Surface-modified upconverting microparticles and nanoparticles for use in click chemistries

Article abstract of DOI:10.1002/chem.201000117

A method is described for modifying the surface of upconverting microparticles (UCμPs) and nanoparticles (UCNPs) such that they become amenable to click chemistry. Respective reagents are presented and used in both kinds of particles, either directly or in combination with tetraethoxysilane. The particles also were labeled by using the click reaction, a) with fluorophores to yield materials that have emission colors that depend on the wavelength of excitation ; b) with maleinimido groups (so to obtain labels for thiols), and c)with biotin (to make them useful for affinity studies based on the biotin-streptavidin system). We believe that both the UCμPs and UCNPs have the potential of being used in numerous areas including upconversion imaging, biolabeling, and derivatization, but also in encoding and security.

Full text of DOI:10.1002/chem.201000117

DOI: 10.1002/chem.201000117
Surface-Modified Upconverting Microparticles and Nanoparticles for Use in
Click Chemistries
Heike S. Mader, Martin Link, Daniela E. Achatz, Katrin Uhlmann, Xiaohua Li, and
Otto S. Wolfbeis*[a]
Abstract: A method is described for
modifying the surface of upconverting
microparticles (UCmPs) and nanoparti-
cles (UCNPs) such that they become
amenable to click chemistry. Respec-
tive reagents are presented and used in
both kinds of particles, either directly
or in combination with tetraethoxysi-
lane. The particles also were labeled by
using the click reaction, a) with fluoro-
phores to yield materials that have
emission colors that depend on the
wavelength of excitation; b) with male-
inimido groups (so to obtain labels for
thiols), and c) with biotin (to make
them useful for affinity studies based
on the biotin–streptavidin system). We
believe that both the UCmPs and
UCNPs have the potential of being
used in numerous areas including up-
conversion imaging, biolabeling, and
derivatization, but also in encoding and
security.
Keywords: biotin · click chemistry ·
fluorescence
upconversion
·
nanoparticles
·
Introduction
vantage of being photoexcitable at wavelengths at which the
auto-absorption of any biological matter is quite weak,
thereby reducing background of both absorption and lumi-
nescence (which would occur, along with Raman scatter, at
wavelengths of >980 nm anyway) to virtually zero.
Upconverting microparticles (UCmPs), as opposed to
UCNPs, clearly are much larger, but more efficient in terms
of upconversion. They are commercially available and used,
for example, in security inks or for the visualization of IR
radiation. UCmPs also have been employed in homogeneous
immunoassays[14] and enzyme activity assays,[15] following
bead-milling to reduce the particle size to the sub-micron
range. Low-energy laser diodes are adequate for photo-exci-
tation, and their (visible) emission is rather bright. Unlike
UCNPs, they cannot be well suspended (as a kind of colloi-
dal dispersion) in aqueous or organic solutions.
To be of use in affinity studies (such as in high-throughput
screening) and in bioassays, the surface of UCNPs and
UCmPs has to be functionalized to facilitate covalent immo-
bilization of biomolecules on their surfaces. Such surface
chemistries are expected to be versatile, to enable immobili-
zation of proteins, receptors, enzymes, or nucleic acid oligo-
mers, to mention a few. Moreover, UCNPs can be suspend-
ed fairly well in certain organic solvents, but not in water
unless their surface is appropriately modified. This is crucial,
however, with respect to many bioapplications.[16] Only if
proper surface modification is accomplished, can their bio-
analytical potential can be fully exploited.
Upconverting nanoparticles (UCNPs), sometimes also re-
ferred to as upconverting nanophosphors display the unique
property of emitting photoluminescence at visible wave-
lengths under near-infrared (NIR) excitation.[1,2] Unlike con-
ventional fluorophores, UCNPs convert low-energy NIR ra-
diation to higher energy (visible) light by multiphoton ab-
sorption and subsequent emission of dopant-dependent lu-
minescence. The large anti-Stokes shift allows easy separa-
tion of the discrete emission peaks from the excitation
source. UCNPs are chemically stable and do not bleach,
which is in striking contrast to most organic fluorophores.
Applications of UCNPs (which are virtually invisible in low
concentrations) include authentication in general, security,[3]
counterfeit,[4,5] brand protection,[6] flow cytometry,[7] point-
of-care diagnostics,[8] and bioarrays.[9] In bioanalytical terms,
they have been demonstrated to be useful in immunoas-
says,[10] as luminescent labels,[11] in sensing pH,[12] and in
imaging of cells and small animals.[13] UCNPs have the ad-
[a] H. S. Mader, M. Link, D. E. Achatz, K. Uhlmann, X. Li,
Prof. Dr. O. S. Wolfbeis
Institute of Analytical Chemistry, Chemo- and Biosensors
University of Regensburg,93040 Regensburg (Germany)
Fax : (+49)941-943-4064
E-mail: otto.wolfbeis@chemie.uni-r.de
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201000117.
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The most common method to improve dispersibility in-
volves the coating of NPs with a thin shell of silica (SiO2).
The resulting silica NPs are chemically stable, fairly biocom-
patible, nontoxic, and can be prepared in narrow size distri-
bution. Silica is well documented as a coating agent for
quantum dots,[17] metal oxides,[18] lanthanide nanoparticles,[19]
and even upconverting particles.[11,20] In fact, NPs and mPs
made from, or coated with silica have been used in various
kinds of sciences including nanomedicine,[21] nanobiotech-
nology,[22] and fluorescence imaging.[23] Yet another benefit
of silica-coated particles is based on the different types of
functional groups that can be attached to the particle sur-
face using appropriate silane reagents.[23,24]
The introduction of functional groups to the surface of
almost any kind of micro- and nanoparticles is also required
to enable bioconjugation. Various kinds of functionalized
particles have been reported in the literature. Generally,
linkers with terminal amino, thiol, or carboxy groups are
preferred.[11,19,25] However, the functional groups required
for these kinds of conjugation are quite abundant in protein-
ic biomolecules, a fact that compromises selective conjuga-
tion. Moreover, amino groups and carboxy groups are
charged in pH 6–8 solution and thus give rise to electrostatic
(i.e. unspecific) interactions including adsorption and parti-
cle aggregation. The so-called “click-chemistry” is an attrac-
tive alternative because the functional groups involved
(azido and alkyne) are hardly present in biomolecules in-
cluding proteins and oligomers. It is therefore said to be
lar studies. Such nanoparticles are believed to represent an
attractive (and bioorthogonal) alternative to acid-functional-
ized upconverting nanophosphors.[32]
Results
Choice of micro- and nanoparticles: The upconverting mi-
croparticles used here (referred to as mP-1 and mP-2) are
commercially available and may be ball-milled to various
sizes to facilitate their use in bioassays, as shown by Kunin-
gas et al.[14] The surface click chemistry reported here was
performed with un-milled particles, mainly for the purpose
of completeness, as the main focus of this work is on nano-
particles (UCNPs). The two kinds of nanomaterials (re-
ferred to as NP-1 and NP-2) were synthesized as described
in the Experimental Section. Table 1 summarizes figures of
merit of the materials used.
Table 1. Micro- and nanoparticles used in this work.
Code
Net formula
Size
Emission color[a]
Dispersible
mP-1
mP-2
NP-1
NP-2
La2O2S: Yb,Er
Y2O2S: Yb,Tm
NaYF4: Yb,Er
NaYF4: Yb,Tm
5–15 mm
5–15 mm
60–90 nm
50–90 nm
green and red
blue and NIR
green and red
blue and NIR
no
no
fairly well
fairly well
[a] Also see Figures 1 and 3.
“bio
N
Synthesis of reagents for surface modification: The reagents
shown in Scheme 1 were used to introduce an azido group
or a terminal alkyne group onto the surface of both the
UCmPs and UCNPs. Similar (but more complex) reagents
have been used in the past[33] to modify the surface of silica
nanoparticles. The introduction of biotin by means of re-
agents 3 and 4 warrants access to the biotin–(strept)avidin
affinity system and the maleinimide 5 allows for selective
binding to thiol moieties.
The click reaction, otherwise known as the Huisgen liga-
tion, involves the dipolar cycloaddition of an organic azido
group to an alkyne group.[27] The catalytic effect of Cu+ ions
on this cycloaddition was independently discovered by
Meldal and co-workers[28] and by Sharpless and co-work-
ers,[29] and this has provided an enormous stimulus to the ap-
plications of this reaction.[30] The reagents used are often
available in a reasonable number of synthetic steps. Cyclo-
addition proceeds in high yields, occurs at room temperature
in many organic solvents and—most notably in terms of bio-
logical applications including bioconjugation[31]—also in
aqueous solution at near-neutral pH. Generally, only simple
purification steps are required, as a result of almost com-
plete and regioselective conversion into the 1,4-disubstituted
1,2,3-triazole.[30] Furthermore, no protecting groups are
needed for the click reaction, as it tolerates a variety of
functional groups and shows high kinetic stability.
We perceived that the combination of click chemistry
with upconversion technology will lead to new avenues in
terms of affinity studies, sensing and labeling, and are re-
porting here a) on reagents for preparing NPs and mPs for
use in click conjugation, b) on respective NPs and mPs with
upconversion capability, and c) on how such particles can be
labeled with fluorophores and other species by means of the
click reaction. We believe that such particles have the poten-
tial of being used in numerous areas including upconversion
imaging and assays, but can also be modified with various
biomolecules to act as vectors for directed labeling in cellu-
Synthesis and characterization of surface-modified upcon-
verting microparticles (UCmPs): The UCmPs were function-
alized by using silanes 1 and 2. This is possible because of
the (partially) oxidic nature of practically all microparticles.
Metal oxides at the surface of solid materials usually are
present in hydrated form (more precisely as hydroxides; see
Scheme 2) in a way comparable to silicium dioxide. Silane
reagents, such as aminopropyl-triethoxysilane (APTS),[34]
have previously been allowed to react with numerous surfa-
ces including uncommon ones, such as titanium, graphite,
iron, or materials used for medical implants, to render them
reactive towards biomolecules. This is a quite direct way of
functionalization and also does not cause any aggregation of
particles.
Figures of merit of the UCmPs thus obtained (type mP-1
or mP-2) are given in Table 2. The UCmPs were character-
ized by SEM, fluorescence spectroscopy, and IR spectrosco-
py. Both kinds of particles have a diameter of about 12 mm,
but are rather irregular in shape and size. Surface modifica-
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O. S. Wolfbeis et al.
vibration of the urethane car-
bonyl group, and a peak at
1538 cmÀ1 characteristic of the
amide. The weak peak at
2126 cmÀ1 is assigned to the C
ꢀ
C
stretching vibrations, and
peaks at 2977, 2944, and
2887 cmÀ1 to C H stretches.
À
The strong green and red in-
trinsic emission of mP-1 upon
excitation with
a l=980 nm
diode laser is clearly visible to
the naked eye. The emission
peaks of mP-1 are found at l=
546 and 660 nm (Figure 1, solid
line). Strong blue (along with
considerable
near-infrared
emission) is observed for micro-
particles of type mP-2, with
peaks located at l=475 and
800 nm, respectively (Figure 1,
dashed line). The rather sharp
and discrete emission lines of
the upconverters represent an-
other attractive feature that has
a large potential in terms of
multiplex signaling and sensing.
Scheme 1. Click reagents used in this work.
Synthesis and characterization
of surface-modified upconvert-
ing nanoparticles (UCNPs):
The surface chemistry of
UCmPs was extended to the re-
Scheme 2. Synthetic route to azido-modified and alkyne-modified upconverting microparticles (UCmPs), re-
spectively.
Table 2. Overview on the surface-modified upconverting microparticles
(mPs) used in this work.
Code
Functionality
Preparation
mP-1-A
azido
mP-1 reacted with 1
mP-2-B
alkyne
mP-1 reacted with 2
mP-1-A-4
mP-1-A-6
biotin
fluorescent label
mP-1-A clicked with 4
mP-1-A clicked with label 6
tion virtually had no effect on the size or morphology of the
particles. As can be seen from the SEM image shown in the
Supporting Information, the UCmPs are not spherical and
show a wide variety in terms of size and shapes.
Figure 1. Emission spectra of UCmPs of type mP-1 (solid line) and mP-2
(dashed line) following excitation with a 10 mW 980 nm diode laser.
Infrared spectra were acquired next in order to verify the
presence of azido or alkyne groups on the modified UCmPs
(see Figure S2 in the Supporting Information). Those of the
azido-modified mP-1 showed a strong peak at 2098 cmÀ1,
which is characteristic of the asymmetric stretch vibration of
the azido group.[35] Further peaks are found at 2965 and
2879 cmÀ1 for the asymmetric stretching vibrations of the
spective nanoparticles. The highest upconversion efficiencies
to date have been observed for doped hexagonal phase
NaYF4.[36] The co-precipitation[37] method chosen here yields
cubic NaYF4, which has a moderate upconversion efficiency.
A (partial) phase transfer to the more efficiently upconvert-
ing hexagonal form was accomplished by tempering the ma-
terial at 4008C, as described in the literature.[37] The UCNPs
À
C H bonds. The IR spectrum of the alkyne-modified mP-2
(see Figure S2 in the Supporting Information) reveals a
strong peak at 1710 cmÀ1 that is attributed to the C=O bond
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were first directly functional-
ized, as described for the
UCmPs, owing to the presence
of hydroxy functions on the sur-
face of an otherwise non-oxidic
material. It is found, however,
that UCNPs coated with a (hy-
drophilic) silica coating are
more stable in dispersion.
Therefore, the UCNPs were si-
Scheme 3. Synthetic route to azido-modified nanoparticles (NP-1-A1 and NP-2-A1) possessing a silica shell,
multaneously coated with silica
and click-functionalized by em-
ploying a mixture of tetraethox-
yorthosilicate (TEOS) and the
respective triethoxysilane (1 or
2) by using a modified Stçber
method.[38] A schematic of this
one-step procedure[39] for simul-
taneous coating and functionali-
zation of UCNPs with either
azide or alkyne is given in
Schemes 3 and 4, respectively.
The UCNPs were character-
ized by using TEM, elemental
analysis, IR spectroscopy, and
fluorescence spectroscopy. The
diameter of the nanoparticles
(see Table 1) after the anneal-
ing process is 60–90 nm for NP-
1 and 50–90 for NP-2 as deter-
mined by TEM (Figure 2, left
and Table 3). They are crystal-
line and almost spherical in
shape. Owing to their small size
and respective click conjugations.
Scheme 4. Synthetic route to alkyne-modified core–shell nanoparticles (NP-1-B1 and NP-2-B1), and respective
click conjugations.
Table 3. Figure of merit of the surface-modified UCNPs of type A1 and B1.
Code
Functionality
Size [nm]
Preparation
NP-1-A1
NP-1-B1
NP-2-A1
NP-2-B1
azido
alkyne
azido
70–100
80–130
60–100
90–130
NP-1 coated with a mixture of TEOS and 1
NP-1 coated with a mixture of TEOS and 2
NP-2 coated with a mixture of TEOS and 1
NP-2 coated with a mixture of TEOS and 2
alkyne
Figure 2. TEM images of annealed UCNPs of type NP-1 (left) and of silica-coated and azido-modified UCNPs of type NP-1-A1 (right).
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O. S. Wolfbeis et al.
they can be dispersed in water or ethanol, but they tend to
aggregate in water solution. The silica-coated UCNPs are
almost spherical and uniform in size (Figure 2, right). They
can be easily dispersed in water and, in contrast to particles
not coated with silica, show little tendency towards aggrega-
tion. The particles are stable for several months in aqueous
or ethanol dispersions.
The core–shell structure of the particles is visible in the
upper right insert of Figure 2. The thickness of the SiO2
coating is about 10 nm for NP-1-A1 (azido-modified), 20 nm
for NP-1-B1 (alkyne-modified), 20 nm for NP-2-A1 (azido-
modified) and 20 nm for NP-2-B1 (alkyne-modified) accord-
ing to TEM measurements. The thickness of the shell seems
to depend on the quantities of TEOS and ammonia em-
ployed, but no systematic study was performed on this.[40]
The presence of the azido or alkyne groups was verified
by elementary analysis. The fractions of C, H, and N, respec-
tively, are 3.1, 1.2, and 2.4% for NP-1-A1, and 3.5, 1.3, and
0.7% for NP-1-B1. The high nitrogen content of NP-1-A1
clearly indicates the presence of azido groups. The presence
of nitrogen in NP-1-B1 results from the presence of the ure-
thane group in the linker. This is equivalent to around
0.57 mmol of azido groups, and to around 0.50 mmol of
alkyne groups, respectively, per gram of particles.
Functionalization of the particles by using click chemistry:
Next, the nanoparticles were functionalized with groups to
make them conjugatable to biomolecules. Such “particle
labels” are highly-attractive alternatives to the widely used,
but toxic quantum dots of the CdSe type. Two versatile
functions were chosen. The first is the maleinimide group,
which undergoes addition to thiol groups at room tempera-
ture and in aqueous solution. It is widely used to label pro-
teins. The second is the biotin group which is the “work
horse” in numerous labeling protocols, because of its very-
strong interaction (KD ꢁ10À14 m) with (strept)avidins. The
functionalized UCNPs listed in Table 4 were obtained by
clicking the nanoparticles of Table 3 to the click reagents 3–
5 of Scheme 1.
Table 4. Figure of merit of the bioreactive UCNPs.
Code
Functionality
Reaction
NP-1-B2-3
NP-1-A2-4
NP-1-A2-5
mP-1-A-4
biotin
biotin
maleinimide
biotin
NP-1-B1 clicked to 3
NP-1-A1 clicked to 4
NP-1-A1 clicked to 5
mP-1-A clicked to 4
The azido-modified biotin 3 was clicked to alkyne modi-
fied NP-1-B1 to give NP-1-B2–3. The presence of the func-
tional groups was verified by means of diffuse reflectance
FT-IR spectroscopy, as shown in Figure S4 in the Supporting
Information. The spectra were acquired by using the respec-
tive alkyne- or azido modified NP-1 as a reference material.
The negative peak at 2131 cmÀ1 in the difference spectrum
(see Figure S4a in the Supporting Information) indicates
As in case of microparticles, IR spectra were acquired to
detect the presence of functional groups. The spectra of the
azido-NPs (NP-1-A1), and the alkyne-NPs (NP-1-B1) are
shown in Figure S3 in the Supporting Information. The
peaks for the azido group, and the alkyne group, respective-
ly are found at the same wavenumbers as discussed for the
microparticles.
À
The emission spectra of the modified nanoparticles were
acquired using a 5 W, l=980 nm continuous wave diode
laser as the excitation light source. Particles doped with yt-
terbium and erbium (type NP-1) give green and red emis-
sions (Figure 3), those doped with ytterbium and thulium
(type NP-2) give blue and NIR emission (Figure 3). Peaks
maxima are at the same wavelengths as those of the UCmPs
with surface modification (type mP-1 and mP-2). The
UCNPs require stronger laser excitation than the UC-mPs in
order to cause efficient upconversion.[41]
that the alkyne functionality has disappeared. The C H
stretch vibration is visible at 2983 and 2908 cmÀ1. The peaks
at 1665 and 1598 cmÀ1 are assigned to the C=O and C N
À
amide stretches.
Alkyne-modified biotin 4, in turn, was clicked to azido-
modified NP-1-A1 to give NP-1-A2–4. The disappearance
of the azido group is indicated by the negative peak at
2108 cmÀ1.The C H stretches are found at 3003 and
À
2906 cmÀ1. The C=O stretch vibration is located 1709 cmÀ1,
À1
À
and the C N amide stretch at 1542 cm (see Figure S4b in
the Supporting Information).
Finally, the alkyne-modified maleinimide 5 was clicked to
azido-modified NP-1-A1 to give NP-1-A2–5. The absence of
the azido functionality is indicated by the negative peak at
À1
À
2114 cm . The C H stretches are located at 3998 and
2906 cmÀ1. The maleinimidic C=O stretches are found at
1788 and 1724 cmÀ1. The C=C stretching peak is located at
1582 cmÀ1 (see Figure S4c in the Supporting Information).
The alkyne-modified biotin 4 also was clicked to azido-
modified microparticles (mP-1-A) to give mP-1-A-4. Its dif-
ferential IR spectrum (see Figure S5 in the Supporting Infor-
mation) indicates that the same surface chemistry has been
accomplished as with nanoparticles NP-1-A2–4.
Figure 3. Emission spectrum of upconverting nanoparticles of type NP-1-
A1 (solid line) and NP-2-A1 (dashed line) suspended in aqueous solution
and photoexcited with a 980 nm laser.
Fluorescent labeling of the particles by using click chemis-
try: Azido- and alkyne-modified particles offer another at-
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Microparticles and Nanoparticles for Use in Click Chemistries
FULL PAPER
tractive possibility in that they may be click-labeled with or-
ganic fluorophores to obtain particles that emit various
colors depending on whether the upconverting (inorganic)
core is photoexcited, or the (organic) fluorophores of the
shell. The luminescence of the upconverter is generated
under NIR excitation, here at l=980 nm, whereas the lumi-
nescence of the organic fluorophore appears under excita-
tion with visible light. Dually excitable particles with dual
emissions are likely to have even more sophisticated appli-
cations than those outlined in the Introduction for conven-
tional upconverters.
spectrum of the organic label (under l=520 and 590 nm ex-
citation, respectively) are given in Figure 4. The absorption
and emission of fluorophore 6 (like that of all oxazines of
the Nile Red type) is strongly solvatochromic, but if cova-
lently bound to the surface of particles of type NP-1-A2, the
excitation and emission maxima are rather invariable and
found at l=590 and 625 nm, respectively, both in water and
ethanol suspension, indicating stronger interaction with the
surface than with the solvent.
To investigate this, two clickable fluorescent labels (see
Scheme 5) were chosen for performing the following experi-
ments. First, the alkyne-modified purple fluorescent dye 6
(Table 5) was clicked to azido-modified UCmPs of type mP-
Discussion
The method described here for chemically modifying the
surface of upconverting mPs and NPs is straightforward and
versatile. It is likely to be appli-
cable to various other materials
possessing oxidic (and thus hy-
droxylic) surfaces. It makes sur-
faces compatible with click
chemistry, which again is highly
versatile, particularly with re-
spect to bioconjugation because
it is bioorthogonal and thus is
not troubled by functional
groups often present in biologi-
cal samples. In fact, click
chemistry has been applied to
NPs made from silica,[24,42]
gold,[43] iron oxide,[44] and vari-
ous polymers.[45,33] The one-step
method introduced here is pri-
marily intended for uses in con-
Scheme 5. Alkyne-modified fluorescent dyes for click labeling of UCmPs and UCNPs.
Table 5. Figure of merit of the fluorescently labeled UCmPs and UCNPs.
Code
Functionality
Preparation
mP-1-A-6
NP-1-A2-6
NP-1-A2-7
fluorophore 6
fluorophore 6
fluorophore 7
mP-1-A clicked with 6
NP-1-A1 clicked with 6
NP-1-A1 clicked with 7
1-A to give mP-1-A-6, which was investigated by fluores-
cence microscopy and fluorescence spectroscopy. A refer-
ence sample, obtained without using the copper(I) catalyst,
showed no trace of the dye after washing the particles. In
contrast, the UCmPs clicked to the fluorophore 6, display
the typical orange fluorescence of the oxazine label. A fluo-
rescence microscopy image of the labeled microparticles
under visible-light excitation (l=550 nm) is given in Fig-
ure S6 in the Supporting Information. The orange-colored
emission of the fluorophore covalently attached to the sur-
face of the particles is clearly visible.
Figure 4. Dual emission of nanoparticles of type NP-1-A2–6 and NP-1-
A2–7. a) Upconversion emission (at 980 nm excitation); b) emission of
label 6 (photoexcitation at 590 nm); c) emission of label 7 (photoexcita-
tion at 520 nm).
text with bioassays. It is much simpler than two-step meth-
ods, such as those used for modifying UCNPs with amino
groups.[11] The purification of UCNPs by size exclusion chro-
matography (SEC) is another attractive feature of the work
presented here. When applying SEC, the NPs can be kept in
solution at any time and aggregation is minimized.
Similarly, the azido-modified nanoparticles of type NP-1-
A1 were clicked to 6 and 7 to give NP-1-A2–6 and NP-1-
A2–7, respectively. The upconversion emission spectrum
(under l=980 nm excitation) and the conventional emission
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O. S. Wolfbeis et al.
in the filtrate was then removed under reduced pressure. The crude prod-
uct was then purified by column chromatography using a gradient of
methylene chloride and methanol as eluents to yield propargylbiotin (4)
as a white solid. The mass and NMR spectra were in agreement with lit-
erature data.[51]
Click reaction of the reactive micro- and nanoparticles
gave particles that can be conjugated to a) (strept)avidin[39,
(via the biotin group), and b) thiols[47] (via the maleini-
46]
mide groups). In a further version, it leads to particles carry-
ing organic fluorescent labels. Thiol labeling is clearly to be
favored in bioconjugation over amine labeling, simply be-
cause most proteins possess numerous free amino groups
(this leading to random labeling and to varying dye-to-pro-
tein ratios), but often only one to three thiol groups. Human
serum albumin (HSA), for example, has one free thiol
group only, but more than 50 amino groups that may be la-
beled.
Propargyl-maleimide (5): Compound 5 was synthesized from maleic acid
anhydride and propargylamine in a two-step synthesis.[52] Maleic acid an-
hydride (4.9 g, 50 mmol) was dissolved in 25 mL of acetone and heated
to reflux. Propargylamine (2.75 g, 50 mmol) was separately dissolved in
acetone and added dropwise to the refluxing solution of the anhydride.
The solution was stirred under reflux for 1 h and the solvent then re-
moved on a rotary evaporator to give a purple solid. The crude product
was recrystallized from a 1:4 mixture of diethyl ether and methanol to
give the respective (open-chain) amidic acid as a colorless solid. Yield:
1
4.58 g (60%). H NMR (300 MHz, [D6]DMSO): d=13.88 (s, 1H), 9.15 (s,
The UCmPs and UCNPs have attractive spectral features
in giving dual emissions depending on the wavelength of ex-
citation. If excited with NIR light, dual emission of the inor-
ganic core is observed (green and bright red, or blue and
dark red). If excited in the visible, the (single) emission of
the organic fluorophore (green or orange) is being seen
(Figure 4). Moreover, by varying the quantity of organic
label, a wide range of intensities can be adjusted for both
the “inorganic” and “organic” emission, thus giving a 2-di-
mensional matrix of two (or three) intensities (and possibly
also lifetimes) that enables unambiguous encoding of parti-
cles. We assume that if organic dyes are used that absorb at
one of the two emission bands of the upconverters, various
ratios of intensities of the dual emission may be adjusted.
1H), 6.32 (d, J=12.3 Hz, 1H), 6.26 (d, J=12.1 Hz, 1H), 3.97 (q, J=
2.5 Hz, 2H), 3.19 ppm (t, J=2.6, 1H); CI-MS: m/z: MÀ calculated: 153.1,
found 153.0.
The ring closure of N-propargylmaleamic acid to give maleimide 5 was
accomplished by dissolving N-propargylmaleamic acid (3.5 g, 23.5 mmol)
in xylene (100 mL) and stirring under reflux for 8 h by using a water trap.
The solution was allowed to cool to room temperature and filtered. The
solvent was removed on a rotary evaporator to obtain the product as a
pale-yellow solid. Yield: 0.97 g (31%). 1H NMR (300 MHz, CDCl3): d=
6.76 (s, 2H), 4.29 (d, J=2.5 Hz, 2H), 2.21 ppm (t, J=2.6 Hz, 1H);
13C NMR (300 MHz, CDCl3): d=169.1, 134.5, 71.6, 26.8 ppm. EI-MS: m/
z: M+ calculated: 135.1, found 135.1.
Upconverting microparticles (UCmPs): Two types of commercially avail-
able UCmPs, referred to as mP-1 and mP-2 (see Table 1) were used. Mi-
croparticles of type mP-1 are composed of La2O2S and doped with yt-
terbium and erbium ions, those of type mP-2 consist of Y2O2S and are
doped with ytterbium and thulium ions. Both were obtained from Mol-
Tech GmbH Berlin (www.mt-berlin.com). Owing to their relatively large
size, they cannot be solubilized in water or organic solvents. Therefore,
when using the microparticles in solution, vigorous stirring is required to
prevent sedimentation of the particles. Alternatively, the particles may be
placed in viscous aqueous solutions of hydrogels such as polyurethane,
poly(vinyl alcohol), or polyacrylamide.
Experimental Section
(3-Azidopropyl)triethoxysilane (1): Dry(!) sodium azide was added to a
solution of (3-chloropropyl)triethoxysilane in dry acetonitrile. Tetrabuty-
lammonium azide[48] was added, as phase transfer catalyst, and the mix-
ture was stirred under reflux for 70 h. The solid was filtered off and the
solvent removed on a rotary evaporator. The colorless residue was dis-
solved in dichloromethane and washed three times with doubly distilled
water. The organic phase was dried over disodium sulfate and the solvent
removed on a rotary evaporator to give the product as a colorless liquid.
Yield 3.46 g (76%). 1H NMR (300 MHz, CDCl3): d=3.82 (q, J=7.0 Hz,
6H), 3.26 (t, J=6.9 Hz, 2H), 1.71 (m, 2H), 1.23 (t, J=7.0 Hz, 9H),
0.67 ppm (m, 2H); 13C NMR (300 MHz, CDCl3): d=58.5, 53.8, 22.7, 18.3,
Surface modification of upconverting microparticles (UCmPs): The
UCmPs were washed with acetic acid prior to silanization to remove am-
monium ion (which is ubiquitous on the surfaces of particles). For exam-
ple, UCmPs (200 mg) of type mP-1 were rinsed three times with acetic
acid (2 mL, 0.1m) and washed several times with doubly distilled water.
The particles were filtered by using a suction filter and then dried over-
night in a drying furnace. The activated UCmPs were then suspended in
dry toluene (20 mL) in a 100 mL Schlenk flask and flushed with dry ni-
trogen. The respective silane (1 or 2, 200 mL) were added and the mixture
was stirred for 48 h at 908C. The mixture was allowed to cool and the
particles were separated by centrifugation for 15 min at 4000 rpm. After
removal of the supernatant, the particles were washed several times with
ethanol and acetone, and finally dried in a furnace at 608C.
7.6 ppm; IRACHTUNGTRENNUNG
(neat): n˜ =2978, 2934, 2892, 2095 cmÀ1; CI-MS: m/z: M+ cal-
culated: 247.1; found MH+ 248.1.
O-(Propargyloxy)-N-(triethoxysilylpropyl)urethane (2): Compound 2 was
purchased from ABCR (www.abcr.de) and used without further purifica-
tion.
Synthesis of upconverting nanoparticles (UCNPs): The UCNPs (referred
to as NP-1 and NP-2; see Table 2) were synthesized using the estab-
lished[37] co-precipitation method. Solutions (0.2m) of the trichlorides of
the ions YIII and YbIII, along with either ErIII or TmIII were combined
with an EDTA solution to form the respective EDTA complexes. This so-
lution (around 40 mL) was injected quickly into a 3.5% aqueous solution
of sodium fluoride. The resulting mixture was stirred for 1 h at room tem-
perature to give a colorless precipitate that was separated by centrifuga-
tion at 4000 rpm for 30 min, washed 3 times with water and then once
with ethanol. The precipitate was dried in a drying furnace and under
vacuum. The colorless powder was tempered at 4008C for 4.5 h under
argon atmosphere.
Azido-biotin (3): Compound 3 was synthesized by a modification of the
method described in the literature[49] using 2-azidoethylamine[50] as the
amino linker. Commercially available biotin NHS ester was dissolved in
dry dimethylformamide (DMF), and 2-azidoethylamine was added. Trie-
thylamine was added and the solution stirred overnight at room tempera-
ture. The solvent was evaporated to give the crude product as a yellow,
oily substance. It was crystallized from ethanol to give the product 2 as a
colorless solid. The mass and NMR-spectra are in agreement with litera-
ture data.
Propargyl-biotin (4): Compound 4 was synthesized by dissolving biotin,
dicyclohexylcarbodiimide, and 1-hydroxybenzotriazole in equimolar
quantities in dry DMF. After stirring for 10 min, an equimolar quantity
propargylamine was added dropwise and the mixture stirred for another
4 h at room temperature. The solvent was removed under reduced pres-
sure to give an orange oil. Addition of little methanol resulted in the pre-
cipitation of N,N’-dicyclohexylurea, which was filtered off. The methanol
Coating and surface modification of UCNPs: The particles were coated
with a silica shell by means of a modified Stçber process.[38] In a typical
experiment, UCNPs (75 mg, NP-1 or NP-2) were dispersed in ethanol
(10 mL) by ultrasonication. Subsequently, the mixture was heated to
408C, and water (500 mL) and ammonia (500 mL, 25 wt%) were added,
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Microparticles and Nanoparticles for Use in Click Chemistries
FULL PAPER
followed by tetraethoxysilane (TEOS, 375 mL). The solution was stirred
for 30 min. The respective functionalized silane (1 or 2; 25 mL) was
added and the mixture stirred for another 2.5 h. After cooling to room
temperature, the particles were purified by size exclusion chromatogra-
phy (on Sephadex LH-60) by using ethanol as the eluent to remove un-
reacted TEOS and ammonia. The proper fraction can be recognized by
its slight turbidity and by illuminating it with a l=980 nm laser, which re-
sults in visible luminescence. The resulting silica-coated UCNPs were
stored in ethanol solution to avoid aggregation. To transfer the particles
into aqueous solution, the reaction mixture from the Stçber coating pro-
cess was diluted with water and purified again by size exclusion chroma-
tography using Sephadex G-75 (which is better water-compatible than
LH-60) with distilled water as the eluent. The resulting suspensions in
either ethanol or water are stable for several months.
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Received: January 16, 2010
Published online: April 1, 2010
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Chem. Eur. J. 2010, 16, 5416 – 5424