Versatile synthesis strategy for carboxylic acid-functionalized upconverting nanophosphors as biological labels

Article abstract of DOI:10.1021/ja076151k

Up-converting rare-earth nanophosphors (UCNPs) have great potential to revolutionize biological luminescent labels, but their use has been limited by difficulties in obtaining UCNPs that are biocompatible. To address this problem, we have developed a simple and versatile strategy for converting hydrophobic UCNPs into water-soluble and carboxylic acid-functionalized analogues by directly oxidizing oleic acid ligands with the Lemieux-von Rudloff reagent. This oxidation process has no obvious adverse effects on the morphologies, phases, compositions and luminescent capabilities of UCNPs. Furthermore, as revealed by Fourier transform infrared (FTIR) and NMR results, oleic acid ligands on the surface of UCNPs can be oxidized into azelaic acids (HOOC(CH₂) ₇COOH), which results in the generation of free carboxylic acid groups on the surface. The presence of free carboxylic acid groups not only confers high solubility in water, but also allows further conjugation with biomolecules such as streptavidin. A highly sensitive DNA sensor based on such streptavidin-coupled UCNPs have been prepared, and the demonstrated results suggest that these biocompatible UCNPs have great superiority as luminescent labeling materials for biological applications.

Full text of DOI:10.1021/ja076151k

Published on Web 02/16/2008
Versatile Synthesis Strategy for Carboxylic
Acid-functionalized Upconverting Nanophosphors as
Biological Labels
Zhigang Chen, Huili Chen, He Hu, Mengxiao Yu, Fuyou Li,* Qiang Zhang,
Zhiguo Zhou, Tao Yi, and Chunhui Huang*
Department of Chemistry & Laboratory of AdVanced Materials, Fudan UniVersity, 220 Handan
Road, Shanghai 200433, People’s Republic of China
Received August 15, 2007; E-mail: fyli@fudan.edu.cn; chhuang@pku.edu.cn
Abstract: Up-converting rare-earth nanophosphors (UCNPs) have great potential to revolutionize biological
luminescent labels, but their use has been limited by difficulties in obtaining UCNPs that are biocompatible.
To address this problem, we have developed a simple and versatile strategy for converting hydrophobic
UCNPs into water-soluble and carboxylic acid-functionalized analogues by directly oxidizing oleic acid ligands
with the Lemieux-von Rudloff reagent. This oxidation process has no obvious adverse effects on the
morphologies, phases, compositions and luminescent capabilities of UCNPs. Furthermore, as revealed by
Fourier transform infrared (FTIR) and NMR results, oleic acid ligands on the surface of UCNPs can be
2 7
oxidized into azelaic acids (HOOC(CH ) COOH), which results in the generation of free carboxylic acid
groups on the surface. The presence of free carboxylic acid groups not only confers high solubility in water,
but also allows further conjugation with biomolecules such as streptavidin. A highly sensitive DNA sensor
based on such streptavidin-coupled UCNPs have been prepared, and the demonstrated results suggest
that these biocompatible UCNPs have great superiority as luminescent labeling materials for biological
applications.
1
. Introduction
Organic dyes have a number of known drawbacks, such as weak
photostability, and broad absorption and emission bands.1
Semiconductor nanocrystals are still controversial due to their
inherent toxicity and chemical instability, although they exhibit
high photostability, size-dependent emissions, high quantum
Conventional downconversion fluorescent materials, including
1
organic dyes, semiconductor nanocrystals (such as CdS, CdSe,
CdTe), and dye-coupled hybrid materials (carbon nanotubes
and mesoporous silica ), are the commonly used fluorophores
for biological studies and clinical application due to their unique
features. However, they also have many intrinsic limitations.
2
3
4
5
yields, and narrow emission bands. In addition, these down-
conversion fluorescent materials usually emit one lower-energy
photon after absorption of a higher-energy ultraviolet (UV) or
visible photon. The use of higher-energy light is associated with
several significant disadvantages, such as low light-penetration
depth, possible severe photodamage to living organisms, and
the autofluorescence (noise) of some biological samples.
Although some organic dyes and semiconductor nanocrystals
can emit higher-energy photons through two-photo absorption
processes, quantum efficiencies are very low and an expensive
(
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pulsed laser is necessary. To solve these problems, the
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005, 4, 435-446. (e) Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay,
development of alternative biological luminescent labels through
the use of up-converting rare-earth nanophosphors (UCNPs) has
attracted a tremendous amount of attention due to the unique
luminescence properties of rare-earth nanocrystals, such as sharp
absorption and emission lines, high quantum yields, long
J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.;
Weiss, S. Science 2005, 307, 538-544. (f) Zhou, D. J.; Piper, J. D.; Abell,
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3) For recent examples: (a) Becker, M. L.; Fagan, J. A.; Gallant, N. D.; Bauer,
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7
lifetimes, and superior photostability. In particular, upconver-
sion is a process where low-energy light, usually near-infrared
(NIR) or infrared (IR), is converted to higher-energy light (UV
2
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1
10.1021/ja076151k CCC: $40.75 © 2008 American Chemical Society
J. AM. CHEM. SOC. 2008, 130, 3023-3029
9
3023

A R T I C L E S
Chen et al.
or visible), through multiple photon absorptions or energy
transfers. In comparison with downconversion fluorescent
involving ligand exchange for the preparation of water-soluble
and functionalized UCNPs has been reported, owing to dif-
ficulties in obtaining versatile substitutional ligands. Therefore,
it is necessary to develop new and simple methods for
synthesizing hydrophilic and functionalized UCNPs.
8
materials, up-converting rare-earth materials have many advan-
tages in biological applications, such as noninvasive and deep
penetration of NIR radiation, the absence of autofluorescence
of biological tissues, and feasibility of multiple labeling by
To date, oleic acid, containing a -CHdCH- group, has been
widely used as the capping ligand in most successful synthesis
7
UCNPs with different emissions under the same excitation.
9
a,9d,10
17
Unfortunately, despite recent advances in the synthetic methods
for controlling the size and shape of UCNPs,9-11 the UCNPs
with hydrophobic organic ligands (such as oleic acid) coating
their surface cannot be used directly in biological applications
because of their very low solubility in water and unfavorable
surface properties. So, a prerequisite for the development of
UCNP-based biological labels is to gain access to water-soluble
nanoparticles bearing appropriate functional groups (such as
approaches to UCNPs
and other nanocrystals. The
Lemieux-von Rudloff oxidation method is well-known to
oxidize selectively a carbon-carbon double bond (R-CHd
1
8
CH-R′) to give two carboxylic acids. These features trigger
our interest in the novel concept of converting hydrophobic
UCNPs into hydrophilic ones. Herein, we introduce a new,
simple and versatile strategy for synthesizing water-soluble and
carboxylic acid-functionalized UCNPs by directly oxidizing
oleic acid ligands to azelaic acid (HOOC(CH ) COOH) with
-COOH, -NH2, or -SH) on their surface for conjugation of
2
7
biomolecules.
the Lemieux-von Rudloff reagent. Moreover, owing to the
presence of free carboxylic acid groups on their surface, the
oxidized UCNPs can be directly conjugated with proteins (such
as streptavidin). A DNA sensor based on streptavidin-coupled
UCNPs has been successfully fabricated.
Generally, some applications of nanosized materials greatly
depend on surface functionalization, and two main strategies
have been developed for converting hydrophobic nanocrystals
(including semiconductor and rare-earth materials) into ones that
can be dispersed in water and also have some functional
chemical groups on their surfaces. One strategy is based on the
2
. Experimental Section
2a,2d,2e,7b,7e,9a,12
encapsulation of hydrophobic nanocrystals with SiO2
2
.1. Materials. All of the chemicals used were of analytical grade
or amphiphilic copolymer.2
c,2d,7a,13
However, these processes
and were used without further purification. NaOH, NaF, ethanol, tert-
butanol, K CO , cyclohexane, acetone, hydrochloric solution, KMnO
and NaIO were purchased from Sinopharm Chemical Reagent Co.
(China). Oleic acid was obtained from Alfa Aesar. Rare earth chlorides
LnCl , Ln: Y, Yb, Er, Ho, Tm) were prepared by dissolving the
corresponding oxides (Y , Yb , Er , Ho , and Tm from
may be costly and/or complicated, and precise control of some
factors (such as the thickness of encapsulation layer) may be
difficult. For example, Zhang et al.14 reported a useful approach
2
3
4
,
4
(
3
for preparing biocompatible silica-coated polyvinylpyrrolidone/
NaYF4 nanocrystals, but it was found that several NaYF4
nanocrystals could be easily incorporated into one silica shell,
probably resulting in some difficulties in biological applications.
The other strategy involves replacing the original organic layer
2
O
3
2
O
3
O
2 3
O
2 3
2 3
O
Beijing Lansu Co. China) in 10% hydrochloric solution and then
evaporating the water completely.
The oligonucleotides 5′-(biotin)-GATGAGTATTGATGC-3′ (as
Capture-DNA), 5′-CGAATAGTTCCATTG-(TAMRA)-3′ (as Report-
DNA) and 5′-CAATGGAACTATTCG GCATCAATACTCATC-3′ (as
Target-DNA) were purchased from Sangon Biotechnology Co. Ltd of
Shanghai, China. Lysine, 1-ethyl-3-(3-dimethylaminopropyl)carbodi-
imide hydrochloride (abbreviated as EDC), N-hydroxysulfosuccinimide
sodium salt (abbreviated as sulfo-NHS), morpholine ethanesulfonic acid
with hydrophilic ligands.2
b,2d,2e,15
For example, Doris et al.
16
recently reported a versatile strategy for semiconductor quantum-
dot ligand exchange. However, up to now, no approach
(
7) (a) Wang, L. Y.; Yan, R. X.; Hao, Z. Y.; Wang, L.; Zeng, J. H.; Bao, J.;
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(
abbreviated as MES) and tris(hydroxymethyl)methanamine (abbrevi-
ated as Tris) were obtained from Sinopharm Chemical Reagent Co.
China).
2.2. Synthesis of Upconverting Nanophosphors. 2.2.1. Synthesis
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modified hydrothermal process.9a In a typical synthesis, NaOH (1.2 g,
30 mmol), water (9 mL), ethanol (10 mL), and oleic acid (20 mL)
were mixed under agitation to form a homogeneous solution. Then 0.6
(
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mmol (total amounts) of rare-earth chloride (1.2 mL, 0.5 mol/L LnCl ,
Ln: Y or 78 mol%Y + 20 mol%Yb + 2 mol%Er (Ho or Tm)) aqueous
solution was added under magnetic stirring. Subsequently, 1.0 M
aqueous NaF (4 mL) solution was added dropwise to the above solution.
The mixture was agitated for about 10 min, then transferred to a 50
mL autoclave, sealed, and hydrothermally treated at 160 °C for 8 h.
The system was cooled to room-temperature naturally, and the products
7
445.
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102-2105. (b) Lehmann, O.; Meyssamy, H.; Kompe, K.; Schnablegger,
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Upconverting Nanophosphors as Biological Labels
A R T I C L E S
were deposited at the bottom of the vessel. Cyclohexane was used to
dissolve and collect the products. The products were subsequently
deposited by adding ethanol to the sample-containing cyclohexane
solution. The resulting mixture was then centrifuged to obtain powder
samples. Pure powders could be obtained by purifying the samples with
ethanol several times to remove oleic acid, sodium oleic, and other
remnants.
through two membrane filters with 0.45 µm nominal pore size connected
in series. The size distribution was measured at 25 °C with a detection
angle of 90°. Luminescence spectra were measured with an Edinburgh
LFS-920 fluorescence spectrometer. Up-conversion fluorescence spectra
were obtained using an external 0-800 mW adjustable continuous-
wave laser (980 nm, Beijing Hi-Tech Optoelectronic Co., China) as
the excitation source, instead of the Xenon source in the spectropho-
tometer. UV-visible absorption spectra were measured using a
Shimadzu UV-2550 ultraviolet-visible-near-infrared spectrophotom-
eter.
2
.2.2. Converting Hydrophobic UCNPs into Hydrophilic Ones.
A mixture of as-prepared UCNP sample (0.1 g), cyclohexane (100 mL),
tert-butanol (70 mL), water (10 mL) and 5 wt % K CO aqueous
solution (5 mL) were stirred at room temperature for about 20 min.
Then 20 mL of Lemieux-von Rudloff reagent (5.7 mM KMnO and
aqueous solution ) was added dropwise. The resulting
2
3
3. Results and Discussion
4
18
3.1. Synthesis and Characterization of UCNPs. In the
0.105 M NaIO
4
present work, NaYF4: 20 mol%Yb, 2 mol % Er (Tm or Ho),
was synthesized by a hydrothermal route in the presence of oleic
acid, since NaYF4 has been reported to be the most efficient
host material for Yb/Er (or Yb/Tm or Yb/Ho) co-doped NIR-
mixture was stirred at about 40 °C for over 48 h. The product was
then isolated by centrifugation and washed with deionized water,
acetone, and ethanol. Subsequently, the product was dispersed in
hydrochloric acid (50 mL) of pH 4-5, and the mixture was stirred for
2
0
3
0 min. At last, the oxidized product was obtained by centrifugation,
washed twice with deionized water and dried under vacuum.
.3. Preparation of a DNA Sensor. 2.3.1. Preparation of Strepta-
vidin-Functionalized UCNPs. To activate the surface carboxylic
to-visible upconversion phosphors. In the course of the
reaction, oleic acid molecules were coated onto the outer face
of the in-situ generated UCNPs through the interaction between
2
3+
rare-earth ions (Ln ) and carboxyl groups of oleic acids, with
1
9
-5
acid groups, EDC (5 mg, 2.6×10 mol) and sulfo-NHS (15 mg,
.9×10 mol) were added to MES buffer (0.1 M, 20 mL, pH 6.0)
containing the oxidized NaYF :Yb,Er nanoparticles (5 mg). The mixture
9,10
the hydrophobic alkyl chains left outside.
All as-prepared
-
5
6
samples were easily dispersed in a nonpolar solvent (such as
hexane or chloroform) and then deposited by the addition of a
polar solvent (such as ethanol or acetone). To convert hydro-
phobic oleic acid-capped UCNPs into carboxylic acid-function-
alized derivatives, we used the Lemieux-von Rudloff reagent
to oxidize the as-prepared samples (Figure 1).
4
was then stirred for 8 h at room temperature. After centrifugation and
washed with water, the precipitate was added into PBS buffer solution
-
8
(
0.1 M, 5 mL, pH 7.2) containing streptavidin (2 mg, ∼3×10 mol).
The linkage reaction was allowed to proceed at 4 °C for 48 h. Then
-4
lysine (20 mg, 1.4×10 mol) was added to remove any unreacted sulfo-
NHS. Finally, streptavidin-functionalized NaYF :Yb,Er nanoparticles
were obtained by centrifugation, and washed three times with water.
.3.2. Detection of DNA. The sensor system consisted of strepta-
vidin-functionalized UCNPs (0.1 mg/mL), Report-DNA (100 nM) and
Capture-DNA (100 nM) in a buffered (100 mM Tris-HCl, 3 mM MgCl
4
Figure 2 presents representative TEM images of the as-
prepared and oxidized NaYF :Yb,Er samples. The monodisperse
4
2
nanocubes of the as-prepared samples with sizes of 8-14 nm
suggest that the long-chain oleic acid ligands on the crystal
surface prevent aggregation (Figure 2A). After the Lemieux-
von Rudloff oxidation, the oxidized samples were seen to still
consist primarily of single nanocubes, but the formation of
nanoarrays was prevented due to interaction effects of the
oxidized ligands (Figure 2B). Both samples exhibit very similar
particle size distributions (10.8 ( 1.3 nm, s.d.: 4.2% for the
as-prepared sample and 4.5% for the oxidized sample, as shown
in Figure S1 of the Supporting Information). Similar TEM
results were also observed for other samples (NaYF4:Yb,Tm
and NaYF4:Yb,Ho as shown in Figure S2 and S3 of the
Supporting Information). These observations show that such
oxidation has no obvious effects on the size and shape of UCNPs
except for their array. In this respect, our oxidation method has
an advantage over other strategies; for example, the previously
reported encapsulation strategy (with SiO2 or amphiphilic
copolymer) usually increases the particle size and changes the
shape of nanocrystals.
EDXA patterns and XRD patterns of both the as-prepared
and oxidized NaYF4:Yb,Er samples were also studied to
investigate the effects of the oxidation process. EDXA patterns
(Figure S4 of the Supporting Information) confirm the presence
of Na, Y, Yb, and F in both samples, and no obvious changes
in the composition are observed. XRD patterns (Figure S5 of
the Supporting Information) also demonstrate that both samples
have similar phases (dominant cubic phase accompanied by
small amounts of hexagonal phase). Therefore, it can be
concluded that the oxidation has no obvious adverse effects on
the compositions and phases of UCNPs.
2
,
pH 8.0) solution. The detecting experiment was performed by keeping
the concentration of sensor constant while varying the concentration
of Target-DNA. Aliquots of stock solutions of Target-DNA were titrated
into the above solution. After each addition of Target-DNA, the mixture
was stirred at 37 °C for 20 min to facilitate the hybridization reaction.
After slowly cooling to room temperature (25 °C), the luminescence
spectrum of the system was measured under continuous-wave excitation
at 980 nm.
2
.4. Characterization. Sizes and morphologies of UCNPs were
determined at 200 kV using a JEOL JEM-2010F high-resolution
transmission electron microscope (HR-TEM). Samples of the as-
prepared and oxidized UCNPs were prepared by placing a drop of dilute
dispersions in cyclohexane and ethanol, respectively, on the surface of
a copper grid. Energy-dispersive X-ray analysis (EDXA) of the samples
was also performed during HR-TEM measurements. X-ray diffraction
(
XRD) measurements were made with a Bruker D4 X-ray diffractometer
1
using Cu KR radiation (λ ) 0.15418 nm). H NMR spectra were
recorded on a Varian Mercury 400 spectrometer. Proton chemical shifts
are reported in ppm downfield from tetramethylsilane (TMS). Fourier
transform infrared (FT-IR) spectra were measured using an IRPRES-
TIGE-21 spectrometer (Shimadzu) from samples in KBr pellets.
Thermogravimetric analysis (TGA) curves were recorded on a DTG-
60H (Shimadzu) at a heating rate of 10 °C/min. Dynamic light scattering
(DLS) experiments were carried out on an ALV-5000 spectrometer-
goniometer equipped with an ALV/LSE-5004 light scattering electronic
and multiple tau digital correlator and a JDS Uniphase He-Ne laser
(632.8 nm) with an output power of 22 mW. To ensure the accuracy
of DLS measurement, great care was taken to eliminate dust from the
samples. The aqueous solution of the oxidized UCNPs was filtered
(
19) Dumelin, C. E.; Scheuermann, J.; Melkko, S.; Neri, D. Bioconjugate Chem.
(20) Kramer, K. W.; Biner, D.; Frei, G.; Gudel, H. U.; Hehlen, M. P.; Luthi, S.
2
006, 17, 366-370.
R. Chem. Mater. 2004, 16, 1244-1251.
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A R T I C L E S
Chen et al.
Figure 1. Synthesis mechanism of carboxylic acid-functionalized UCNPs from oleic acid-capped precursors.
and 1448 cm- are also found in the spectrum of the as-prepared
sample, associated with the asymmetric (Vas) and symmetric (Vs)
stretching vibrations of the carboxylic group of the bound oleic
acid. However, in the case of the oxidized sample, bands
corresponding to the carboxylic group are observed at 1638 and
1
-
1
1
532 cm . The evolution of ligands on the surface was further
1
investigated by H NMR analysis of the undoped NaYF4
samples (Figure S6 of the Supporting Information). The H
1
NMR spectrum (Figure S6A) of the as-prepared NaYF4 sample
dispersed in CDCl3 is consistent with previously reported data
1
0
for oleic acid-capped nanocrystals, and thus confirms the
presence of oleic acid ligands in the as-prepared samples. The
H NMR signals of the bound oleic acid molecules on the
Figure 2. TEM images of the as-prepared (A) and oxidized (B) NaYF4:
Yb,Er samples.
1
surface of nanoparticles are broadened with respect to those of
2
1a
the free oleic acid, which results from an inhomogeneous
distribution of the magnetic environments due to site variations
on the nanoparticle surface as well as a decrease in the rotational
22
1
freedom of the ligands. The H NMR spectrum of the oxidized
NaYF4 sample dispersed in DMSO-d6 was also recorded (Figure
S6B), and proved to be similar to the standard spectrum of
2
1b
azelaic acid, apart from the broadening. In contrast, a peak
centered at δ ) 5.34 ppm (peak d, assigned to -HCdCH-)
and a peak centered at δ ) 0.85 ppm (peak a, assigned to -CH3)
disappeared after the oxidation, indicative of cleavage of the
carbon-carbon double bond in oleic acid ligands. On the basis
of the above-described FTIR and NMR results, it can be deduced
that the oleic acid ligands on the surface of UCNPs are oxidized
to azelaic acids.
Figure 3. FTIR spectra of the as-prepared (a) and oxidized (b) NaYF4:
Yb,Er samples.
To evaluate the ligand content in both the as-prepared and
oxidized NaYF4:Yb,Er samples, thermogravimetric analysis
(
TGA) was performed (Figure S7 of the Supporting Informa-
tion). TGA curves show two weight loss stages in the range of
0 to 600 °C. The first weight loss stage in the temperature
The capping ligands on the surface of UCNPs were identified
by FT-IR spectra (Figure 3). Both the as-prepared and oxidized
NaYF4:Yb,Er samples exhibit a broad band at around 3450
4
range of 40-200 °C is due to the loss (2-3 wt %) of absorbed
water from both samples. The second stage from 200 to 600
°
-1
cm , corresponding to O-H stretching vibration. The transmis-
sion bands at 2924 and 2854 cm- in both samples are
respectively assigned to the asymmetric (Vas) and symmetric (Vs)
stretching vibrations of methylene (CH2) in the long alkyl
1
C is attributed to the combustion of the organic groups in the
samples, and the weight loss in this stage reflects the ligand
9d
-1
chain. A peak at 3007 cm attributed to the )C-H stretching
vibration can clearly be seen in the spectrum of the as-prepared
sample (Figure 3a),9d but this feature is apparently lost in the
spectrum of the oxidized sample (Figure 3b), suggesting
cleavage of the -HCdCH- group. In addition, bands at 1564
(21) (a) Standard NMR of oleic acid: www.sigmaaldrich.com/spectra/ fnmr/
FNMR004904.PDF; (b) Standard NMR of azelaic acid: www.sigmaald-
rich.com/spectra/fnmr/FNMR004172.PDF.
(22) (a) Sudarsan, V.; Sivakumar, S.; van Veggel, F. C. J. M.; Raudsepp, M.
Chem. Mater. 2005, 17, 4736-4742. (b) Kuno, M.; Lee, J. K.; Dabbousi,
B. O.; Mikulec, F. V.; Bawendi, M. G. J. Chem. Phys. 1997, 106, 9869-
9882.
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Upconverting Nanophosphors as Biological Labels
A R T I C L E S
Figure 4. Colloidal solutions of the oxidized NaYF4: 20 mol%Yb, 2 mol
%
Er sample in water: (A) The solution showing its transparency; (B) Total
upconversion under continuous-wave excitation at 980 nm; (C and D)
upconversion viewed through red and green filters, respectively.
Figure 5. Luminescence spectra of aqueous solutions of NaYF4: 20
mol%Yb, 2 mol % Er nanoparticles (0.4 mg/mL) excited with a 980-nm
laser: (A) As-prepared sample in cyclohexane; (B) the oxidized sample in
water.
content. It is clear that the content of oleic acid ligands in the
present as-prepared samples is about 11.32 wt %, and that the
content of azelaic acid ligands decreases to 7.55 wt %, which
chiefly results from the change in molecular weight of ligands
change of ligands. Similar effects are also found for other
samples (NaYF4:Yb,Tm and NaYF4:Yb,Ho as shown in Figure
S10 of the Supporting Information). In addition, colloidal
aqueous solutions of UCNPs exhibit similar luminescent spectra
as the pH of the solution is adjusted over a wide range (pH
(from 282.46 for oleic acid to 188.22 for azelaic acid). The
results also suggest that almost all of the ligands are still attached
to UCNPs during the oxidization.
As a result of the ligand evolution from oleic acid to azelaic
acid on the surface of UCNPs, all of the oxidized samples can
be readily dispersed in water (Figure 4A) and in some polar
organic solvents such as DMF, or DMSO (Figure S8 of the
Supporting Information) due to the presence of free carboxylic
acid groups. Dynamic light scattering analysis (Figure S9 of
the Supporting Information) further indicates that most of the
oxidized UCNPs in aqueous solution are about 12.5 nm in
diameter with excellent dispersion, and only a very small amount
of UCNP aggregates of diameter 70-120 nm can be observed,
correlating nicely with the particle size determined by TEM
study (Figure 2B). The luminescent properties of the oxidized
samples in water were investigated, and Figure 4B-D shows
the upconversion luminescence photographs of a colloidal
aqueous solution of the oxidized NaYF4:20 mol%Yb, 2 mol %
Er sample under continuous-wave excitation at 980 nm. The
total luminescence (Figure 4B) appears yellow-green in color
2
∼11, Figure S11 of the Supporting Information), indicating
excellent pH-independent luminescent properties of UCNPs as
compared to organic dyes and semiconductor quantum dots.
1,2
3
.2. Applications as Luminescent Biological Labels. Most
importantly, the presence of free carboxylic acid groups on the
surface of azelaic acid-capped UCNPs facilitates further con-
jugation with various biomolecules. Typically, the terminal
carboxylic acid groups allow the immobilization of amine-
containing proteins and DNA. In view of the highly specific
-15
interaction (high affinity, Kd ) 10 ) between streptavidin and
biotin, in the present study, streptavidin was chosen as the
23
target biomacromolecule to attach to the oxidized UCNPs. As
shown in Figure 6A, covalent coupling of streptavidin to the
surface of UCNPs was facilitated by cross-linkers (EDC and
sulfo-NHS) which activated the surface carboxy groups on
UCNPs and led to the formation of amide bonds with strepta-
vidin.
Furthermore, as an applied example of UCNP-based lumi-
nescent biological labels, the feasibility of designing a DNA
sensor based on streptavidin-functionalized UCNPs was dem-
onstrated through the specific interaction between streptavidin
and biotin. The design principle of the DNA sensor is shown
in Figure 6B. By using two shorter oligonucleotides to capture
a longer target oligonucleotide, a sandwich-type hybridization
format is adopted in this DNA sensor. One of the shorter
oligonucleotides is labeled with biotin (DNA1-biotin, as capture-
DNA) (Figure 6B), and the other is labeled with TAMRA
3
+
due to a combination of green and red emissions from the Er
ion. This is confirmed by Figure 4, parts C and D, where the
solution under the same excitation conditions is viewed through
red and green filters, respectively. The corresponding upcon-
version spectrum of the oxidized sample in water and that of
the as-prepared samples in cyclohexane under continuous-wave
excitation at 980 nm are shown in Figure 5, and are similar to
1
0b,10c,11a,14
previously reported spectra for these materials.
Both
spectra feature three distinct Er3 emission bands. The green
+
emissions between 514 and 534 nm and between 534 and 560
2
4
4
nm are the result of transitions from H11/2 and S3/2 to I15/2 of
(DNA2-TAMRA, as reporter-DNA) whose excitation spectrum
3+
Er , respectively. A red emission is observed between 635 and
80 nm due to the transition from F9/2 to I15/2. It is well-known
overlies the green emission band of the NaYF4:Yb,Er nanopar-
ticles (Figure S12 of the Supporting Information). Under
illumination with a 980-nm laser, only luminescent signals of
NaYF4:Yb,Er nanoparticles were observed from a mixture of
DNA1-biotin, DNA2-TAMRA, and streptavidin-functionalized
NaYF4:Yb,Er nanoparticles (Figure 7A). This indicates that the
nanoparticle and DNA2-TAMRA are far apart in the absence
of target-DNA, and that energy transfer from the nanoparticles
4
4
6
that the crystallinity and surface properties of UCNPs may alter
the intensity of the fluorescence emission peaks of the doping
lanthanide ions. For example, NaYF4:Yb,Er samples prepared
under different conditions exhibit different intensity ratios of
the green and red emission peaks.1
0b,10c,11a,14
In this case of
NaYF4:Yb,Er, a slight decrease in this ratio (I540/I654) is
observed, from 0.93 for the as-prepared sample to 0.87 for the
oxidized sample (Figure 5), which may be attributed to the
(23) Wilchek, M.; Bayer, E. A. Methods Enzymol. 1990, 184, 14-45.
J. AM. CHEM. SOC.
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A R T I C L E S
Chen et al.
Figure 6. Synthesis of streptavidin-functionalized UCNPs (A) and
schematic of DNA nanosensors based on UCNPs (B).
to TAMRA is negligible under continuous-wave excitation at
980 nm. Upon addition of target-DNA, a broad and featureless
emission band (∼580 nm) gradually appears, corresponding to
the TAMRA emission, and signal intensities of green emissions
between 514 and 560 nm of UCNPs decreases significantly
while those of red emissions between 635 and 680 nm remain
largely unchanged (Figure 7A), thus indicating energy transfer
from the green emission of UCNPs to TAMRA. The effective
energy transfer implies that the resulting assembly brings the
fluorophore (energy acceptor) and the rare earth nanoparticle
Figure 7. Luminescence spectra of a mixture of streptavidin-functionalized
NaYF4:Yb,Er nanoparticles, capture-DNA, and reporter-DNA in the pres-
ence of different concentrations of target-DNA under continuous-wave
excitation at 980 nm (A), and the linear relationships between target-DNA
concentration and the intensity ratios I540/I654 (B) or I580/I540 (C).
(energy donor) into close proximity. In addition, the lumines-
cence intensity ratio (I580/I540 or I540/I654) is found to vary linearly
with the concentration of target-DNA in the measured range of
4. Conclusions
In summary, we have demonstrated a new, efficient and
10-50 nM (Figure 7, parts B and C), suggesting high sensitivity.
versatile procedure for converting hydrophobic UCNPs into
water-soluble and carboxylic acid-functionalized derivatives by
directly oxidizing oleic acid ligands to azelaic acid. No obvious
adverse effects of the oxidation on the morphologies, phases,
compositions and upconversion luminescence of UCNPs are
observed. It should be noted that our conversion procedure is
very simply, and do not change the size and shape of particles
in contrast with the previously reported encapsulation strategy.
Moreover, this procedure is not limited to hydrophobic UCNPs,
and it can easily be applied to other hydrophobic nanoparticles
Thus, we can quantitatively detect the target oligonucleotide
by monitoring the intensity ratio upon continuous-wave excita-
tion at 980 nm. The successful fabrication of a DNA sensor
also indicates that streptavidin molecules on the surface of
NaYF4:Yb,Er nanoparticles remain highly active.
The highly specific interaction between streptavidin and biotin
has been widely applied in the studies of modern biological
24
and medical fields, and a wide variety of biotinylation reagents
are commercially available.25 Consequently, streptavidin-func-
tionalized UCNPs may be used for detecting and/or binding to
a broad range of biotinylated proteins and DNA. Therefore, we
anticipate that this streptavidin coating surface chemistry
provides a novel approach for the production of biocompatible
photoluminescent UCNP probes, giving UCNPs great potential
as luminescent labeling materials for biological applications.
(including rare earth, semiconductor and metal nanoparticles)
where only surface ligands can be oxidized.
More importantly, the presence of free carboxylic acid groups
on the surface of azelaic acid-capped UCNPs allows further
conjugation with various biomolecules, and streptavidin-func-
tionalized UCNPs derived from azelaic acid-capped precursors
provides a novel approach for detecting and/or binding to a
broad range of biotinylated proteins or antibodies. The successful
fabrication of a DNA sensor based on streptavidin-functionalized
UCNPs suggests that it might be possible to expand the
application of these UCNPs as luminescent labels into other
biological fields such as bioimaging.
(
24) (a) Pinaud, F.; King, D.; Moore, H.-P.; Weiss, S. J. Am. Chem. Soc. 2004,
26, 6115-6123. (b) Ramanathan, K.; Bangar, M. A.; Yun, M.; Chen,
W.; Myung, N. V.; Mulchandani, A. J. Am. Chem. Soc. 2005, 127, 496-
97. (c) Oh, E.; Hong, M.-Y.; Lee, D.; Nam, S.-H.; Yoon, H. C.; Kim,
1
4
H.-S. J. Am. Chem. Soc. 2005, 127, 3270-3271. (d) Zhang, C.-Y.; Yeh,
H.-C.; Kuroki, M. T.; Wang, T.-H. Nature Mater. 2005, 4, 826-931.
25) For example, see: www.invitrogen.com.
(
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Upconverting Nanophosphors as Biological Labels
A R T I C L E S
1
Acknowledgment. The authors thank National High Technol-
ogy Program of China (2006AA03Z318), National Science
Foundation of China (20490210 and 20501006) and Shanghai
Sci. Tech. Comm. (05DJ14004 and 06QH14002) for financial
support.
and luminescence spectra of UCNPs; H NMR spectra of the
undoped NaYF4; Photographs of colloidal solutions of
UCNPs in DMF and DMSO; absorption and emission spectra
of aqueous solution of TARMA-labeled reporter DNA. This
material is available free of charge via the Internet at
http://pubs.acs.org.
Supporting Information Available: Size distribution, TEM
characterization, EDXA spectra, XRD patterns, TGA curves
JA076151K
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