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CdSeZnS Nanocrystals with Dye-Functionalized Polymer Ligands Containing Many Anchor Groups.

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Angewandte
Chemie
Fluorescent Nanocrystals
CdSe/ZnS Nanocrystals with Dye-Functionalized
Polymer Ligands Containing Many Anchor
Groups**
Inga Potapova, Ralf Mruk, Christian Hbner,
Rudolf Zentel, Thomas Basch, and Alf Mews*
Colloidal semiconductor nanocrystals (NCs) are color tunable inorganic fluorophores, which consist of an inorganic core
and an outer shell of molecular ligands.[1] While the optical
properties can be adjusted by the composition[2] and the size
of the core,[3] the ease with which the NCs can be chemically
processed is mainly governed by the molecular structure of
the ligands. In this context we define a ligand as consisting of
three parts: a functional anchor group that attaches to the
inorganic NC surface, an outer functional group, and a spacer
in between. The architecture of the ligand is of major
importance for assembling the NCs into larger supramolecular structures[4] or conjugating them to biological samples
where they serve as fluorescence labels.[5] For these purposes
it is essential that both the anchor and outer functionality can
form strong chemical bonds. We will address this problem by
introducing a novel type of polymer ligand, where the bond
strength is increased by the presence of multiple anchor
groups. Since our model ligands are decorated with dye
molecules, we can employ fluorescence resonance energy
transfer (FRET) to study the formation and stability of the
NC–dye couples, where the NCs are the donors and the dyes
the acceptors[6, 7] . Based on the ensemble absorption and
fluorescence spectra, the amount of dye attached to a given
NC[6] and the average distance between the NC and the dye
molecules can be estimated.[8] However, since the relative
orientation and structure of the FRET couples can differ
significantly from one complex to the next, we extend our
measurements to single NC–dye complexes. Our experiments
clearly show that static and dynamic heterogeneities occur in
the ensemble of NC–dye couples, a result which is of
importance for a FRET-based analysis of intermolecular
distances.
The CdSe/ZnS core–shell NCs are synthesized by an
established method[3, 9] in the presence of phosphonic acids[10]
and trioctylphosphine oxide (TOPO) as ligands. The surface
molecules can be subsequently cross linked to form multianchor ligands to stabilize the ligand shell.[11, 12] Herein we
follow a different strategy by prefabricating a multianchor
[*] Dr. I. Potapova, Dr. C. Hbner, Prof. Dr. T. Basch, Dr. A. Mews
Institute for Physical Chemistry
University of Mainz
55099 Mainz (Germany)
Fax: (+ 49) 6131-39-23953
E-mail: alf.mews@uni-siegen.de
Dr. R. Mruk, Prof. Dr. R. Zentel
Institute for Organic Chemistry
University of Mainz, 55099 Mainz (Germany)
[**] This work was supported by a grant from the SFB 625 (A10 and B7).
Angew. Chem. Int. Ed. 2005, 44, 2437 –2440
DOI: 10.1002/anie.200462236
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2437
Communications
Scheme 1. Stepwise build-up of a dye-labeled multianchor ligand. The reactive ester groups of the polymer chain (n 200) are successively
exchanged by (x 1) Texas Red molecules (TexRd), and (y 100) Boc-protected butyldiamine and hexylamine groups. After deprotection the functional ligand carries amino groups as anchors for the NCs, alkyl chains for solubility, and on average one dye molecule. DMF = N,N-dimethylformamide.
ligand, which is based on a statistical copolymer. The synthesis (Scheme 1) starts from a polymer chain of reactive
esters (n 200),[13] which is labeled with one dye molecule on
average by reaction with the amino group of Texas Red
cadaverine.[14] The next step is the replacement of approximately 50 % of the reactive esters within each polymer
molecule with mono-Boc (Boc = tert-butoxycarbonyl) protected diamine groups, subsequently the remaining ester
groups are substituted by mono-amines. After deprotection,
the polymer ligands have amine groups as anchors for the NC
surface, long alkyl chains to provide solubility in hydrophobic
solvents, and about one dye molecule for optical tracing and
energy-transfer experiments.
Although we had evidence that the functionalized ligands
can also bind to the TOPO covered NCs (see below), we
performed experiments where the attachment can be monitored directly by a solubility change. In these experiments we
stripped most of the TOPO molecules from the NC surface by
several cycles of successive precipitation and dissolution of
the particles in pyridine.[15] These particles could only be
dissolved in chloroform upon addition of the dye-labeled
polymer. This behavior is direct evidence for the attachment
of the polymer to NCs, because the alkyl chains of the
polymer provide solubility in chloroform and not in pyridine.
Another indication for the formation of the NC–dye
complexes comes from the spectroscopic observation of
FRET between the NCs and the dyes. The FRET process,
which is operative on a length scale of several nanometers,
leads to a decrease of the donor (NC) fluorescence FD to a
fluorescence intensity FDA in the presence of an acceptor, and
an increase of the acceptor (dye) fluorescence. From the
spectral overlap between the emission spectrum of the NCs
and the absorption spectrum of the dyes (gray shaded area in
Figure 1 a) a Frster radius of R0 = 4.6 nm can be calculated at
which the transfer efficiency should be E = 1 FDA/FD, as
described in detail in ref. [6] This calculation has assumed a
dipole orientation factor between the dyes and the NCs of
k2 = 2=3 ,[16, 17] which is not justified in advance.
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2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. a) Absorption (solid lines) and fluorescence (dotted lines)
spectra of the NC (green) and the dye (red) in solution. The spectral
overlap of the NC fluorescence (dotted, green) and the dye absorption (solid, red) is shaded in gray. b) Fluorescence spectra of the
NCs (green), dye (red), NC–polymer–dye (dark blue), and
NC–dye (light blue), respectively, upon excitation at l = 488 nm. Dye
fluorescence can only be observed upon energy transfer within the
NC–dye and NC–polymer–dye complexes.
Whereas the absorption spectra of NC–dye and NC–
polymer–dye are just superpositions of the spectra of the
respective compounds, the corresponding fluorescence spectra shown in Figure 1 b change by complex formation. In
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Angew. Chem. Int. Ed. 2005, 44, 2437 –2440
Angewandte
Chemie
Figure 1 b the fluorescence spectra of the pure NCs (green)
and dyes (red) are compared to those of an NC–polymer–dye
(dark blue) and an additional composite of NCs and Texas
Red without the polymer linker (NC–dye, light blue). Without the polymer linker the dye either binds to the NC surface
through its amino group or by nonspecific binding.[18] The
NC–dye composites were prepared by mixing the TOPO–
NCs and the dye ligands in chloroform with NC:dye concentration ratios of 1:1. These ratios were judged from the UV/
Vis spectra of the isolated compounds.[19, 20] The fluorescence
spectra were taken at an excitation wavelength of 488 nm,
where the absorption is strong for the donor (NC, green) and
weak for the acceptor (dye, red), as can be seen in Figure 1 a.
This approach leads to a strong NC fluorescence and a weak
dye fluorescence for the isolated compounds. In contrast, the
NC–dye composites show enhanced dye fluorescence and
reduced NC fluorescence, which is a signature of electronic
excitation-energy transfer from the NCs to the acceptor dye(s).
Although the spectra presented in Figure 1 b serve as a
clear indication for a FRET-type process, quantitative information is difficult to extract. Besides uncertainties in the
calculation of the Frster radius R0, the transfer efficiencies,
as estimated from the spectra, depend on an accurate
determination of the respective NC:dye ratios,[6] on the
specificity of the anchor group,[7, 18] and even on the reaction
time.[21] We found that the measured transfer efficiency
increases within the first minutes, probably owing to the
diffusion-limited reaction between the dyes and the NCs. This
increase is followed by a decrease over the next few hours.
Because the dye absorption also decreases over this time, this
change is most likely a result of a time-dependent degradation
of the dyes. As a general trend, the NC–polymer–dye
composite was much more stable than the NC–dye composite
against dye degradation. The decomposition of the dye is
thought to be induced by charge (hole)-transfer process from
the NCs to the dyes,[19] which should strongly depend on the
NC–dye separation. Thus the polymer backbone that
increases the NC–dye separation, leads to a spatial and
energetic barrier to the charge transfer. In the same way the
polymer spacer also leads to a reduced FRET efficiency which
can be seen in the spectra in Figure 1.
In addition to the complications mentioned above, there
could be several sources of inhomogeneity within the
ensemble of FRET pairs, which can only be addressed by
investigating individual luminescing complexes.[22] We therefore performed fluorescence measurements with a scanning
confocal optical microscope on single NC–dye and NC–
polymer–dye structures blended in thin polymer films.[23] The
image shown in Figure 2 a represents the total fluorescence of
individual NC-dye complexes without the polymer spacer. To
visualize the energy-transfer efficiency, we plotted the
dichroic ratio (D = (INC Idye)/(INC+Idye)) of the same area
(Figure 2 b), that is, emission predominantly from the NCs
appears in green, whereas red spots reflect emission of the
dyes. The red spots in this image are attributed to dye
molecules, that were excited by energy transfer from the NCs,
because samples of the dye molecules alone showed no
detectable fluorescence signal under the same experimental
conditions.
Angew. Chem. Int. Ed. 2005, 44, 2437 –2440
Figure 2. a) Fluorescence image and b) dichroic ratio of the NC–dye
complexes upon excitation with l = 488 nm (red: high-intensity dye
fluorescence; green: high-intensity NC fluorescence). The transfer efficiency can vary greatly between single complexes. c) Integral transient
fluorescence intensity of a single NC–polymer–dye complex and
d) comparison of the transient total intensity (black), dye fluorescence (red), and NC fluorescence (green). The off periods of the NCs and
dyes occur simultaneously and the transfer efficiency fluctuates during
the on time.
Clearly there are several complexes where energy transfer
cannot be observed at all, which is most likely due to the
stochastic labeling process. Following Poisson statistics about
37 % of the particles contain no dye molecule at all and about
8 % are labeled with more than two dye-molecules and
therefore show a higher transfer efficiency.[6] The variation of
transfer efficiency can also be attributed to the formation of
small aggregates, variations in ligand geometries,[24] or the
random distribution of transition dipole alignments between
the NCs and dyes.[25] Further it can be seen in Figure 2 b that
sometimes the lower part of the spots is red and the upper
part is green, which is due to dye degradation during the scan
(the image is built up line-by-line from bottom to top). This
behavior could not be seen for the NC–polymer–dye complexes, highlighting again the higher photostability of this
compound. Otherwise, the fluorescence images of the NC–
polymer–dye composites were almost identical to those in
Figure 2 b and are not shown.
Only the confocal fluorescence microscopy experiments
on single fluorescing complexes allowed the temporal fluctuations of the FRET efficiencies to be investigated. These
experiments were only performed with NC–polymer–dyes,
owing to their higher photostability. The transient total
emission intensity shown in Figure 2 c exhibits the typical
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2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2439
Communications
fluorescence on–off blinking behavior, which can also be
observed for isolated NCs not interacting with dye molecules.[26] The off periods of the fluorescence of isolated NCs
are explained by a very fast quenching process of the excited
state, possibly a result of temporary charging of the NCs.[26]
Since this quenching process (tnr 100 ps)[27] is much faster
than the estimated energy-transfer time, which is in the range
of the NC fluorescence lifetime (tfl 10–30 ns),[28, 29] the off
states of NC and dye in a complex appear simultaneously. In
addition it can be seen from Figure 2 d that even within the on
state of an individual NC–polymer–dye donor–acceptor pair
the transfer efficiency is fluctuating in time. Although such
fluctuations can be readily observed by studying single
complexes, it is difficult to determine their origin because a
number of parameters (e.g. separation, orientation, spectral
overlap) can influence the energy-transfer efficiency.
In summary we have introduced a versatile ligand which
can be modified by a large number of anchors and functional
groups. Decoration of the ligand with dye molecules allowed
us to investigate electronic excitation energy transfer between
the NCs and the attached chromophores. NC–polymer–dye
composites were found to be of higher photostability than
NC–dye composites which allowed FRET experiments on
single composites to be performed. These experiments
revealed substantial inhomogeneities within the ensemble of
NC–polymer–dye complexes and even dynamic fluctuations
of the electronic interactions for single NC–polymer–dye
composites, which must be taken into account when interpreting FRET results for separation determinations.
Experimental Section
Synthesis of aminopolymers: Poly(N-acryloyloxysuccinimide)
(245 mg; 1.45 mmol repeating units) was solved in DMF (12 mL). A
solution of Texas-red cadaverine (5.0 mg; 0.007 mmol) in DMF
(4 mL) was added. The resulting solution was stirred for 5 h at 50 8C.
After that a solution of 1-Boc-1,4-diaminobutane (136.5 mg;
0.725 mmol) in DMF (4 mL) was added and the solution again stirred
for 5 h at 50 8C. After addition of hexylamine (2.0 mL) the solution
was stirred for further 16 h at 50 8C. The solution was concentrated to
about 5 mL in vacuum and poured onto water. After the precipitation
of the polymer the mixture was centrifuged and the solvent was
decanted. After drying, 286 mg of a red solid was obtained. 1H NMR
(CDCl3, 200 MHz): d = 3.07 (CH2 a to NH), 2.48 (CH, main chain),
1.47 (CH2, main chain), 1.40 (CH3, Boc group), 1.25 (CH2, side chain),
0.85 ppm (CH3, hexyl chain).
Cleavage of the Boc group: The polymer was dissolved in CH2Cl2
(30 mL). After that trifluoroacetic acid (2.0 mL) was added. The
mixture was stirred at room temperature for 2 h. After that 2 n HCl
(40 mL) was added and the phases separated. The organic phase was
extracted with 2 n HCl (2 20 mL). The combined aqueous phases
were adjusted to pH 13 by addition of 4 n NaOH solution. After
heating, the polymer precipitated as a red solid that was purified by
repeated stirring in hot water. The reaction yields 102 mg of the
polymer. 1H NMR (CDCl3, 200 MHz): d = 3.11 (CH2 a to NH), 2.69
(CH, main chain), 1.49 (CH2, main chain), 1.26 (CH2, side chain),
0.86 ppm (CH3, hexyl chain).
Optical Characterization: Confocal fluorescence images and
spectra were obtained at room temperature using a home-built
microscope equipped with a piezo scanner (Physical Instruments) and
a high NA microscope objective (100 , NA = 1.4). Circularly
polarized laser light (l = 488 nm) was focused down to a diffractionlimited spot size corresponding to an excitation intensity of
2440
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
200 W cm 2. To obtain the images, the sample was raster-scanned
through the objective and the fluorescence light was guided to EG&G
avalanche photo diodes with optical filters being used to eliminate the
scattered light. The images are of size 256 256 pixels obtained with
an integration time of 10 ms/pixel.
Received: October 7, 2004
Published online: March 17, 2005
.
Keywords: cadmium selenide · dye labeling · energy transfer ·
nanocrystals · polymer ligands
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