Biotinylated CdSe/ZnSe nanocrystals for specific fluorescent labeling

Article abstract of DOI:10.1039/b403714f

A set of two new surface ligands is presented for the preparation of water-soluble, biotinylated CdSe/ZnSe core/shell nanocrystals, suitable for fluorescent biological labeling. It consists of a thiolated diethyleneglycol derivative and an alkylthiol substituted biotin molecule, which replace the initial capping ligands at the nanocrystal surface. Successful ligand exchange and long-term photostability of the modified nanocrystals as well as their highly specific binding to neuronal cells are demonstrated in different labeling experiments.

Full text of DOI:10.1039/b403714f

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A R T I C L E
Biotinylated CdSe/ZnSe nanocrystals for specific fluorescent
labeling
Nicolas Charvet,a Peter Reiss,*a Andre´ Roget,a Alain Dupuis,b Didier Gru¨nwald,b
Sophie Carayon,a Fre´de´ric Chandezona and Thierry Livachea
aCEA Grenoble, De´partement de Recherche Fondamentale sur la Matie`re Condense´e,
17 rue des Martyrs, 38054 Grenoble, France. E-mail: preiss@cea.fr; Fax: 133 438 78 51 13;
Tel: 133 438 78 97 19
bCEA Grenoble, De´partement Re´ponse et Dynamique Cellulaires, 17 rue des Martyrs,
38054 Grenoble, France
Received 10th March 2004, Accepted 27th May 2004
First published as an Advance Article on the web 18th June 2004
A set of two new surface ligands is presented for the preparation of water-soluble, biotinylated CdSe/ZnSe
core/shell nanocrystals, suitable for fluorescent biological labeling. It consists of a thiolated diethyleneglycol
derivative and an alkylthiol substituted biotin molecule, which replace the initial capping ligands at the
nanocrystal surface. Successful ligand exchange and long-term photostability of the modified nanocrystals as
well as their highly specific binding to neuronal cells are demonstrated in different labeling experiments.
Introduction
Results and discussion
Fluorescent semiconductor nanocrystals have attracted much
attention in the field of biological labeling due to their unique
optical properties combined with high photo-stability. Core/
shell structures of CdSe/ZnS, CdSe/ZnSe or CdSe/CdS type are
of special interest, as they exhibit efficient photoluminescence
in the visible part of the spectrum.1–4 Synthesized by organo-
metallic routes in non-aqueous solvents, these nanocrystals are
hydrophobic because surfactant molecules (e.g. trioctylpho-
sphine oxide, TOPO) cap their surface and prevent them from
aggregating. In order to achieve nanocrystal hydrosolubility –
indispensable for their use in biological labeling – different
strategies have been applied in the literature:
1.) Substitution of the original surface ligands by (di)mer-
captocarboxylic acids; nanocrystals can be dispersed in water
after deprotonation of the carboxylic acid group by increasing
the pH.5,6 Disadvantages of this procedure are the pH-
sensitivity of the colloidal dispersion and the high tendency
for non-specific interactions of the (charged) carboxylate
groups with biomolecules such as proteins, DNA, etc.
2.) Growth of an additional silica shell on the semiconductor
shell.7,8 Though providing very high nanocrystal stability, a
rather complicated series of synthesis and purification steps
and a difficult control of the resulting surface chemistry are
drawbacks of this approach.
3.) Encapsulating the nanocrystals with micelles,9 cross-
linked polymers10 or charged proteins.11 These techniques do
not replace the original surface ligands and therefore preserve
essentially the initial fluorescence quantum yield (Q.Y.). On the
other hand, the diameter of the nanocrystals increases
drastically and lies typically in the range of 12–25 nm. This
may be too bulky for certain applications due to the reduced
capability of the nanocrystals to access target systems. Further-
more, as a consequence of their large size, the number of
nanocrystals which can bind to a target molecule is reduced,
which may decrease imaging sensitivity.
In this article, we present a simple and straigthforward
method to overcome the cited problems. Two new surface
ligands are synthesized, which minimize non-specific inter-
actions because of uncharged solubilization functions, while
maintaining essentially the original nanocrystal diameter.
CdSe/ZnSe nanocrystals12 are dispersed in water after function-
alization with the specially designed ligand 1 (Scheme 1),
namely a derivative of diethyleneglycol where one alcohol
group has been replaced by a thiol group. The latter binds to
the nanocrystal surface, while the two ether groups as well as
the remaining alcohol function ensure hydrosolubility. The
substantial role of the ether groups becomes clear in control
experiments where 1-mercaptohexanol is used instead of ligand
1, leading to nanocrystals which can be dispersed in alcohols
but not in water. The neutral alcohol group in 1 minimizes non-
specific electrostatic interactions, but does not allow for the
conjugation of nanocrystals with biomolecules via crosslinking
agents. For this reason we synthesized ligand 2 (Scheme 1), an
alkylthiol substituted biotin molecule.
Once ‘‘biotinylated’’ by means of this ligand, nanocrystals
can easily be conjugated with biomolecules, using the specific
and strong interaction between biotin and streptavidin. As a
result of the relatively short chain length of ligand 1, the biotin
moieties of ligand 2 can stick out the organic capping layer,
enhancing its accessibility by streptavidin binding sites. In
order not to affect the hydrosolubility of the nanocrystals, we
used low molar fractions (typically 5 mol%) of ligand 2 with
respect to ligand 1 during nanocrystal functionalization. For
molar fractions exceeding 10%, nanocrystals do not redisperse
completely in buffer solutions.
Functionalization of CdSe/ZnSe nanocrystals with ligand 1
enables their dispersion in water or in buffer solutions. To
study the influence of the buffer system, different 50 mmolar
solutions have been compared. Our results indicate that TRIS
(pH 8), HEPES (pH 7.4) and especially borate buffers (pH 8.5)
provide better colloidal and photophysical stability of the
functionalized nanocrystals than phosphate based buffers such
as phosphates (pH 7.4 and 8.5) and phosphate buffered saline
(PBS; pH 7.4, 10 mmolar solution). Colloidal stability has been
verified by repeated centrifugation of the samples in intervals of
several days in order to detect partial precipitation. After one
month keeping the functionalized nanocrystals in borate buffer
solution, no precipitate and no altering of the fluorescence
properties could be observed. It should be underlined here that
the pH of the solution can be chosen in a range varying from
2 6 3 8
J . M a t e r . C h e m . , 2 0 0 4 , 1 4 , 2 6 3 8 – 2 6 4 2
T h i s j o u r n a l i s ß T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4

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Scheme 1 Synthesis pathways of ligands 1 and 2, used for the preparation of water-soluble, biotinylated core/shell nanocrystals. For details and
characterization data see Experimental section.
slightly basic to slightly acidic values (pH 5–9) without pre-
cipitation of the nanocrystals. This is an important advantage
over methods where hydrosolubility is assured by charged
groups, such as carboxylate or ammonium groups. On the
other hand, a decrease of the fluorescence Q.Y. of the CdSe/
ZnSe nanocrystals upon ligand exchange and dispersion in
water is observed.13 We attribute this to hole trapping by the
thiol ligands, which could be additionally favoured by the
relatively weak confinement of the holes in the CdSe/ZnSe core/
shell structure. As will be shown in the following, the fluo-
rescence efficiency remains however largely sufficient and stable
for labeling experiments. In Fig. 1, optical properties of the
nanocrystals before and after functionalization are displayed.
If the nanocrystals are successfully functionalized with both
ligand 1 and ligand 2, they can selectively bind to streptavidin.
As a control experiment, biotinylated agarose beads with a
mean size of 50 mm are first incubated with streptavidin and
then with the functionalized nanocrystals. After thoroughly
rinsing, the agarose beads exhibit a bright fluorescence, which
clearly proves the successful binding of the nanocrystals to the
beads. To exclude non-specific interaction of the nanocrystal
surface ligands with the beads, a large excess of biotin has been
added without any detectable decrease of the beads’ fluores-
cence. If, however, nanocrystals carrying exclusively ligand 1
and not ligand 2 (no biotin groups) are used for incubation, no
fluorescence of the beads is detectable after washing. In an
additional experiment the stability against photobleaching of
the nanocrystal labeled beads is compared to the one of beads,
which are labeled with R-Phycoerythrin, one of the most
commonly-used fluorescent dyes. Both samples are continu-
ously irradiated and photos are taken at different time intervals
(Fig. 2). While the fluorescence of the Phycoerythrin labeled
beads is practically extinguished after 15 minutes, the emission
of the nanocrystal labeled ones can still be detected after
8 hours, demonstrating a significantly higher photostability.
Finally we present first results where the water-soluble,
biotinylated nanocrystals are used for labeling of nerve cells in
primary cell culture. For this issue, neuronal cells from mouse
embryo cerebellum (stage E13), grown for 5 days on polylysin
glass covers, are fixed in 4% paraformaldehyde and are made
permeable with triton X100. The primary antibody TuJ1 from
neuronal microtubules is reacted with a biotinylated secondary
antibody, which allows, after incubation with streptavidin, the
binding of biotinylated nanocrystals. Positive control experi-
ments are carried out either by replacement of the streptavidin–
biotin–nanocrystal complex by Cy3 labeled streptavidin or by
direct revelation of TuJ1 antibody with a common fluorescent
dye (Alexa-488, Molecular Probes), fixed on the secondary
antibody. Negative control experiments are realized by omis-
sion of the TuJ1 antibody from the experiment. Fig. 3a reveals
J . M a t e r . C h e m . , 2 0 0 4 , 1 4 , 2 6 3 8 – 2 6 4 2
2 6 3 9

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Fig. 3 (a) Fluorescence image of nerve cells in primary culture
labeled with biotinylated nanocrystals (excitation wavelength 458 nm).
(b) Control experiment with Alexa-488 labeled cells.
fluorescent labeling occurs also on the cell interior, where the
TuJ1 antibodies are located. This underlines the potential of
functionalized nanocrystals to act as efficient intracellular
fluorescent probes after triton X100 permeabilization.
Conclusion
We have presented a set of two new ligands, specially designed
for the use of CdSe/ZnSe and similar types of nanocrystals in
biological labeling: the first one providing hydrosolubility and
the second one allowing for specific binding to streptavidin via
a biotin group. The functionalized nanocrystals exhibit long-
term colloidal stability in common buffer systems such as
borate solutions. Successful functionalization of nanocrystals
with the two ligands has been shown by labeling of streptavidin
modified agarose beads. The stability of the fluorescence of
these beads against photobleaching is increased by more than
one order of magnitude as compared to R-Phycoerythrin
labeled ones. Staining of nerve cells demonstrates the very low
tendency for non-specific binding of the biotinylated nano-
crystals as a consequence of the use of uncharged groups to
assure hydrosolubility. This underlines the high potential for
their use in intracellular labeling.
Fig. 1 (a) UV-vis absorption spectra of the CdSe/ZnSe core/shell
nanocrystals used in this study. (b) Photoluminescence spectra of the
same nanocrystals (excitation wavelength 365 nm).
unequivocally the specific targeting of the nerve cells by the
nanocrystals, as can be concluded from the comparison with
the control (Fig. 3b). In contrast to a similar experiment where
carboxylated CdS nanocrystals were attached to neurons,14
Experimental
Synthesis of ligand 1 (8-thio-3,6-dioxaoctanol) (ref. 15)
In a 50 mL two-neck flask equipped with a condenser, 5 g
(29.6 mmol) of 2-(2-(2-chloroethoxy)ethoxy)ethanol (Aldrich,
Ref. 16,297-3, 98%) and 2.97 g (39 mmol) of thiourea (Aldrich,
Ref. 24,025-7, 991%), dissolved in 20 mL of ultrapure water,
are stirred under reflux for 2 hours. The mixture becomes
homogeneous after a few minutes. A solution of 2.5 g of
sodium hydroxide in 25 mL of water is added and the mixture is
refluxed for a further 2 hours. After cooling, the aqueous layer
is acidified with a solution of 6 mL of concentrated sulfuric acid
in 4 mL of water, and extracted with ethyl acetate (5 6 25 mL).
The collected organic phases are dried over sodium sulfate,
filtered, evaporated under reduced pressure and purified by
flash chromatography on silica (chloroform/methanol, 97.5/
2.5, Rf: 0.5) to give compound 1 as a colourless and odourless
oil (2.43 g, 50%).
d1 (200.13 MHz, CDCl3) 3.76–3.60 (10H, m, CH2O), 2.71
H
(2H, dt, J ~ 8.06 Hz and J ~ 6.19 Hz, H6), 2.52 (1H, m, OH),
1.60 (1H, t, J ~ 8.06 Hz, SH).
d13C (56.32 MHz, CDCl3) 72.87 (C2), 72.48 (C5), 70.36 (C3),
70.27 (C4), 61.78 (C1), 24.21 (C6).
Fig. 2 Fluorescence images of agarose beads labeled with biotinylated
nanocrystals (top row) and with biotinylated R-Phycoerythrin (bottom
row). The mean size of the beads is 50 mm. Under continuous
irradiation with a mercury lamp (100 W), photos are taken at different
time intervals using an Olympus Microscope equipped with an
U-MWU band pass filter (cut-off 330–385 nm).
m/z (Fab1) 167 (M 1 H)1, 184.
C6H14O3S, found: C, 43.32%; H, 8.32%; S, 19.30%, requires
C, 43.35%; H, 8.49%; S, 19.29%.
2 6 4 0
J . M a t e r . C h e m . , 2 0 0 4 , 1 4 , 2 6 3 8 – 2 6 4 2

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Synthesis of ligand 2
(dichloromethan/methanol, 90/10, Rf: 0.26). The pure product
is isolated as a colorless oil which solidifies with time (187 mg,
30%).
Biotin N-hydroxysuccinimide ester A (ref. 16). To a stirred
solution of biotin (Sigma, Ref. B-4501, 99%, 5 g, 20.47 mmol)
and N-hydroxysuccinimide (Aldrich, Ref. 13,067-2, 97%,
2.36 g, 20.47 mmol) in anhydrous DMF (150 mL), 1.3 eq. of
1,3-dicyclohexylcarbodiimide (Aldrich, Ref. D8,000-2, 99%,
5.49 g, 24.44 mmol) is added. The mixture is stirred at room
temperature for 48 h. The formed dicyclohexylurea is filtered
off and the solvent is evaporated under reduced pressure to
dryness. 500 mL of diethyl ether are added to the residue and
the solution is stirred for 2 h, giving a white precipitate. After
filtration, this solid is recrystallized in isopropanol, yielding the
pure product as a white powder (6.5 g, 93%).
d1 (200.13 MHz, CDCl3) 6.75, 6.55, 6.44, 5.93 (4 6 1H,
H
4 6 br, NH, SH), 4.52 (1H, m, H1), 4.35 (1H, m, H4), 3.70–3.50
(12H, m, HG–L), 3.40–3.30 (4H, m, HN 1 HE), 3.16 (1H, m,
H3), 2.96–2.88 (2H, m, H2), 2.52 (2H, m, HX), 2.17 (2H, m, HO),
1.85–1.50 (14H, m, HA,B,C,F,M,P,W), 1.50–1.20 (12H, m, HQ–V).
d13 (56.32 MHz, CDCl3) 173.4, 173.2, 172.6, 164.0, 70.4,
C
70.0, 69.8, 61.9, 60.3, 55.5, 40.5, 37.8, 37.6, 36.8, 35.9, 34.0,
29.4, 29.3, 29.1, 29.0, 28.3, 28.1, 28.0, 25.8, 25.5, 25.4, 24.6.
m/z (DCI NH3 1 Isobutane) 647 (M 1 H)1.
d1 (200.13 MHz, CDCl3) 4.31 (1H, m, H1), 4.16 (1H, m,
Preparation of water-soluble, biotinylated nanocrystals
H
Ligand 2 (20 mL, 250 g L21 in dry chloroform) and ligand 1
(24.4 mg) are mixed with CdSe/ZnSe nanocrystals3 (core
diameter 3.7 nm, shell thickness 0.7 nm; 500 mL, 8 g L21 in dry
chloroform) in a 2 mL microtube under argon (molar ratio ~
95 mol% of ligand 1 and 5 mol% of ligand 2). The mixture is
stirred for 48 h at 30 uC in the dark. Then water (500 mL) and
acetone (100 mL) are added and the microtube is vigorously
shaken for 1 h. The aqueous layer is separated and evaporated
to dryness to give a colored solid, which is redispersed in water
(200 mL) and centrifuged (45 s, 10,000 rpm). The supernatant is
filtered with a 30000 d Vivaspin# filter, rinsed twice with water
(100 mL), vacuum-dried and stored at 4 uC.
H4), 3.10 (1H, m, H3), 2.89 (1H, m, H2), 2.81 (5H, s, HE 1 H2’),
2.67 (2H, m, HD), 1.80–1.35 (6H, m, HA,B,C).
m/z (DCI NH3 1 Isobutane) 342 (M 1 H)1.
N-(13-Amino-4,7,10-trioxatridecanyl) biotinamide B (ref. 17).
Biotin N-hydroxysuccinimide ester A (4 g, 11.71 mmol) is
dissolved in 100 mL of dry DMF. Under inert atmosphere, this
solution is added dropwise within 1 h to a solution of 4,7,10-
trioxa-1,13-tridecanediamine (Aldrich, Ref. 36,951-9, 97%,
12.91 g, 58.6 mmol) in 4 mL of triethylamine. After stirring
the reaction mixture at room temperature for 72 h, the solid
formed is filtered off and DMF is evaporated under reduced
pressure. The resulting oil is added dropwise to 1 L of hexane.
A white precipitate is formed after a few minutes. It is
recrystallized in isopropanol (2 h at reflux, 12 h without heating
and stirring), yielding 3.55 g of the pure product as white
crystals (67%).
Labeling of agarose beads with biotinylated nanocrystals
Biotin–agarose beads (Sigma, Ref. B 0519) in PBS (50 mL) are
rinsed twice with 100 mL of PBS (Sigma, Ref. P 4417). After
centrifugation, the surpernatant is removed. A solution of
streptavidin (Sigma, Ref. S 4762) in PBS (50 mL, 2.5 mg mL21
)
is added to the beads and the reaction mixture is kept at room
temperature during 15 minutes. The beads are then rinsed twice
with PBS (100 mL). Biotinylated nanocrystals are dispersed
in 50 mL of ultrapure water. 5 mL of this dispersion are
poured into the solution of streptavidin-conjugated beads.
After 5 minutes, the beads are rinsed twice with PBS (30 mL)
and placed on microscopy slides.
Fp: 111–113 uC
d1H (200.13 MHz, D2O) 4.40 (1H, m, H1), 4.25 (1H, m, H4),
3.60–3.35 (12H, m, HG–L), 3.25–3.10 (3H, m, H3 1 HE),
2.90–2.81 (2H, m, H2), 2.67 (2H, m, HN), 2.12 (2H, m, HD),
1.75–1.50 (8H, m, HA,B,C,M), 1.42 (2H, m, HF).
m/z (DCI NH3 1 Isobutane) 447 (M 1 H)1.
11-Mercaptoundecanoyl-N-hydroxysuccinimide ester C (ref. 18).
N-Hydroxysuccinimide (Aldrich, Ref. 13,067-2, 97%, 1.00 g,
8.7 mmol) is stirred in dichloromethane (500 mL) for 30 min.
11-Mercaptoundecanoic acid (MUA) (1.88 g, 8.62 mmol),
dissolved in 10 mL of dichloromethane, is poured into the first
solution. Then 1,3-dicyclohexylcarbodiimide (DCC) (Aldrich,
Ref. D8,000-2, 99%, 1.96 g, 9.5 mmol), dissolved in 50 mL
of dichloromethane, is added dropwise within 30 min. The
resulting mixture is stirred at room temperature for 24 h. The
dicyclohexylurea formed is filtered off and the solvent is
evaporated under reduced pressure. The resulting residue is
purified by flash chromatography (pentane/diethyl ether, 50/50,
Rf: 0.4) to yield compound C as a white solid (1.63 g, 60%).
d1H (200.13 MHz, CDCl3) 2.84 (4H, s, H12), 2.60 (2H, t, J ~
7.40 Hz, H2), 2.52 (2H, pseudo dt, H11), 1.85–1.50 (4H, m,
H10 1 H3), 1.50–1.10 (12H, m, H4–H9).
R-Phycoerythrin labeled agarose beads are prepared in the
same way, but using 2 mL of a solution of R-Phycoerythrin-
biotin conjugate (Molecular Probes, Ref. P 811) instead of
biotinylated nanocrystals.
Labeling of neuronal cells with biotinylated nanocrystals
Neuronal cells from mouse embryo cerebellum (stage E14) are
grown for 5 days on polylysin glass covers in neurobasal medium
supplemented with glutamine. The cells are then fixed for
20 minutes in 4% paraformaldehyde in phosphate-buffered saline
(PBS in mM: 3.16 NaH2PO4, 6.84 Na2HPO4, 0.15 NaCl, pH 7.2).
Fixation and all subsequent steps are performed at room
temperature. After permeabilization for 30 minutes in PBS 1
0.1% Triton X-100, the cells are blocked for 2 h with reagent 2
from a CSA amplification kit (DAKO), supplemented with
0.01 mg mL21 streptavidin. Subsequently, the cells are washed
3 times with PBS 1 0.1% Tween20 and incubated for 30 minutes
with the neuron specific anti-class III b-tubulin antibody (TuJ1,
BAbCO, dilution 1/500 in PBS 1 0.1% Tween20 1 5 mM biotin).
In the following, the buffer used for all incubation and washing is
PBS 1 0.1% Tween20. The cells are washed again 3 times and
incubated for 30 minutes with biotinylated anti-(mouseIgG)-
antibody (reagent 4 of CSA kit, DAKO). After three additional
washes, the cells are incubated either with Cy3-streptavidin
(Amersham) or with unlabeled streptavidin (10 mg mL21) for
30 minutes and washed again 3 times. Cells labeled with Cy3-
streptavidin can then be mounted in an antifading medium
(DAKO) and directly observed, whereas cells incubated with
unlabeled straptavidin are further incubated for 30 minutes with
d13C (56.32 MHz, CDCl3) 169.1, 168.6, 34.0, 30.9, 29.3, 29.2,
29.0, 28.7, 28.3, 25.6, 24.6, 24.5.
m/z (DCI NH3 1 Isobutane) 218 (MUA), 236 (MUA 1
NH41), 316 (M 1 H)1, 333 (M 1 NH41).
Ligand 2 (ref. 18). Under inert atmosphere, N-(13-amino-
4,7,10-trioxatridecanyl) biotinamide B (446 mg, 1 mmol) is
added to a solution of 11-mercaptoundecanoyl-N-hydroxy-
succinimide ester C (315 mg, 1 mmol) in 50 mL of dry
chloroform and 200 mL of triethylamine. The reaction mixture
is stirred at room temperature for 1.5 h (control by thin layer
chromatography). Chloroform is evaporated under reduced
pressure and the residue is purified by flash chromatography
J . M a t e r . C h e m . , 2 0 0 4 , 1 4 , 2 6 3 8 – 2 6 4 2
2 6 4 1

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in the first labeling step.
3
4
Labeling of neuronal cells with Alexa-488
5
6
After permeabilization for 30 minutes in PBS 1 0.1% Triton
X-100, the cells are blocked for 2 hours with reagent 2
from a CSA amplification kit (DAKO), supplemented with
0.01 mg mL21 streptavidin. Subsequently, the cells are washed
3 times with PBS 1 0.1% Tween20 and incubated for
30 minutes with the neuron specific anti-class III b-tubulin
antibody (TuJ1, BAbCO, dilution 1/500 in PBS 1 0.1%
Tween20 1 5 mM biotin). The cells are washed again 3 times
and then incubated for 30 minutes with Alexa488-anti-
(mouseIgG)-antibody (Molecular Probes) (1/1000 dilution in
PBS 1 0.1% Tween20). Finally, the cells are washed 3 times
and are mounted in an antifading medium (DAKO).
7
8
9
10 Quantum Dot Corporation, www.qdots.com.
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12 CdSe/ZnSe core/shell nanocrystals used in this study were
prepared following ref. 3.
13 The Q. Y. of CdSe/ZnSe nanocrystals (60–85% in chloroform)
decreases to the percent range after functionalization with a
mixture of 95 mol% of ligand 1 and 5 mol% of ligand 2 and
dispersion in water or buffer solutions.
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Acknowledgements
Dr. N. Charvet gratefully acknowledges the Direction des
Objectifs Mate´riaux (DOB) for his post-doctoral grant. This
work was supported by the ‘‘Action Concerte´e Nanosciences -
Nanotechnologies’’ program (Ministe`re de la Recherche - CNRS
- CEA) and by the region Rhoˆne-Alps through the ‘‘The´matique
Prioritare Cancer’’ program (grant 03 013689 01).
17 D. S. Wilbur, D. K. Hamlin, R. L. Vessela, J. E. Stray,
K. R. Buhler, P. S. Stayton, L. A. Klumb, P. M. Pathare and
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2 6 4 2
J . M a t e r . C h e m . , 2 0 0 4 , 1 4 , 2 6 3 8 – 2 6 4 2