrum and fluorescence emission spectra of Tren-QDs
Figure 4.4 shows a plot of the fluorescence intensity versus concentration of QDs
with a good linear relationship (inset in Figure 4.4). The result demonstrated that
functionalized QDs were uniformly dispersed in PBS 7.4 buffer in a large
concentration range. Furthermore, there was no fluorescence emission spectra band
shift along with a serial representative emission spectrum at different concentration
of QDs even at the lowest concentration of QDs. Tren-QDs can disperse uniformly
and stably in aqueous solution for around one week without precipitation when
preserved in the dark and under ambient conditions.
- 86 -
1000
Fluorescence Intensity
900
800
700
[Tren-QDs]
600
500
400
300
200
100
0
400
450
500
550
600
650
Wavelength (nm)
Figure 4.4: The concentration-dependent of fluorescence property of QDs.
4.3.3 Sensing of transition metals The effect of pH in a range between 4.0 and 9.0
was studied in order to find out the optimum conditions for the sensing of transition
metals with Tren-QDs. The results obtained from this study showed Tren-QDs
behave their highest stability between PBS 7.0 and PBS 7.5 (results not shown). A
final PBS 7.4 buffer was selected for further experiments. Besides, pH 7.4 is also
physiological pH and Tren-QDs system might be functioned in vivo in the future for
sensing either transition metals or radicals.
Responses to metal ions at concentration of 1.3 x 10-5 M
except for Na(I), K(I),
Mg(II) and Ca(II) of 13 x 10-5 M in 0.02 x 10-6 M Tren-QDs in PBS 7.4 buffer are
shown in Figure 4.5. From Figure 4.5, it could be easily found that the fluorescence
emission of Tren-QDs was quenched significantly by Co(III) and Cu(II) respectively.
Although other transition metals (Ni(II), Fe(III), Fe(II), Mn(III), Mn(II), Cr(III),
- 87 -
Cr(II), VO2+, Ru(III), Ru(II) Ag(I), Pd(II)) also quenched the fluorescence of
Tren-QDs to different extents but their quenching effects were not as strong as those
of Co(III) and Cu(II). On the other hand, the fluorescence emission was slightly
enhanced by Zn(II) and Cd(II) respectively. Furthermore, the effect of interferences
of foreign substances such as Na(I), K(I), Mg(II) and Ca(II) was investigated as well.
The result shows that they have hardly effect on Tren-QDs fluorescence even in high
concentration. Therefore, the sensing result demonstrates that this method has high
sensitivity and selectivity towards Co(III) and Cu(II). Hopefully, Tren-QDs system
could be utilized to sense Co(III) and Cu(II) in biological system in the future since
it would not be affected by the presence of interfering substances even in high
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
Ca(Ⅱ)
Mg(Ⅱ)
K(Ⅰ)
Pd(II)
Na(Ⅰ)
Ag(I)
Cd(Ⅱ)
Ru(Ⅱ)
Ru(Ⅲ)
VO2+
Cr(Ⅱ)
Cr(Ⅲ)
Mn(Ⅱ)
Mn(Ⅲ)
Fe(Ⅱ)
Fe(Ⅲ)
Co(Ⅲ)
Ni(Ⅱ)
Cu(Ⅱ)
Zn(Ⅱ)
0.0
Control
Relative Fluorescence Intensity
concentration.
Metal Ions
Figure 4.5: Effect of metal ions on the fluorescence of Tren-QDs. Concentrations of
transition metal ions are all 1.3 x 10-5 M, and other alkali and alkali earth metal ions
are 13 x 10-5M. Concentration of Tren-QDs is 2.0 x 10-8 M.
The result of slight fluorescence enhancement of Tren-QDs by Zn(II) and Cd(II) is
- 88 -
consistent with Chen et al. reports[30] except they used L-cysteine functionalized CdS
QDs instead of Tren-CdSe QDs . From the fluorescence spectra, these ions enhanced
the band edge fluorescence intensity of the Tren-QDs upon introduction of salts of
these ions, therefore it is attributed to some form of passivation of the surface trap
states of QDs[31].
Although Co(II) source was used in this study,
but Co(III) is likely the right
candidate which coordinated with Tren-QDs. The high-spin Co(II) will readily
become low-spin Co(II) when coordinated to amine ligands. The resulting Co(II)
complex is known to react with oxygen and become Co(III). Indeed, we observed
color changes of the solution upon addition of Co(II) solution to the Tren-QDs.
Figure 4.6 displays the absorption behavior of Co(III) which coordinated with Tren
ligands of various concentration in aqueous solution. There is an intense absorption
occurred around 559 nm (i.e. the fluorescence emission maximum wavelength) when
Co(III) was coordinated with Tren ligands. Therefore the absorption spectrum
effective overlap with the band edge of fluorescence emission which ranging from
around 500 – 600 nm (see Figure 4.3). This feature suggests that the energy transfer
via Förster mechanism would be favorable. That is the energy transfer occurs from
Tren-QDs donor to Co(III) acceptor via space in which the acceptor absorbs energy
at the emission wavelength of the donor and quenching is observed when the donor
does not re-emit the energy fluorescently itself. Apart from the spectral overlap
between the donor emission and the acceptor absorption spectrum, energy transfer
- 89 -
also depends on the relative orientation and the distance between the donor and
acceptor[32].
0.20
Co(III)
0.15
1 eq Co(III):1 eq Tren
Absorbance
1 eq Co(III):2 eq Tren
1 eq Co(III):3 eq Tren
1 eq Co(III):4 eq Tren
0.10
1 eq Co(III):5 eq Tren
1 eq Co(III):6 eqTren
1 eq Co(III):7 eq Tren
1 eq Co(III):8 eq Tren
0.05
0.00
400
500
600
700
800
900
Wavelength (nm)
Figure 4.6: Electronic spectra of Co(III) complexes of various concentrations in aqueous
solution.
The absorption of Cu(II) complexes was also investigated and shown in Figure 4.7.
There is only a slight absorption occurred around 559 nm (i.e. weak overlap with the
band edge of fluorescence emission which ranging from around 500 – 600 nm) when
Cu(II) was coordinated with Tren. Hence the quenching of Tren-QDs upon the
addition of Cu(II) is only minor attributed by the energy transfer. Besides, from the
absorbance spectrum of Cu(II) alone, there is a peak arises at around 780 nm which
is attributed to the d-d transition of Cu(II) which energy gap is complementary to
blue light and therefore contribute to the blue color of
the copper(II) sulphate
pentahydrate (source of Cu(II)). However, there is another peak arises at the shorter
- 90 -
wavelength upon the addition of Tren ligands. It is likely due to d-d transition as well.
The blue shift is attributed to stronger ligand field of Tren than water. Since the
absorbance coefficiency is still small judging from the UV-Vis spectrum and it can
not be a charge transfer band.
0.20
d-d transition
Absorbance
0.15
Cu(II)
1eq Cu(II):1 eq Tren
1eq Cu(II):2 eq Tren
1eq Cu(II):3 eq Tren
1eq Cu(II):4 eq Tren
1eq Cu(II):5 eq Tren
1eq Cu(II):6 eq Tren
1eq Cu(II):7 eq Tren
1eq Cu(II):8 eq Tren
0.10
d-d transition
0.05
0.00
400
500
600
700
800
900
Wavelength (nm)
Figure 4.7: Electronic spectra of Cu(II) complexes of various concentrations in aqueous solution.
Apart from the minor attribution of energy transfer, electron transfer pathway can
also apply on the observed quenching effect of Tren-QDs by Cu(II). This is because
Cu(II) is d9 ion and therefore Cu(II) tends to receive one additional electron in order
to occupy d10 stable valence configuration. Hence, electron transfer is initiated from
Tren-QDs to Cu(II) upon the coordination of Cu(II) with Tren-QDs. Future work
shall be targeted at elucidating the fluorescence quenching mechanism.
- 91 -
However, the other quenching pathway proposed by Xie et al. group could also be
plausible. Small transition metal ion Cu(II) could pass through the shell layer and
interact with core if the QDs used were not perfectly capped with Tren ligands,
resulting in the chemical displacement of Cd(II) by Cu(II). The displacement can be
achieved because the solubility of CuSe is extremely low if compared to that of
CdSe. So the quenching result could be explained in terms of strong binding of Cu(II)
onto the surface of Tren-QDs. As shown in Figure 4.8, when the CuSe particles grew,
its energy levels will become lower than that of pure QDs, facilitating the electron
and hole transfer from the CdSe to CuSe energy levels rather then the process of
fluorescence generation in the CdSe. Consequently, the fluorescence of Tren-QDs
was quenched[18]. However, this assumption will be proved using high resolution
transmission electron microscopy (TEM) in the future. By using TEM, one could
image the surface structure of Tren-QDs.
e-
X
h+
Tren-QDs
CuSe
Figure 4.8: Energy scheme of the Tren-QDs in the presence of Cu(II).
- 92 -
Figure 4.9 describes the effect of Co(III) and Cu(II) concentration dependence on the
fluorescence intensity of Tren-QDs. For each of the transition metal ions, the
fluorescence intensity all decreases greatly in exponential behavior along with the
increase of the ion concentrations. The Tren-QDs in PBS 7.4 buffer are most likely
to coordinate with Co(III) and Cu(II) respectively because the amount of those metal
ions used was in excess with respect to the molar concentration of Tren-QDs.
Furthermore, each molecule of QDs consists of many Tren ligands on its surface.
Taking into account the reaction of Tren with each Tren ligand consists of more than
one amine group, which therefore brings up the number of coordination atoms of
Tren-QDs with metal ions. In the presence of Tren, the quenching mechanism of
Tren-QDs is more of a surface alteration rather than formation of a completely new
product. Consequently, the fluorescence intensity does not decrease linearly with
increasing concentrations of the quencher (i.e. Co(III)/Cu(II)).
Relative Fluorescence
1.20
1.00
0.80
Cu(II)
0.60
Co(III)
0.40
0.20
0.00
0
5
10
15
20
25
30
Concentration of ions (μM)
Figure 4.9: Effect of Cu(II) and Co(III) concentration on the fluorescence intensity
of Tren-QDs.
- 93 -
Furthermore, the Stern-Volmer quenching equations for Co(III) and Cu(II) were
studied. Prior to discussing the results, it is important to introduce on the general
characteristic of dynamic and static quenching which can be explained by
Stern-Volmer plot. Basically, dynamic quenching occurs only when the excited
fluorophore experiences contact with an atom or molecule that can facilitate
non-radiative transitions to the ground state (i.e. diffusive encounters). In the
simplest case of dynamic quenching, Stern-Volmer equation was held: I0/I = 1 +
KSV[Q]
= τo/τ where I0 and I are the fluorescence intensities observed in the
absence and presence, respectively, of quencher, [Q] is the quencher concentration,
KSV is the Stern-Volmer quenching constant, τo and τ are the excited state lifetime in
the absence and presence of the quencher. In the situations where the fluorophore
forms a ground-state complex with the quencher, and the complex causes quenching,
then the static quenching occurs. In such a case, equation I0/I = 1 + Ka [Q] is held
where Ka is the association constant of the complex. However, it is possible for both
static and dynamic quenching to occur simultaneously. In Tren-QDs system, the
Tren-QDs are fluorophores whereas the transition metals, Co(III) and Cu(II) are
quenchers.
Figure 4.10 shows Stern-Volmer quenching curves describing I0/I as a function of
transition metal concentration. Both Co(III) and Cu(II) curves can fit into
conventional linear Stern-Volmer equation with linear correlation coefficient of
0.978 and 0.910 respectively. Besides, the Stern-Volmer quenching constant (Ksv) are
- 94 -
found to be 0.2124 μM-1 and 0.1366 μM-1 for Co(III) and Cu(II) respectively. The
higher the value of KSV, the more efficient the quenching effect by a quencher.
Therefore in this case, Co(III) exhibits higher quenching ability than that of Cu(II). A
linear Stern-Volmer plot is also indicative of a single class of fluorophores, all
equally accessible to quencher. With the linearity, the concentration of transition
metal ions can be monitored by measuring the fluorescence intensity of Tren-QDs.
However, with the present data of Co(III) and Cu(II) study, it is not possible to
definitely state which mechanism (dynamic or static) predominates since the lifetime
study is not conducted in this experiment. The Stern-Volmer plots of other transition
metals also can fit into linear Stern-Volmer equation (data not shown) but with lower
KSV values than that of Co(III) and Cu(II), showing that they are poorer quenchers.
7.00
6.00
Io/I = 0.2124[Q] + 1
Io/I
5.00
2
Cu(II)
Co(III)
R = 0.978
4.00
3.00
Linear (Cu(II))
Linear (Co(III))
Io/I = 0.1366[Q] + 1
2
2.00
R = 0.910
1.00
0.00
0
5
10
15
20
25
30
Concentration of ions
(μM)
Figure 4.10: Stern-Volmer plot of Cu(II) and Co(III) concentration dependence of the
fluorescence intensity of Tren-QDs.
4.3.4
Tren-QDs-CS2-Fe(II)
complex
system
Tren-QDs
was
successfully
coordinated with transition metal by our coworker. Tren-QDs-Cu(II) and
Tren-QDs-Co(III) have been applied for sensing radicals and other oxygen species.
- 95 -
By screening with UV-Vis spectrum trough three metal ions Fe(II), Fe(III) and
Cu(II), we try to design and synthesis another Tren-QDs metal complex which is
sensitive to biologically important hydrosulfide anion and nitric oxide.
The effect of carbon disulfide on QDs-metal ions complex The addition of carbon
disulfide (CS2) was based on the idea that the free primary amino group of Tren
ligand on Tren-QDs surface could react with CS2 to form a dithiocarbamate group,
which can strongly chelate the transition metal ions. By using this chelating ability
we can coordinate more metal ions on the surface of Tren-QDs and therefore create
more sensitive fluorescent Tren-QDs-CS2-M+ metal complexes which can be applied
to the sensing of hydrosulfide anion and nitric oxide. CS2 was know to have the
ability to quench QDs fluorescence to a moderate degree, its synergetic effect with
metal ions on Tren-QDs could severely quenching its fluorescence and compromise
the sensitivity of the complex. Therefore, effect of metal ions and CS2 on the
fluorescence of Tren-QDs is our first step of to study.
- 96 -
1.2
Relative Fluorescence Intensity
1
0.8
0.6
0.4
0.2
0
Na(Ⅰ)
Co(Ⅱ)
Ag(Ⅰ)
Mn(Ⅲ)
Fe(Ⅱ)
Fe(Ⅲ) Ni(Ⅱ)
Metal Ions
Cd(Ⅱ)
Zn(Ⅱ)
Cu(Ⅱ) Control
Figure 4.11 Effect of metal ions and CS2 on the fluorescence of Tren-QDs. Concentration of
metal ions are all 90.9×10-6M. Concentration of CS2 is 2.48×10-6M. Concentration of Tren-QDs
is 24.8×10-9M.
Fluorescent response to metal ions at concentration of 90.9×10-6M Tren-QDs
( 24.8×10-9M) and CS2 (2.48×10-6M) mixture are shown in Figure 4.11. CS2 was
thoroughly mixed with Tren-QDs and left in darkness for 30 minutes, followed by
adding of 10 kinds of metal ions water solution at the concentration of 90.9×10-6M.
From Figure 4.11, it could be found that the fluorescence emission of Tren-QD-CS2
was quenched by Co(II) and Cu(II) most obviously. Although other transition metals
(Mn(III), Fe(II), Fe(III), Ag(I), Ni(II) and Zn(II)) also quenched the fluorescence to
different extents but their quenching effects were not as strong as those of Co(II) and
Cu(II). Furthermore, the effect of interferences of foreign substances Na(I) was also
investigated. The result shows it has hardly any effect on the fluorescence comparing
to the control group. This result is in agreement with our previous observation in
- 97 -
PBS buffer without CS2 (Figure 4.5) except the fluorescence emission enhancement
from addition of Zn(II) and Cd(II) was not reproduced. Instead a slight quenching
was observed after addition of each ion. The addition of CS2 did not directly affect
optical properties of Tren-QDs, which make it possible that CS2 serves as a bridging
unit between Tren-QDs and transition metal ions.
Synthesis of Tren-CS2-metal complex and its reaction with NaSH Copper(II),
Iron(II) and Iron(III) was chosen for the first batch UV-Vis screening for selection of
complex which has strong absorption peaks at Tren-QDs emission area (i.e.
500-600nm). The synthetic procedure was simply mixing Tren solution (10mL,
2.67mM) and CS2 (10mL, 8.32mM) solution was thoroughly and stirred vigorously
overnight. When the reaction was stopped, each 5mL of the mixture was added into
1mL of copper(II), Iron(II) and Iron(III) water solution (6mM) respectively. The four
mixtures were thoroughly vortexed, followed by the addition of 20uL NaSH
(200mM) water solution respectively. The final concentration of component in each
mixture was Tren: CS2: M+: HS-=0.51mM: 1.59mM: 0.91mM: 1.98mM. All mixture
was 2 times diluted with methanol and studied by UV-Vis spectrum scan
(200-900nm). Control group was the one without metal ion addition. The UV-Vis
spectrum of Tren-CS2-Fe(II) complex with and without hydrosulfide are shown in
Figure 4.12.
- 98 -
3.0
Fe(II)+SH
Fe(II)
2.5
Absorbance
2.0
1.5
1.0
0.5
0.0
-0.5
-1.0
200
300
400
500
600
700
800
900
Wavelength (nm)
Figure 4.12 UV-Vis spectrum of Tren-CS2-Fe(II) complex before (red line) and after (black line)
NaSH addition. (Tren: 0.255mM, CS2 :0.795mM, M+: 0.455mM, HS- :0.99mM.)
Copper(II), Iron(II) and Iron(III) all caused drastic absorbance increase at 550nm,
which is the maximum of Tren-QDs emission peak, when they were added into the
mixture of Tren and CS2. In Figure 4.13, Fe(II) has the highest absorbance after HSaddition among all three metal ions tested, followed by Fe (III) and Cu (II). But 10
minutes after the UV-Vis essay, Fe(III) and Cu (II) complexes precipitated out from
the solvent. Fe (II) complex, however, was able to sustain in solution several days
after HS- addition.
- 99 -
1
0.9
0.8
Normalized Absorbance
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Control
Control+HS-
Cu(Ⅱ)
Cu(Ⅱ)+HS-
Fe(Ⅲ)
Fe(Ⅲ)+HS-
Fe(Ⅱ)
Fe(Ⅱ)+HS-
Figure 4.13 Effect of hydrosulfide anion on the UV-Vis absorbance of Tren-QDs-CS2-metal
complex at 550nm. Concentration of each component are Tren: 0.255mM, CS2 :0.795mM, metal
ions: 0.455mM, HS- :0.99mM.
The precipitate of the metal complex could be seemed as the proof of successful
coordination (because Tren itself would not form insoluble precipitate with these
metal ions in methanol), but the ultimate goal of this research is to coordinate metal
ions on Tren-QDs surface, and precipitation of metal complex could result in
precipitation of QDs itself from the solution. Therefore, only Iron(II) was suitable
candidate for further investigation. In Figure 4.12, the UV-Vis spectrum of
Tren-CS2-Fe(II) before and after NaSH addition was compared. It was clearly found
that after HS- addition UV-Vis absorbance mainly increased at 200-600nm area.
Hence the complex has the potential to block both the excitation light and the
- 100 -
emission of Tren-QDs. This observation is consistent with the Chen et al.’s report. In
their publication the quenching effect of Fe3+ on the luminescence of L-cysteine and
thioglycerol-capped was categorized as “inner filter effect”, which resulting from the
strong absorption of excitation wavelength at 400 nm[10]. And the interference could
be eliminated by adding fluoride ions to form a colorless complex FeF63-.
4.3.5 Tren-QDs-CS2-Fe(II) complex synthesis and its application With
understanding of the optical properties of Tren-CS2-Fe(II), the surface of Tren-QDs
was modified by the formation of
Tren -CS2-Fe(II) complex. The synthesis step
was simply started by mixing saturated Tren-QDs deionized water solution (43nM,
10mL) and CS2 methanol solution (52μM, 2mL) together and fully vortexed for 30
seconds. Following that, Fe(OAc)2 water solution (135μM, 1mL) was added to the
mixture. The ratio of each component is Tren-QDs: CS2: Fe(II)=1:242:314.The
reaction solution was vigorous stirred overnight in darkness. After reaction, the
product mixture was purified by dialysis in Sigma-Aldrich dialysis membrane
against deionized water for one hour to remove unreacted reagents in order to
improve the sensitivity of the Tren-QDs-Fe(II) complex. The proposed complex
structure was shown Figure 4.14. The purified complex was sealed in PTFE vials
and kept in darkness at room temperature for further dilution or applications.
- 101 -
S
Fe
S
HN
H
N
S
Fe S
N
Fe
S
NH
S
S
-
NH
S
ZnS
H
N
N
S
-
S
S
Fe
S
S
-
CdSe
NH
S
S
Fe
S
HN
N
N
H
HN
-
S
S
S
S
S
Fe
HN
N
H
N
Fe
S
NH
S
S
Fe
Figure 4.14 Proposed surface structure of CdSe QDs capped by Tren ligands and their
coordination complexes with Iron (II) ions.
Tren-QDs-CS2-Fe(II) complex sensing hydrosulfide anion The purified
Tren-QDs-CS2-Fe(II) complex was diluted 2 times into PBS 7.4 buffer. Hydrosulfide
anion was provided by NaSH deionized water (0.189M) solution. Each time 20μL of
NaSH solution was added into 3mL of the complex, the working solution was fully
vortexed and left in darkness for 5 minutes before fluorescence intensity
measurement. Totally 140μL NaSH solution was added into the complex. Figure
4.15 describes the effect of HS- anion on the fluorescence emission spectrum of
Tren-QDs-CS2-Fe(II) complex.
- 102 -
Tren-QDs-CS2-Fe(Ⅱ)
146.9
140
130
Tren-QDs-CS2-Fe(Ⅱ)+1.25 mM NaSH
120
Tren-QDs-CS2-Fe(Ⅱ)+2.49 mM NaSH
110
Tren-QDs-CS2-Fe(Ⅱ)+3.70 mM NaSH
100
Tren-QDs-CS2-Fe(Ⅱ)+4.91 mM NaSH
90
Tren-QDs-CS2-Fe(Ⅱ)+6.10 mM NaSH
80
Tren-QDs-CS2-Fe(Ⅱ)+7.27 mM NaSH
70
Tren-QDs-CS2-Fe(Ⅱ)+9.57mM NaSH
60
50
40
30
20
10
6.3
500.0
510
520
530
540
550
560
570
580
590
600
610
620
630
640
650
660
670
680
692.5
nm
Figure 4.15 Spectra of Tren-QDs-CS2-Fe(II) complex quenched by NaSH solution
Figure 4.16 shows the Effect of HS- concentration on the fluorescence intensity of
Tren-QDs-CS2-Fe(II)
complex.
From
this
result,
it
is
clear
that
the
Tren-QDs-CS2-Fe(II) complex exhibits sensitivity to HS- anion. After addition of
NaSH solution, the fluorescence intensity was quenched moderately (42.2%) by HSanion.
- 103 -
Normalized Fluorescence
Intensity
1
Normalized Fluorescence Intensity
0.95
0.9
0.85
1
0.95
0.9
0.85
0.8
0.75
0.7
0.65
0.6
0.55
0.5
y = -0.0604x + 0.9868
2
R = 0.9832
0
1
2
3
4
5
6
7
NaSH concentration(mM)
0.8
0.75
0.7
0.65
0.6
0.55
0.5
0
1
2
3
4
5
6
NaSH Concentration(mM)
7
8
9
10
Figure 4.16 Effect of HS- concentration on the fluorescence intensity of Tren-QDs-CS2-Fe(II)
complex
To further understand the quenching mechanism and improve the sensitivity to HSanion, we modified component ratio of the complex Tren-QDs-CS2-Fe(II). The
synthetic procedure was the same as the previous step, but the amount of CS2 (1mL,
430mM in methanol) and Fe(OAc)2 (460mM, 1mL in water) was significantly
increased. Hence, the final ratio of the Tren-QDs-CS2-Fe(II) complex reaction
mixture is QDs: CS2: Fe(II)=1:1000:1069. When the reaction was stopped, the
mixture was withdrew and purified and the purified new complex was also 2 times
diluted in PBS 7.4 buffer and sent for NaSH quenching test.
- 104 -
Normalized Fluorescence
Intensity
1.00
Normalized Fluorescence Intensity
0.90
0.80
0.70
1.00
0.90
0.80
y = -0.1332x + 1.0094
R 2 = 0.9742
0.70
0.60
0.50
0.40
0.30
0.20
0
0.60
2
4
6
NaSH concentration (mM)
0.50
0.40
0.30
0.20
0
1
2
3
4
5
6
7
8
9
10
NaSH Concentration (mM)
Figure 4.17 Effect of hydrosulfide anion concentration on the fluorescence intensity of
Tren-QDs-CS2-Fe(II) complex (excited at 400nm)
From above Figure 4.17, the fluorescence of new Tren-QDs-CS2-Fe(II) complex was
more effectively quenched
(73.6%) after addition of same amount NaSH solution
(0.189M, 0-140μL). By comparing the slope of quenching plots with previous
complex in the 0 mM to 6 mM range, the change of slope from 0.0604 (Figure 4.18
inserted graph) to 0.1332 (Figure 4.18 inserted graph) clearly indicates the
improvement of sensitivity to HS- anion. On the other hand, this result proves our
successful removal of extra Fe(II) ions from the reaction mixture. The addition of
NaSH solution immediately turned the colorless new Tren-QDs-CS2-Fe(II) into
yellow-brown colored solution.
The UV-Vis absorption of this new Tren-QDs-CS2-Fe(II) complex was investigated
and results are shown in Figure 4.18. Before addition of NaSH (blue solid line), the
- 105 -
complex itself has a strong absorption peak at around 340 nm, which is the same as
Tren-Fe(II) complex (Figure 4.13 ), but it has no major absorption from 400 nm to
600 nm, which is the Tren-QDs excitation (400 nm) and fluorescence emission (550
nm-590 nm)area. After addition of NaSH, however, the absorption drastically
increases in the area below 500 nm, and it has slight increased the absorbance from
500
nm
to
600
nm.
From
this
absorption
increase
we
speculate
a
Tren-QDs-CS2-Fe(II)-SH complex with strong inner filter effect on Tren-QDs was
formed. This sulfide containing complex has strong absorbance at 400 nm and
greatly reduced the excitation light of Tren-QDs and consequentially resulted in low
fluorescence emission. But the absorption at around 600 nm (Figure 4.18 insert)
could also play some role in the quenching mechanism.
0.025
3
0.02
Absorbance
Absorbance
2.5
2
0.015
0.01
0.005
1.5
0
500
510
520
530
540
550
560
Wavelength (nm)
570
580
590
600
1
0.5
0
300
350
400
450
500
550
600
650
Wavelength (nm)
Figure 4.18 UV-VIS spectrum of Tren-QDs-CS2-Fe(II) complex before (solid line)and
after(dotted line) NaSH addition
- 106 -
To confirm this hypothesis, the experiment was repeated at the excitation wavelength
480 nm, at which the Tren-QDs-CS2-Fe(II)-SH complex does not have absorption
peaks. The results shown in Figure 4.19 demonstrates the fluorescence emission of
Tren-QDs-CS2-Fe(II) was also quenched at excitation wavelength 480 nm (42.2%),
but the quenching was not as effective as the complex exited at 400 nm (73.6%). By
this comparison, we can conclude that the addition of NaSH solution caused
formation of Tren-QDs-CS2-Fe(II)-SH and this complex quench fluorescence of
Tren-QDs by strong inner filter effect which blocks both its excitation and emission
wavelength of Tren-QDs.
1
Normalized Fluorescence Intensity
0.9
0.8
0.7
0.6
Before NaSH addition
Affter NaSH addition
0.5
0.4
0.3
0.2
0.1
0
Excited at 400nm
Excited at 480nm
Different Excitation Wavelength
Figure 4.19 Quenching Tren-QDs-CS2-Fe(II) complex by NaSH at different excitation
wavelength
- 107 -
Because Tren-QDs fluorescence emission is pH sensitive, the pH value of the
complex solution was measured to exclude other possibilities of the emission
quenching. After addition, the original PBS 7.4 buffer solution was turned to a pH
10.2 solution. Since our previous study shows Tren-QDs are stable in pH ﹥7.4
environment, we can conclude the pH change is not responsible for the quenching.
Tren-QDs-CS2-Fe(II) was also found to be sensitive to another biologically
important specie nitro monoxide. The preliminary study showed addition of 0.5mL
saturated NO water solution (2× 10-6M) can efficiently quench Tren-QDs-CS2-Fe(II)
by 66.7% (Figure 4.20). The quenching was at first attributed to the formation of
Tren-QDs-CS2-Fe(II)-NO complex. But the pH value of final solution was found to
be acidified to pH 2.7, at which Tren-QDs’ hybrid structure is not stable, and
therefore makes the quenching mechanism complicated.
108.2
Tren-QDs-CS2-Fe(Ⅱ) complex
105
100
95
90
85
80
75
70
Tren-QDs-CS2-Fe(Ⅱ) complex+0.06 μM NO
65
60
55
50
Tren-QDs-CS2-Fe(Ⅱ) complex+0.12 μM NO
45
40
Tren-QDs-CS2-Fe(Ⅱ) complex+0.18μM NO
35
30
Tren-QDs-CS2-Fe(Ⅱ) complex+0.24 μM NO
25
20
Tren-QDs-CS2-Fe(Ⅱ) complex+0.28μM NO
15
10
7.3
500.0
510
520
530
540
550
560
570
580
590
600
nm
610
620
630
640
650
660
670
680
690
700.0
Figure 4.20 Spectra of Tren-QDs-CS2-Fe(II) complex quenched by nitric oxide solution
- 108 -
Tren-QDs-CS2-Fe(II) still requires investigation on application of other metal ions;
further characterization methods, like FT-IR may provide us more information of the
complex bonding; its components ratio could also be optimized to be a more
sensitive sensing probe; and the development of other primary amine containing
ligands other than Tren would also be necessary for more effective coordination of
metal ions on QDs surface.
Further investigation in our lab is on going to screen for possible alternatives on
Tren-QDs-CS2-Metal system. We believe more transition metal ions like Fe(II) could
be used to coordinate with Tren-QDs. Rational design and fabrication of these
QD-metal ion complex as fluorescent probes would be promising approach to radical,
hydrosulfide and NO sensing.
4.4 Conclusion
Fluorescent quantum dots (QDs) are an attractive alternative to fluorescent organic
dyes due to their broad excitation spectra, narrow symmetric and tunable emission
spectra, and excellent photostability. The functionalization of QDs confers this
promising labeling material unique properties which can be applied in special areas.
The present chapter described the application of tris(2-aminoethyl)amine (Tren)
functionalized quantum dots and its metal complex in ions and
hydrosulfide anion
sensing. Water-soluble Tren-QDs were found to be a kind of satisfactory selective
- 109 -
sensing fluorescence probe in physiological buffer solution. Fluorescence response
of Tren-QDs to sixteen physiologically important transition metal ions (Zn(II),
Cu(II), Ni(II), Co(III), Fe(III), Fe(II), Mn(III), Mn(II), Cr(III), Cr(II), VO2+, Ru(III),
Ru(II), Cd(II), Ag(I) and Pd(II)) was investigated. The fluorescence intensity of
Tren-QDs was significantly quenched by Co(III) and Cu(II) respectively. The
fluorescence quenching by Co(III) could be explained by Förster resonance energy
transfer between QDs and the surface Tren-Co(III) complex
upon the coordination
of Co(III) with Tren-QDs. Besides, the probable fluorescence quenching by Cu(II)
can be explained by a combination of energy transfer (minor) and electron (major)
effects. However, the other quenching pathway could be plausible, that is the
chemical displacement of surface Cd(II) by Cu(II) and the CuSe particles which can
be formed at the surface of CdSe core will quench the recombination fluorescence of
Tren-QDs by facilitating non-radiative electron and hole annihilation. Apart from
fluorescence quenching, fluorescence enhancement of Tren-QDs was achieved by
Zn(II) and Cd(II) respectively which is attributed to some form of passivation of the
surface trap states of QDs. Based on the distinct fluorescence response of Tren-QDs
to different transition metal ions, Tren-QDs can be developed to be a sensitive
fluorescent
probe
for
transition
metal
ions
detection.
Furthermore,
the
Tren-QDs-Fe(II)hybrid materials were used as hydrosulfide anion (HS-) sensing. The
fluorescence intensities of Tren-QDs-Fe(II) were further quenched significantly by
NaSH. And preliminary study has indicated this quenching was due to the formation
of excitation and emission blocking metal complex. Therefore, hybrid materials
- 110 -
could be developed as potential HS- sensors. In future work, the factors causing the
further significant quenching of fluorescence of transition metal and Tren-QDs
hybrid material would be investigated. Also, lifetimes study for Stern-Volmer
quenching and characterization of Tren-QDs by TEM would be carried out.
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[...]... labeling in bioanalysis and sensing The nature of the ligands being coordinated to the QDs surface and the particular type of bonds which it forms with the nanocrystal surface atoms are of great importance to quantum dots researchers By ligand designing and fabrication, several important properties of quantum dots can be tuned for specific purpose: processibility, reactivity and stability All of these... nitric oxide solution 108 -X- Chapter 1 General Introduction 1.1 Quantum Dots and Its Properties 1.1.1 Introduction to quantum dots Quantum dots (QDs) are nanostructured semiconductor materials[1] These colloidal nanocrystalline semiconductors comprising elements from the periodic groups II-VI, III-V or IV-VI, are featured with roughly spherical and with typical sizes (diameter) in the range 1-12 nanometer... - Chapter 2 Thiolated Caffeic Acid Functionalized Quantum dots 2.1 Introduction Semiconductor nanocrystals (or quantum dots, QDs) have size-dependent fluorescent emissions wavelengths with high quantum yields and superior photostability comparing to organic dyes[1] Thus QDs have great potential in various biological imaging applications, including labeling cells and other biological components However,... visible signal when it reacts with free radicals or other species and serves as a fluorescent probe Our goal is to synthesize and purify thio-caffeic acid for quantum dot ligand exchange and try to replace the original hydrophobic trioctylphosphine oxide (TOPO) ligand of quantum dots with thio-caffeic acid 2.2 Experimental Section 2.2.1 Materials and instruments All solvents used were of reagent grade unless... Quantum dots ligand exchange with thio-caffeic acid The 100 nM quantum dots solution in 2mL chloroform and then added into 1mL 100 times equivalent amount of thio-caffeic acid diethyl ether solution dropwise This mixture was vigorously stirred under nitrogen for 12 hours at room temperature in darkness When the reaction was stopped, it was found the quantum dots precipitated out from solution The quantum. .. thiol ligands were found to undergo a photocatalytic oxidation using CdSe nanocrystals as photocatalysts and form the disulfides during the process But severity of this oxidation varies with different thiol ligands, and some of them can sustain long enough in solution for practical applications[ 19] Another disadvantage of Thiol ligands is that it can cause severe fluorescence quenching of quantum dots. .. from the particle, thus providing a visible signal of its occurrence In this work, we used derivatives of antioxidant caffeic acid and Trolox to study the electron transfer between small molecule ligands and CdSe/ZnS quantum dots 1.2.1 Ions and small molecules sensing Methods based on chemical or physical interactions between target chemical species and the surface of the nanoparticles are very simple... semi-conductor material and nanocrystal size -1- Semiconductor QDs are characterized by a band-gap between their valence and conduction electron bands (Figure 1) When a photon having an excitation energy exceeding the semiconductor band-gap is absorbed by a QD, electrons are promoted from the valence band to the high-energy conduction band The excited electron may then relax to its ground state by the... these nanoparticles behave differently from bulk solids due to quantum confinement effects [2,3] The result of quantum confinement are that the electron and hole energy states within the nanocrystals are discrete, but the electron and hole energy levels and therefore the band-gap is a function of the QDs diameter as well as composition[5] The band-gap of semiconductor nanocrystals increase as their size... neurotransmitter dopamine and CdSe/ZnS QDs[22]; Maurel et al reported a non-linear quenching effects of fluorescent quantum dots by nitroxyl free radicals TEMPO (2,2,6,6-tetramethylpiperidine-N-oxide free radical) and suggested the -7- mechanism would involve electron transfer from the conduction band to the nitroxide (a mild acceptor), and back electron transfer from the nitroxide to the valence band, effectively ... Introduction 1.1 Quantum Dots and Its Properties 1 1.1.1 Introduction to quantum dots 1.1.2 Quantum dots optical properties 1.2 Quantum Dots in Optical Sensing Applications 1.2.1 Anion and small molecules... Pergamon Press, Oxford, 1979 - 34 - Chapter Trolox Functionalized Quantum Dots 3.1 Introduction 3.1.1 Quantum dots and its synthesis Quantum dots (QDs) are spherical semiconductor nanoparticles... several ligands bounding to the surface of ZnS capped CdSe quantum dots (QDs) was studied Different ligands for quantum dots functionalization was synthesized and characterized by ESI-MS, NMR and FT-IR