REVIEW

Biological Applications of Rare-Earth

Based Nanoparticles
Cedric Bouzigues,†,* Thierry Gacoin,‡ and Antigoni Alexandrou†
†
Laboratoire d'Optique et Biosciences, Ecole Polytechnique, CNRS UMR7645 INSERM U696, 91128 Palaiseau Cedex, France and
‡
Laboratoire de Physique de la Matière Condensée, Ecole Polytechnique, CNRS UMR7643 911128 Palaiseau Cedex, France

D
etection of biomolecules is essential
for many applications in biomedi- ABSTRACT Biomedicine and cell and molecular biology require powerful imaging techniques of
cine cell and molecular biology, the single molecule scale to the whole organism, either for fundamental science or diagnosis. These
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in vitro or in living cells and organisms. applications are however often limited by the optical properties of the available probes. Moreover, in
Efficient analytical methods, such as immu- cell biology, the measurement of the cell response with spatial and temporal resolution is a central
nassays or DNA microarrays, and imaging
instrumental problem. This has been one of the main motivations for the development of new
techniques have thus been developed for
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probes and imaging techniques either for biomolecule labeling or detection of an intracellular
decades. These applications are however
often constrained by the available probes, signaling species. The weak photostability of genetically encoded probes or organic dyes has
whose optical properties may limit the imaging motivated the interest for different types of nanoparticles for imaging such as quantum dots,
possibilities. The development of probes for nanodiamonds, dye-doped silica particles, or metallic nanoparticles. One of the most active fields of
labeling or enhancing the efficiency of de- research in the past decade has thus been the development of rare-earth based nanoparticles, whose
tection has thus become essential in order optical properties and low cytotoxicity are promising for biological applications. Attractive properties
to improve sensitivity of analytical devices.
of rare-earth based nanoparticles include high photostability, absence of blinking, extremely narrow
Moreover, in cell biology, the measurement
of the cell response with spatial and tem- emission lines, large Stokes shifts, long lifetimes that can be exploited for retarded detection
poral resolution is a central instrumental schemes, and facile functionalization strategies. The use of specific ions in their compositions can be
problem. These issues have motivated the moreover exploited for oxidant detection or for implementing potent contrast agents for magnetic
development of single biomolecule;such resonance imaging. In this review, we present these different applications of rare-earth
as lipids or membrane receptors;labeling nanoparticles for biomolecule detection and imaging in vitro, in living cells or in small animals.
and tracking in living cells or indicators of
We highlight how chemical composition tuning and surface functionalization lead to specific
intracellular signaling species, such as calcium
properties, which can be used for different imaging modalities. We discuss their performances for
ions1 or reactive oxygen species (ROS).2
The weak photostability of genetically imaging in comparison with other probes and to what extent they could constitute a central tool in
encoded probes such as green fluorescent the future of molecular and cell biology.
protein (GFP) or organic dyes, used for
protein labeling, have however limited their KEYWORDS: rare-earth nanoparticles . surface functionalization . biomolecule
detection . chemical composition tuning . lanthanide
biological applications. For example, the
tracking of membrane proteins labeled with
organic dyes can provide the measurement
of membrane protein diffusion coefficients their fluorescence, the so-called blinking,
but no information on the evolution of their potential cytotoxicity in vivo, and complex
organization. This is due to photobleaching functionalization strategies3,4,7,8 have lim-
that occurs within a few seconds under ited their use.
standard conditions of illumination for sin- These elements justify the interest for
gle molecule detection. The use of semi- different types of nanoparticles for imaging
conductor fluorescent nanocrystals, or or for protein and nucleic acid detection,
quantum dots, has notably extended the such as nanodiamonds,9
11 dye-doped silica * Address correspondence to
particles,12,13 or metallic nanoparticles.14,15 [email protected].
scope of possible biological applications of
fluorescent probes, due to their high quan- One of the most active fields of research in Received for review June 28, 2011
tum yield and high photostability.3,4 Quan- the past decade has thus been the devel- and accepted October 7, 2011.
tum dots have thus, for example, been used opment of rare-earth doped nanoparticles,
Published online October 08, 2011
for in vitro assays, single protein labeling whose optical properties and low cytotoxicity 10.1021/nn202378b
and tracking in living cells,5,6 or imaging in are promising for biological applications.16
19
small animals. However, intermittency of Attractive properties of rare-earth doped C 2011 American Chemical Society

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nanoparticles include high photostability, absence VOCABULARY: surface functionalization
a way to
of blinking, extremely narrow emission lines, large introduce chemical functional groups to a surface. Materi-
Stokes shifts, long lifetimes that can be exploited als with functional groups on their surfaces can be de-
for retarded detection (consisting in luminescence signed from substrates with standard bulk material
detection taking place after the excitation has properties : Stoke's shift
the difference (in wavelength
stopped and after short-lived, approximately nanose- or frequency units) between positions of the band maxima
cond, fluorescence has decayed), and facile functio- of the absorption and emission spectra (fluorescence and
nalization strategies when synthesized in water. Raman being two examples) of the same electronic
Moreover, unique, reversible oxidoreduction pro- transition.
cesses of the rare-earth ions can be exploited for oxi-
dant detection, and the high magnetic moment of the case of Eu2þ and Ce3þ. In these cases, large
certain ions, like Gd3þ, under magnetic field renders absorption cross sections are observed so that these
them potent contrast agents for magnetic resonance ions may be used as sensitizers, with spectroscopic
imaging. properties that may be drastically affected by the host
In this review, we present different applications of matrix through crystal field splitting.
rare-earth based nanoparticles for biomolecule detec- As compared to quantum dots, a major difference
tion and imaging in vitro, in living cells or in small is expected in rare-earth compounds considering that
animals. We will here focus mainly on rare-earth doped the states involved in the transitions are mainly
nanoparticles, which may harbor thousands of ions. localized within the sensitizer and activator species,
Extensive efforts have also been dedicated to rare-earth while energy transfer processes occur through dipolar
ion chelates or organic complexes involving one or a or exchange interactions. The consequence is that
limited amount of ions20 and have managed to exploit these systems are not expected to exhibit quantum
several of the unique rare-earth ion properties.21
23 confinement effects, at least for sizes of more than
Nanoparticles, however, present the additional advan- one or two nanometers. This means that the exact
tage of single-particle detection due to the large control of size distribution is not as critical as for
number of ions present and the absence of blinking quantum dots. On the other hand, homogeneous
renders them competitive with respect to quantum dispersion and optimum concentration of activator/
dots for single biomolecule tracking. Furthermore, the sensitizer ions is of crucial importance due to con-
nanoparticle surface can be seen as a platform where centration quenching effects. Rare-earth distribution
multiple functionalization and targeting groups can be within the host matrix, in addition to the appropriate
easily grafted. control of their oxidation state, is thus a critical issue
to be addressed when considering synthesis of nano-
PREPARATION OF RARE-EARTH-BASED NANO- particles.
PARTICLES Different processes may be used for the elaboration
Synthesis. Elaboration of rare-earth doped nanopar- of nanoparticles in the objective of providing well-
ticles has been the subject of an increasing number of dispersed suspensions. Processes such as laser
publications since about 2000.24,25 Studies usually ablation27 and dispersion from calcinated powders28
concern compositions that are already well-known to would in principle allow access to almost any com-
be highly efficient in the bulk state and developed pound known in the bulk state, but they do not
mainly for lighting and display applications.26 Other yet allow a satisfying control of size and dispersion.
compositions have also been studied to take advan- The colloid chemistry route thus still remains the best
tage of some of the original spectroscopic properties of approach. The basic principle relies on the reaction
rare-earth ions such as upconversion, quantum cut- between precursors in solution, either through copre-
ting, persistent luminescence, etc. cipitation or thermal decomposition. The critical issue
Most compounds usually consist of a host oxide is the ability to obtain particles with sufficient crystal-
matrix-containing species that acts as light absorber linity, despite the low temperatures involved in these
(sensitizer) and the rare-earth light-emitting ions (the soft chemistry routes as compared to bulk material
activator). In some cases, such as in upconversion synthesis, performed at high temperatures (typically
systems, a low phonon
energy matrix may be impor- 900
1400 °C) from solid state reactions. This aspect is
tant, which leads to a preference for fluorides rather probably the main reason for the limited number of
than oxides. The intrinsic nature of the rare earth compounds that have been investigated up to now, as
mainly determines the spectroscopic properties of compared to the numerous known bulk compositions.
the material (in terms of absorption and emission This also explains why initial investigations were
wavelength and transition efficiencies). Exceptions achieved on compounds such as YVO4 and LaPO4
are found in some cases when, instead of f
f electron compositions.24,25,29,30 These materials, which are
configuration transitions, d electrons are involved in efficient phosphors in the bulk state, can indeed be
the absorption or emission transitions, for example, in obtained with a reasonable crystallinity from simple

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Figure 1. (A) Transmission electron microscope image of Gd2O3:Eu nanoparticles. Reprinted with permission from ref 75.
Copyright 2005 SPIE. (B) Absorption (dashed line), luminescence excitation (for emission at 615 nm) and emission (excitation
at 280 nm) spectra (solid lines) for a YVO4:Eu nanoparticle colloidal solution. Reprinted from ref 25. Copyright 2000 American
Chemical Society. (C) Luminescence evolution for NaYF4:Yb,Er particles under 500 mW illumination in a scanning confocal
microscope. Reprinted with permission from ref 76. Copyright 2009 Elsevier. The nanoparticle luminescence level is stable
under excitation in contrast to that of organic fluorophores (DAPI or Alexa532). (D) Luminescence evolution for a single YVO4:
Eu particle under continuous 1.6 kW/cm2 illumination. Reprinted with permission from ref 77. Copyright 2009 Nature. The
luminescence decay under illumination is due to photoreduction and is reversible (see section on oxidant nanoprobes).

precursor salts in water. They also accept a homoge- for a specific application. Among the proposed materials,
neous incorporation of many rare-earth dopants. we can cite without being exhaustive: europium-doped
Nevertheless, recent improvements in colloid yttrium vanadate,24,25,30,32 or gadolinium oxide,33 cerium-
chemistry have allowed the investigation of other and terbium- doped lanthanum fluoride,34 upconver-
systems through the use of hydrothermal and sol- sion particles like erbium- and ytterbium-doped yttrium
vothermal processes.16,31 As compared to conven- oxide,35 yttrium oxysulfide,36 lanthanum molybdate,37
tional coprecipitation routes, reactions are achieved sodium and yttrium fluoride38
40 or yttrium vanadate,41
within high boiling point coordinating solvents at high persistent luminescence particles like europium

temperature (typically 120
350 °C). This allows a good dysprosium-manganese doped magnesium silicate.42
control of nucleation/growth processes, leading to Most syntheses provide particles in the 3
15 nm
particles with an excellent crystallinity and a narrow or in the 20
50 nm size range (Figure 1A). The
size distribution. In the case of rare-earth-doped com- difference mainly originates from the preparation
pounds, such techniques are still limited to a few protocol (for example, solvent used for the synthesis
compounds (phosphates, fluorides), but extension to and/or presence of complexing agents) and the
many other systems can surely be expected in a near corresponding control of nucleation/growth pro-
future. cesses. This size consideration is of high importance
Among the reported rare-earth doped nanoparticles, regarding (i) the number of emitted photons per
many have already been tested for biological applica- particle, which scales as the third power of their
tions. The choice of matrix and rare-earth ion determines diameter and (ii) limiting processes that alter quantum
the chemical, magnetic, and/or spectral properties of efficiency. Smaller particles are indeed clearly limited by
each type of nanoparticle and renders them suitable surface quenching,43 while emission from particles in the

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20
50 nm range appears to be limited mainly by the are typically coated with poly(ethylene glycol)
particle crystallinity.41 (PEG)62,63 or Dextran molecules.64,65
Rare-earth compounds can also be used as a doping
agent in different types of nanoparticles, such as silica PROPERTIES OF RARE-EARTH DOPED NANO-
PARTICLES
particles44
47 or polystyrene beads. The objective of
this approach is to use the high biocompatibility of Optical Properties. The optical properties of rare-
these materials, while taking advantage of the chemi- earth-doped nanoparticles are determined by the
cal properties introduced by the rare-earth com- chemical nature of their constituents (doping ions
pounds. Silica nanoparticles can be produced by and matrix). Their luminescence is in most cases due
incorporating rare earths in the silica framework,44,45,48 to forbidden transitions among different f-electron
by designing core
shell (SiO2-rare earth) particles48,49 or configurations of the rare-earth ion (except for Ce3þ
by the embedding or coating of rare-earth chelates.50,51 and Eu2þ involving d-electrons). These low-lying energy
Silica particles are usually prepared by microemulsion levels are well isolated from the environment, and
methods44,45,48,50 with lanthanides added to the reaction therefore the transition energy varies little with the
mix or by sol
gel processes.47 Polystyrene
acrylic host material and the nanoparticle size. As a conse-
acid particles have also been dyed by rare-earth quence, absorption (typically 1
5 nm broad)25,29 and
chelates52
54 and are commercially available. emission peaks (typically 10 nm broad)25,29,34 are both
Functionalization. For the biological applications, sur- narrow compared to organic dyes or quantum dots
face functionnalization of the particles is a crucial step. (Figure 1B).
The objective is first to ensure good dispersion of the The nanoparticles can be either excited using a
particles in biological media, that is, in water at neutral transition of the matrix (usually in the UV) and subse-
pH and high ionic strength. Second, particles should quent energy transfer to the rare-earth ion or using
bare specific organic or bio-organic groups which aim direct excitation of the rare-earth ion directly respon-
at targeting specific receptor sites, and/or ensuring sible for the emission or able to transfer its energy to
inocuity in the case of in vivo experiments. the emitting ions, in the case of codoping such as
Immediately after synthesis, particles usually have a Ce
Tb. In most of the cases, a large Stokes shift is
specific surface chemistry required to control the involved (∼150 nm for YVO4:Eu nanoparticles), much
nucleation/growth process and appropriate dispersion larger than typical Stokes shifts of organic fluoro-
in the solvent used. Biological applications thus require phores, which further facilitates background elimina-
a step of postsynthesis functionalization. Strategies may tion. UV excitation is not optimal for applications in
be common to those used for other types of particles living cells or organisms and, therefore, direct excita-
such as metallic particles or quantum dots.3,4,55 How- tion of, for instance, Eu3þ has been employed using an
ever, surface derivatization of particles synthesized in argon laser and has allowed single-particle detection.32
water is clearly more straightforward56,57 as compared In this case, the narrowness of the absorption lines
to that of particles, such as semiconductor quantum limits the usable light sources for imaging. However,
dots, synthesized in organic solvents which require a the continuous development of compact, high-power,
hydrophobic to hydrophilic adaptation by elaborate inexpensive solid-state lasers and light-emitting
chemical methods.3,4,7,8 Usually, hydrophobic nano- diodes should provide alternative solutions.66
particles are first coated to ensure their stability in Upconversion particles provide a highly efficient
aqueous medium, by ligand exchange58,59 and/or strategy for background signal elimination since they
silica32,58 or polymer coating.8,60 absorb two or more photons to emit photons of higher
Functionalization strategies consist of a first step energy.67,68 Thus, the nanoparticle emission is anti-
which aims at covering the surface of the particles with Stokes shifted and takes place in a wavelength range
reactive groups (amino, carboxylates, thiols, aldehydes, where endogenous biomolecule emission is absent. In
etc.) so as to ensure, in a second step, the further addition, upconversion nanoparticles are excited in the
coupling of biomolecules of interest such as streptavidin near-infrared allowing deeper tissue penetration. The
or antibodies. Different methods can be used for upconversion from the near-infrared to the visible
the first step, such as direct surface grafting of molecules takes place via energy transfer between different dopant
or encapsulation with a polymer61 or with a thin shell ions and/or excited-state absorption, and single parti-
of organosilane carrying amine or other functional cle imaging has been recently demonstrated.40,63,69
groups.32,36,38,56,57 In the case of oxide nanoparticles, Because these upconversion processes involve real
silica and organosilane coating is quite staightforward long-lived metastable states with lifetimes on the
because of the presence of surface hydroxyle groups microsecond scale, inexpensive continuous-wave la-
that act as a coupling agent with silanes. For in vivo sers can be used, whereas multiphoton excitation
applications, as in the case of other types of nanopar- typically requires expensive, pulsed lasers. Biomedical
ticles, to ensure furtivity in the blood circulation and applications using upconversion nanoparticles have
avoid detection by the immune system, the nanoparticles been demonstrated for erbium- and ytterbium-doped

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a
TABLE 1. Optical Properties of Different Probes

cross-section extinction coefficient quantum photon emission emission time before lifetime
2
1
1
1
emitter (cm ) (M 3 cm ) yield rate (s ) width (nm) photobleaching (s) (ns)

organic dye 3.10
16 ∼75000 0.92 7  105 ∼40 few seconds ∼4 ns

(Alexa 488)
quantum dot ∼1.10
14 (ref 81,82) ∼2  106 0.3
0.5 (ref 81,83) 1  107 ∼30 (ref 81) >1000 s ∼10 ns (ref 84)
rare-earth nanoparticle 2.10
16 ∼50000 0.16 1  105 ∼10 (ref 32) >1000s ∼300 μs (ref 57,80)
(30 nm Y0.6 Eu0.4VO4
particles with
∼60000 emitters)
nanodiamonds 9.10
17 (ref 85) ∼25000 0.99 (ref 86) <3  105 ∼75 (ref 85) >1000 s ∼10 ns (ref 85)
(with 3NV centers)
a
The absorption cross-section for A488, Quantum dots (bare 3 nm CdSe82 or core-shell CdSe/Zns,81 3 nm core radius), YVO4:Eu nanoparticles and nanodiamonds were
measured with respective excitations of 488, 500, 466, and 523 nm. Emission rate and time before photobleaching are estimated for a nonsaturating excitation intensity of
1 kW/cm2 and with a typical number of photons emitted before photobleaching of 106 for A488. Note that A488 is one of the brightest organic fluorophores. All data concerning
A488 are available in the documentation provided by the Invitrogen company. Note that the photon emission rate for quantum dots does not take into account the absence of
emission when the dots are in a dark state (blinking). For nanodiamonds, the quantum yield was measured in bulk material and is probably weaker in nanoparticles.
Furthermore, note that a broad emission spectrum is unfavorable for efficient detection in the presence of a fluorescent background. Thus, the photon emission rates presented
in the table likely notably overestimate the number of photons that can be collected. Finally, these values concern bare particles and fluorophores and may be lower after
functionalization and coupling to biomolecules.

nanoparticles such as NaYF4:Yb,Er or NaGdF4:Yb, For direct excitation of Eu3þ ions at the 7F0,1
5D2
Er.38,39,63 Recently, a highly crystalline YVO4 matrix transition at 466 nm, for example, the extinction
has also proven promising.69 Two-color lymphatic coefficient is 50000 cm
1 3 M
1 for 30-nm Y0.6Eu0.4VO4
imaging was furthermore performed using two rare- nanoparticles.32 For excitation of the matrix, on the
earth dopant pairs Yb/Er and Yb/Tm in NaYF4 other hand, the extinction coefficient is much higher.32
nanocrystals.39 Efforts during synthesis may increase the nanoparticle
The forbidden nature of the transitions among brightness, for example by annealing72,73 or micro-
different f-electron configurations leads to very long wave treatment.74
excited state lifetimes (∼1 ms) compared to the life- One of the main disadvantages of organic dyes is
time of organic fluorescent probes (∼1 ns). As a con- their fast (a few seconds under ∼1 kW/cm2 of resonant
sequence, the brightness (the number of emitted illumination) and irreversible transition to a dark state,
photons per unit time) of a single emitting center is or photobleaching. Quantum dots, on the other hand,
much lower than that of organic fluorophores or show blinking at the single particle level that can only be
quantum dots. However, in a single nanoparticle sev- avoided with highly specific growth procedures.78,79
eral hundreds or thousands of ions contribute to light This blinking feature is completely absent from rare-
emission, so that a single nanoparticle is detectable earth based nanoparticles due to the presence of a
under biologically compatible laser illumination with a large number of emitters in each particle.32,40,63,69 The
CCD camera.32,70,71 In addition, the long lifetimes can luminescence of rare-earth nanoparticles under illumi-
be exploited for easy implementation of retarded nation is higly stable: In most cases the luminescence
detection schemes eliminating undesired signals like remains unchanged (Figure 1C)76 while a partial decay
cell background fluorescence and direct acceptor ex- is observed, for Eu3þ-doped nanoparticles, for example
citation in Förster resonant energy transfer (FRET) (Figure 1D).32,77 This decay has been proven to be
experiments.32,71 Ca0.2Zn0.9Mg0.9Si2O6:(Eu2þ, Dy3þ, reversible and due to the photoinduced reduction of
Mn2þ) nanoparticles present extremely long fluores- Eu3þ to Eu2þ (Figure 1D).77 In both systems, the
cence decay times (∼100 s). The order of magnitude of photostability allows the continuous observation of rare-
the persistence of the luminescence enables experi- earth nanoparticles for arbitrarily long durations.63,76,80,69
ments with extemporaneous excitation of nanoparti- Furthermore, the oxido-reduction processes observed
cles and imaging without excitation. This can be fruitful in YVO4:Eu nanoparticles have been exploited to de-
for in vivo imaging42 to minimize background. monstrate an oxidant sensor for hour-long experi-
Strictly speaking, the brightness limitation is not ments (see subsection Oxidant Nanoprobes and ref
due to the lifetime but to the extinction coefficient and 77). This variability in optical properties (rate of photon
the quantum yield. When one of the electric-dipole emission, photostability, etc.) due to the chemical
forbidden rare-earth ion transitions is used for the nature of the nanoparticles makes them suitable for
excitation, the extinction coefficients are relatively low different applications at the single particle level and as
compared to quantum dots (105
106 cm
1 3 M
1) but volume labels, for example, for in vivo applications.
are comparable to those of organic fluorophores (Table 1). These different applications are detailed below.

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Figure 2. DNA detection with upconversion nanoparticles. (Left) Scheme for the DNA labeling with nanoparticles on microarrays.
These experiments were made with nanoparticles coated with silica and functionalized with amino groups and biotin. Biotin
is indicated in green and neutravidin in blue. cDNA with biotinylated nucleotides is first synthesized by reverse transcrip-
tion (top left). Hybridization with complementary probe DNA grafted on the microarray (bottom left) is then revealed by the
binding of neutravidin-coated fluorescent nanoparticles (center). (Right) Microarray labeled with Cy5 (top) and Y2O2S:Yb,Er
nanoparticles (bottom). Note the three spots indicated by white lines, that are only detected through nanoparticle imaging.
Results reprinted with permission from ref 36. Copyright 2001 Nature.

Magnetic Properties. Several rare-earth ions have a LaF3:Ce3þ,Tb3þ nanoparticles of ∼50 nm radius
large number of unpaired electrons and thus provide coated with polymers are fluorescent mostly due to
high magnetic moments under a magnetic field. Gd3þ to Tb3þ transitions. This fluorescence can be quenched
and Eu2þ ions with seven unpaired electrons yield the by the presence of nucleic acids such as DNA. This is
highest magnetic moments and can thus be used to possibly due to the formation of hydrogen bonds
decrease the longitudinal relaxation time T1 of water between DNA and carboxylic acids of the nanoparticle
protons in a strong static magnetic field after a reso- polymer coating enabling energy transfer from excited
nant radiofrequency excitation. Gd3þ, due to its higher Tb3þ to DNA. It is thus possible to determine DNA
availability and chemical stability, is thus widely used concentration by measuring the fluorescence inten-
as a contrast agent for magnetic resonance imaging sity. Concentrations as low as 2 μg/mL have been
(MRI). Applications of Gd-based nanoparticles to MRI detected:34 this compares favorably to conventional
and their comparison with commercial Gd-complexes measurements performed with a spectrophotometer.
are detailed below in the subsection titled Contrast DNA microarrays or DNA chips are widely used to
Agents for MRI Imaging. study mRNA expression patterns. This technology
Cytotoxicity. The cytotoxicity of Cd-containing quan- relies on the hybridization of probe DNA strands
tum-dots is mostly due to a potential release of free spotted on a chip with a fluorescently labeled target
cadmium and depends on their surface functionaliza- DNA, usually obtained by reverse transcription of
tion.87,88 This is an important limitation of their use for mRNA extracted from cells. The fluorescence intensity
biomedical applications and justifies research for others reveals the quantity of the complementary fragment of
semiconductor compounds (InP, CuInS2). For different each probe. The commonly used fluorophores are
types of rare-earth nanoparticles, with or without cyanine dyes (Cy3 and Cy5). This method has been
functionalization, no cytotoxicity was observed.62,77,89 advantageously replaced by biotinylation of target
For example, gadolinium oxide particles injected in DNA followed by labeling with neutravidin-coated
mice were naturally eliminated by renal excretion after Gd2O3:Eu91 or with upconversion nanoparticles
a few hours without any damage to the animal,62 and Y2O2S:Yb,Er36 (Figure 2). Concentrations of target
YVO4:Eu nanoparticles internalized in vascular cells DNA as low as 1 ng/mL were detected, which is a 5
induce no mortality hours after their internalization.77 times improvement compared to Cy5 labeling. Weakly
The potential toxicity of each type of nanoparticle expressed mRNA, undetectable by usual methods, has
depends on its constituent materials and on the chemical thus been revealed in a sample extracted from a
properties of its surface coating and the size of the bladder carcinoma36 (Figure 2).
particles.90 Therefore, further experiments as a func- Detection of single nucleotide polymorphisms
tion of coating and constituent materials are necessary (SNPs) is important for the diagnosis of different dis-
before any general assessments can be made. eases, such as polycystic kidney disease (PKD), and is
usually based on quantitative RT-PCR (reverse tran-
BIOCHEMICAL APPLICATIONS scription polymerase chain reaction), which is a time-
DNA Assays. Rare-earth nanoparticles have been used consuming and expensive method. Fe3O4/Gd2O3:Eu
for DNA detection following two different approaches: nanoparticles33,91 consisting of a Fe3O4 core and a
the use of DNA-sensitive nanoparticles and the use of Gd2O3:Eu shell are fluorescent due to the presence of
nanoparticles as labels of DNA fragments. Eu3þ ions and magnetic due to the magnetite core, and

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Figure 3. Detection of single nucleotide polymorphisms. (A) Method for purification of target DNA. DNA fragments with
(þPKD) or without a mutation (
PKD) of interest are amplified by PCR then hybridized with Alexa488-labeled DNA and probe
DNA (complementary of the þPKD mutated strand) coupled to Fe3O4/Gd2O3:Eu nanoparticles. After that treatment, only
þPKD fragments are labeled both by the nanoparticle and the fluorophore. Purification is then achieved by a magnet due to
the magnetic properties of the nanoparticles. The ratiometric detection of A488 fluorescence (compared to nanoparticle
fluorescence) in the purified sample then reveals accurately the presence of the target DNA. (B) Fluorescence ratio for þPKD
and
PKD samples: significant detection for [DNA] > 200 ng/mL. Reprinted with permission from ref 92. Copyright 2008 Springer
ScienceþBusiness Media.

they have been successfully used to detect SNPs.92 Europium-doped gadolinium oxide nanoparticles
Nanoparticles were functionalized with neutravidin were used to detect protein micropatterns.75 Biotin
and then coupled to probe DNA fragments (com- was regularly patterned on a silicon wafer by micro-
plementary of the target DNA carrying the þPKD contact printing and nanoparticles were coated with
mutation). Genomic DNA was then extracted from streptavidin, able to specifically bind biotin. After 1 h
tissue and the region of interest containing the poly- incubation and rinsing of the unbound particles, the
morphism was amplified by PCR and hybridized;for pattern was observed by fluorescence microscopy
both
PKD and þPKD samples;with a complemen- (Figure 4A). This experiment demonstrated the feasi-
tary strand labeled by an organic dye (Figure 3A). When bility of efficient protein detection on a substrate and
probe DNA coupled to nanoparticles and sample DNA could be used as a model for biologically relevant
are mixed, hybridization occurs preferentially for the problems using antibody-coated nanoparticles. The
correct SNP. Hybridized fragments containing nano- possibility to use rare-earth nanoparticles in immu-
particles are separated from the solution by a magnet noassays instead of organic dyes was demonstrated by
and the luminescence ratio between Eu3þ and organic labeling mouse IgG with NaYF4:Yb,Er nanoparticles
dye reveals the fraction of bound DNA. The presence of coupled to antimouse antibodies (Figure 4B).38
a given polymorphism is thus detected by the mea- Nanoparticles with organic cores have also been
surement of the fluorescence ratio (Figure 3B). The successfully used for sandwich-type immunoassays.54
combination of magnetism for separation and of fluo- As in standard ELISA protocols, prostate specific antigen
rescence for quantification using the reference signal (PSA) or thyroid stimulating hormone (TSH) were
of the organic dye yields a powerful method for incubated and bound in microwells coated with specific
quantitative detection of SNPs. antibodies. Europium-chelate-doped polystyrene
Protein Detection. In vitro detection of proteins by nanoparticles were coated with specific antibodies against
fluorescent or radioactive labels in immunoassays is PSA or TSH in order to probe the bound antigen in
important either for molecular biology experiments or microwells. Nanoparticle fluorescence was then detected
for diagnosis. Lanthanide chelates or cryptates are at 615 nm through a fluorimeter to reveal the presence
already commonly used and commercially distributed of the antigen. The comparison between nanoparticle
in that context (dissociation-enhanced lanthanide fluo- labeling and soluble dye revealed that specificity is
rescence immunoassay) as an improvement to con- mostly determined by antibodies,54 while sensitivity
ventional ELISA (enzyme-linked immunoabsorbent can notably be increased by the use of nanoparticles.53
assay). These applications rely on the ensemble detec- These applications;DNA detection or protein
tion of fluorescence of europium chelates under UV immunodetection;are routine procedures in biology
excitation to improve sensitivity.93,94 The development laboratories. The advantage of rare-earth nanoparticles
of nanoparticle-based methods is however essential to is to provide a qualitative improvement of existing
achieve single molecule detection either for ultrasen- assays based on the rare-earth photostability, efficient
sitive in vitro detection or for applications where in vivo detection, and facile coupling of magnetic and lumi-
single protein imaging is required (see below). nescent properties.

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Figure 4. Protein detection on micropatterns. (A) Pattern of biotin revealed by the nanoparticle luminescence. Reprinted with
permission from ref 75. (B) Immunoassay: the presence of mouse IgG was revealed by the emission of nanoparticles coupled
to antimouse antibodies (line 4). No nonspecific binding to controls lines (line 1 and 2) was observed. Reprinted from ref 38.
Copyright 2002 American Chemical Society.

FRET. Fluorescence resonant energy transfer (FRET) In the absence of protein, labeled BSA at the surface of
has become a widely used technology in biology using the particle is excited by FRET. The decrease of the FRET
different types of materials for the detection of protein
signal in the presence of proteins replacing labeled
protein interactions.95 This method relies on the effi- BSA is thus a function of protein concentration. Typical
cient energy transfer of an excited fluorophore labeling concentrations of 100 μg 3 L
1 of BSA, γ-globulin or
one protein (the donor) to the fluorophore labeling thyroglobulin were thus detected. These performances
a second protein (the acceptor) and on the ability are comparable with commercial protein titration
to specifically detect the fluorescence resulting from methods. Similarly, europium-chelate-doped polystyr-
this process. It is thus important to efficiently discrimi- ene beads were used for FRET experiments to detect
nate this signal from direct acceptor excitation and cells and probe their viability in solution.100 In that case,
donor emission leaking in the acceptor channel. labeled BSA adsorption is prevented by the presence of
Lanthanide nanoparticles are promising donors for cells, resulting in a FRET signal decrease depending on
FRET applications (more rigorously termed LRET in the number of cells in solution. The sensitivity of this
the case of lanthanide ions for luminescence resonant method was sufficient to count five cells in solution in a
energy transfer) for multiple reasons.21 First, the large microwell, which is significantly less than that of
Stokes shift of lanthanide systems allows excitation at standard automated counters.
much shorter wavelength than the acceptor absorp- Experiments performed with Cy5 acceptors ad-
tion and thus prevents its direct excitation. Second, the sorbed onto YVO4:Eu donor nanoparticles also illu-
narrow emission lines of lanthanide nanoparticles limit strated the efficiency of the use of nanoparticles:
the overlap with the acceptor emission spectrum. FRET was revealed by a significant alteration of both
Third, the absence of photobleaching facilitates the the nanoparticle and the Cy5 fluorescence decay
FRET experiments. Fourth, the long (∼1 ms) lifetime of (Figure 5 panels A and B, respectively).71 These experi-
the excited states in lanthanide systems allows easy ments show the efficiency of rare-earth nanoparticles
detection of donor lifetime changes due to FRET as FRET donors for biological applications by detect-
(Figure 5A). Indeed, a simple setup with a mechanical ing the proximity of a fluorophore commonly used in
chopper and a numerical oscilloscope is sufficient for living systems. Furthermore, the possibility of observ-
these lifetime measurements. In addition, these long ing FRET using a single rare-earth nanoparticle donor
lifetimes lead the acceptor emission due to FRET to was demonstrated.71
occur at longer time scales than direct fluorescence of Upconversion nanoparticles, usually made by co-
an organic fluorophore acceptor (∼1 ns) and enables an doping with Er3þ and Yb3þ, also have an interesting
easy separation in time-resolved FRET experiments.71 potential as donors in FRET applications.101,102 Their
Such properties were already obtained by using soluble anti-Stokes shift indeed allows FRET with an excitation
europium chelates and cryptates as a donor allowing a wavelength longer than that of acceptor emission and
UV excitation in ensemble experiments.21,94,96
98 In thus a clean detection of a specific FRET signal. These
addition, their long lifetimes have been exploited to particles have been used in different biological con-
demonstrate FRET applications with quantum dots texts. Nanoparticles were coated with streptavidin and
(QDs) as acceptors despite the QD broad absorption mixed with a biotinylated protein labeled with a
spectrum thus yielding a robust photostable FRET fluorescent acceptor,101 whose fluorescence was de-
system.22 Micrometric melamine nanoparticles have tected after infrared excitation of the donor nanopar-
been coated by several layers of europium chelate to ticles: this demonstrated the feasibility of protein
detect proteins.99 The proposed method is based on detection with FRET from upconversion nanoparticles.
the competition between adsorption of fluorescently This has been applied in vitro103 and for diagnosis104
labeled BSA and target protein on the nanoparticles. for the detection of estradiol (E2) by a competitive

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Figure 5. Fluorescence resonant energy transfer using YVO4:Eu nanoparticles as donors and Cy5 fluorophores as acceptors.
Direct excitation of Eu3þ ions at 466 nm. (A) Fluorescence decay (617 nm) for bare nanoparticles (NP) or nanoparticles coated
with Cy5 (NP-Cy5). Note the faster decay of fluorescence for NP-Cy5, revealing FRET. (B) Fluorescence decay in the Cy5
emission channel (670 nm) of free or NP-bound Cy5. A pair of synchronized mechanical choppers enables starting the detec-
tion 50 μs after the excitation beam has been blocked. Thus, no signal is observed for a free Cy5 solution under Eu3þ excitation
indicating efficient rejection of direct Cy5 (acceptor) excitation with the donor excitation wavelength. The excitation of NP-
Cy5 shows a long emission lifetime determined by the donor lifetime as expected for FRET emission.21 The absence of signal
for nonresonant nanoparticle excitation (457 nm) confirms the exclusive detection of the FRET signal without any contribu-
tions from direct acceptor excitation. The two zero-signal curves (Cy5 and NP-Cy5 under 457 nm excitation) are indistin-
guishable. The thin black line is a biexponential fit of the NP-Cy5 signal. Reprinted from ref 71. Copyright 2006 American
Chemical Society.

immunoassay. La2O2S:Er3þ,Yb3þ nanoparticles were specific protein imaging in vivo. Upconversion nano-
coated with E2 antibody and mixed with E2 coupled particles are particularly interesting in that perspective,
to an acceptor fluorophore (E2-AF680), which creates due to the absence of fluorescence background com-
FRET signal. After addition to a blood sample, there is a ing from the external medium.39,76,107 Moreover, infra-
competition between native E2 from the blood and red excitation is interesting for imaging of tissues due
labeled E2, causing a reduction of FRET (Figure 6A,B). to their low absorption in biological media. Y2O3:Yb,Er
Concentrations in the nanomolar range have thus nanoparticles have thus been used for imaging of the
been detected.104 This application is particularly inter- Caenorhabditis elegans worm.107 Particles were added
esting for blood testing, because of its sensitivity and to worm food, ingested without damage for the worm,
the small sample volume required for the experiment. and imaged by infrared excitation. This provides
A similar approach was used to quantify an endonu- images of the worm digestive system (Figure 7A).107
clease activity. Nanoparticles were coated with a Experiments combining uptake of nanoparticles
nucleotide coupled to a fluorescent acceptor (AF680) and and confocal microscopy have been proposed76,108 in
a quencher (nonfluorescent acceptor) at its ends.105 cell cultures and in vivo. NaYF4:Yb,Er particles have
Thus, this conjugate does not emit in the acceptor been loaded in HeLa cells108 or mouse skeletal
channel (Figure 6C). When the nucleotide is cleaved by myoblasts76 in culture by incubation. This allows the
the endonuclease, the quencher is released and FRET tracking of single cells in vitro and in vivo after injection
signal is restored depending on the endonuclease of these loaded cells in a mouse vein or intramuscular
concentration. (Figure 6D).105 As a consequence, this injection of nanoparticles (Figure 7B).76 The anti-Stokes
allows a quantitative monitoring of the protein activity. shift of upconversion nanoparticles provides a suffi-
Interestingly, this assay is not a competitive assay and cient contrast for imaging at a depth of ∼2 mm, which
provides a direct activity measurement, without any is comparable to performances obtained in 2-photon
requirement of labeling the protein of interest. microscopy.
Although direct excitation of AF680 as a donor could Intravenous injection of persistent luminescence
allow the same kind of measurements, the use of MgSiO3:Eu2þ,Dy3þ,Mn2þ particles in living mice has
upconversion nanoparticles notably reduces the back- enabled in vivo imaging of different organs during
ground due to their anti-Stokes shift. 1 h without need for excitation after injection.42 Inter-
estingly, the coating of these nanoparticles determines
CELL BIOLOGY AND IN VIVO APPLICATIONS which organs are labeled: this is the first step toward
Nonspecific Imaging. The simplest applications of rare- targeting for specific imaging (Figure 7C).
earth nanoparticles in living systems are nonspecific Specific Protein Targeting. The labeling of proteins
loadings of nanoparticles in living cells or organisms. with fluorescent tags in cells or tissues has become a
These experiments illustrate their remarkable optical major tool of cell biology. It is widely used in fixed cells
properties and their absence of cytotoxicity and pro- in immunocytochemistry for the study of cell organiza-
vide instrumental developments likely to contribute to tion. The existing diversity of organic dyes is rather

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Figure 6. FRET-based estradiol and endonuclease activity detection using upconversion nanoparticles as donors. (A) Upconver-
sion nanoparticle (UCP) functionalized with Anti-E2 antibody. There is a competitive binding of native E2 (black) and acceptor-
coupled E2 (white) responsible for FRET. (B) FRET signal as a function of native E2 concentration. (C) Upconversion nano-
particle coupled to an oligonucleotide containing at its two ends an acceptor (A) and a quencher (Q). Successive energy
transfer from the upconversion nanoparticle to the acceptor and from the acceptor to the quencher leads to fluorescence
quenching for uncleaved oligonucleotides. The fluorescence is restored when the quencher is released by cleaving of the
oligonucleotide. (D) FRET signal as a function of the oligonucleotide concentration with and without endonuclease. Reprinted
with permission from ref 106. Copyright 2008 John Wiley & Sons, Inc.

sufficient for that purpose. However, the visualization of The targeting of a specific protein in a living cell with a
proteins in a living cell is slightly more difficult because nanoparticle (quantum dots or rare-earth doped nano-
of the fast photobleaching of GFP variants or of organic particles) has been, however, so far mostly restricted to
dyes. In this domain, rare-earth-doped nanoparticles membrane proteins. Unbound nanoparticles can indeed
can contribute notably to biology, because of their be rinsed while specifically bound nanoparticles stay at
remarkable photostability and absence of emission the cell membrane by recognizing their target protein.
intermittency. Nanoparticles can be functionalized (with Rinsing is not possible inside the cell which prevents
an antibody or a ligand) to specifically bind a protein. this approach for cytosolic proteins. An interesting
YVO4:Eu nanoparticles functionalized with epoxy groups approach for that purpose is the external nanoparticle
were coupled to guanidinium groups (Figure 8A).32 These labeling of purified proteins followed by the purification
groups are responsible for the inhibition of voltage- of nanoparticles bound to a functional protein and
gated sodium channels by saxitoxin and tetrodotoxin their subsequent injection into the cell. The reliability
(neurotoxins secreted by marine microorganisms).109 of this method depends on the extreme purity of the
When these nanoparticles are added to the culture injected sample: any unbound nanoparticle would
medium of frog cardiomyocytes, they bind to the pore cause an artifact. It has been used only for quantum
opening of sodium channels and thus allow their dots up to now for a very particular system;single
imaging through laser excitation of Eu3þ ions at kinesin imaging;for which specific purification methods
466 nm (Figure 8B). The long lifetime of nanoparticle exist.110 Strictly speaking, this method does not target
fluorescence enabled a time-gated detection: two cytosolic proteins but injects labeled proteins in the
synchronized choppers (one for the source, the other cytosol. The possibility to label intracellular proteins
for the camera) induce a ∼50 μs delay between the end with nanoparticles thus remains a major challenge.
of the illumination and the beginning of the acquisi- Organic fluorophores have been used to target genetically
tion. This eliminates the cell fluorescence and is a major modified cytosolic proteins.111 While such approaches
advantage of long-lifetime fluorescent probes. It has can also be envisaged for nanoparticles, particular cau-
thus been possible to image sodium channels with tion in terms of size is required to avoid artifacts due to
single molecule resolution in living cells (Figure 8B). the molecularly crowded intracellular environment.112

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Single-Protein Tracking. For several years, there has ε-toxin receptors in the membrane of Madin
Darby
been a growing interest in single molecule imaging in canine kidney (MDCK) cells (Figure 9).80,118 Functiona-
living cells, particularly for membrane proteins.5,113
115 lization by amine groups followed by coupling to the
Their tracking indeed reveals important information on toxin56,57 leads to the binding of the nanoparticles to
the cell membrane properties and organization or the the membrane receptors. Single-particle detection has also
interactions with other proteins. The use of quantum dots been demonstrated with upconversion particles;40,63,69 the
has become quite popular, although their blinking causes specific binding to single biomolecules, however, still
the trajectory reconstruction to be complex.116,117 remains to be shown.
Nonblinking YVO4:Eu particles have been used to track The coupling ratio between nanoparticles and pro-
teins is essential to achieve single molecule measure-
ments. Therefore, Casanova et al. proposed a method
based on the single-step photobleaching of proteins
labeled with organic fluorophores to determine the exact
number of proteins bound to single nanoparticles.56
This was demonstrated for YVO4:Eu nanoparticles but
the approach is applicable to any type of particle. In
practice, when the mean ratio is close to 1:1, it is
unlikely for a single particle to bind more than one
receptor.110,118 In that case, a single nanoparticle tra-
jectory reliably reflects the trajectory of a single receptor.
The viscosity difference between the outer medium and
the cell membrane ensures that the observed motions
are not hindered by the size of the nanoparticles. This
was confirmed in different systems by comparing
diffusion coefficients of nanoparticle-labeled and organ-
ic dye-labeled proteins.80,117 Owing to the photostabil-
Figure 7. (A) Nonspecific imaging with upconversion nano-
particles. C. Elegans worm after Y2O3:Yb,Er nanoparticle ity and absence of blinking of the nanoparticles,
ingestion for 24 h (nanoparticles appear in green) revealing several minute-long uninterrupted trajectories with a
the worm digestive system. Reprinted from ref 107. Copy- 20
50 ms resolution and a localization precision down
right 2006 American Chemical Society. (B) Projection of a
2 mm thick stack of a mouse hind limb after injection of to 20 nm were recorded (Figure 9). It should be noted
myoblasts with internalized NaYF4:Er,Yb nanoparticles. that a minimum nanoparticle size is required for these
Reprinted with permission from ref 76. Copyright 2009 Els- applications so that the numbers of photons emitted
evier. (C) Images of carboxyl coated Ca0.2Zn0.9Mg0.9Si2O6:-
(Eu2þ, Dy3þ, Mn2þ) nanoparticles injected to a living during the short acquisition times, which are propor-
mouse. Owing to persistent luminescence, images were tional to the nanoparticle volume,70 are detectable
obtained without excitation. Reprinted with permis- above the background. Typically, YVO4:Eu nanoparti-
sion from ref 42. Copyright 2007 National Academy of
Science, USA. The nanoparticles accumulated preferen- cles with sizes of 30 nm or above were required, sizes
tially in the liver. comparable to those of commercial quantum dots.

Figure 8. Labeling of voltage-gated sodium channels using YVO4:Eu nanoparticles. (A) Nanoparticle functionalized with
guanidinium groups able to bind sodium channels. (B) Fluorescence image of a living frog cardiomyocyte. Yellow dots are
nanoparticles labeling voltage-gated sodium channels. Diffusion limited spots are single nanoparticles. Scale bar: 5 μm.
Reprinted from ref 32. Copyright 2004 American Chemical Society.

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Figure 9. Single toxin receptor tracking using YVO4:Eu nanoparticles.80,118 (A) Fluorescence image of single toxin receptors in
the membrane of MDCK cells labeled by YVO4:Eu nanoparticles. (B) Example of a reconstructed trajectory. Confinement can
be observed. (C) Potential felt by the receptor describing the trajectory shown in panel B.

Using single-molecule tracking data obtained with of a law relating the instantaneous concentration of
rare-earth-doped nanoparticles, it was not only possi- H2O2 to the fluorescence signal:
ble to measure the diffusion coefficient, which is also t
accessible by tracking of organic-dye labeled proteins, S(t)=S(0) ¼ A[C(t)](1
exp(
))
τ[C(t)]
but also to reconstruct the force field of the micro-
domain in which the toxin receptor was moving using This enables the in vitro dynamic and quantitative
a novel approach based on Bayesian inferences80,118 measurement of H2O2 concentration (Figure 10D).
(Figure 9). This is an illustration of the exciting possibi- The response of YVO4:Eu nanoparticles is not specific
lities provided by the use of nanoparticles in biology: to H2O2, and similar fluorescence recovery can be
their remarkable optical properties not only facilitate obtained with other oxidant species. These particles
previously possible experiments but also give access to can thus probably also be used for the detection of
entirely new biologically relevant information. other physiological oxidants such as ClO
or NO.77
Oxidant Nanoprobes. The detection of intracellular Nanoparticles were internalized in vascular smooth
signaling molecules is an essential requirement in cell muscle cells (VSMCs) by pinocytic loading to dynami-
biology. H2O2 is implicated in signaling in a variety of cally measure the intracellular H2O2 concentration
physiological cellular processes (contraction, migra- (Figure 11). No cytoxicity due to the nanoparticles
tion, proliferation, differentiation, apoptosis, etc.) and within the cell was observed: after 18 h, the proportion
plays an important role in pathophysiological condi- of living cells was equal to that obtained in the absence
tions like atherosclerosis, inflammatory processes, and of nanoparticles.77 This highlights one of the major
neurodegenerative and malignant diseases. The ability advantages of rare-earth doped nanoparticles com-
of the cell to produce distinct responses to signals pared to other methods of intracellular labeling en-
sharing the same secondary messenger is currently abling harmless long-term imaging and likely in vivo
poorly understood due to the lack of accurate and applications.
dynamic methods to measure the intracellular response. In response to stimulation by endothelin-1 (ET-1) or
Recent experiments using rare-earth nanoparticle platelet derived growth factor (PDGF), VSMCs produce
imaging have contributed to this field by the quanti- H2O2 through the activation of the NADPH oxidase
tative and dynamic measurement of H2O2 concentra- (NOx)protein complex leading, respectively, to con-
tion in living cells.77 The main emission of YVO4:Eu traction or migration. The produced quantities of
nanoparticles is due to the 5D0
7F2 transition of H2O2 were similar (7 μM). The timing of the production,
dopant Eu3þ ions centered at 617 nm. Eu3þ ions can however, was notably different. These differences are
be reduced to Eu2þ ions through a photoinduced important in relationship with the signal transduction
mechanism (Figure 10A). A relatively high intensity in living cells and more specifically for understanding
resonant illumination for 250 s is thus sufficient to the ability of the cell to discriminate between different
induce an important fluorescence decrease (∼70%, signals sharing common second messengers. These
Figure 10A). The subsequent in vitro application of measurements were enabled by the unique oxido-
H2O2 causes an oxidation of Eu2þ and consequently a reduction properties of rare-earth-based nanoparticles.
recovery of the fluorescence at 617 nm (Figure 10A,B). The H2O2 production in the vascular system was
This recovery amplitude and speed depends on the quantified over time: this result is only accessible by
applied H2O2 concentration (Figure 10B,C). Further- rare-earth nanoparticle imaging. Alternative methods
more, this recovery is reversible (Figure 10A) and no using either organic oxidant indicators such as DCF
aging of the nanoparticles was revealed: their response (DiChloroFluorescein),2 Amplex Red (Invitrogen), or a
is identical whatever the history of the oxidation state genetically encoded sensor (Hyper protein)119 only
was.77 Altogether, these results allow the determination provide qualitative information due to their lack of

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Figure 10. In vitro H2O2 detection using YVO4:Eu nanoparticles. (A) Cycles of photoreduction and chemooxidation. The cycles
number 2 and 4 are obtained for a H2O2 concentration half that for other cycles. (B) Fluorescence evolution after
photoreduction and subsequent addition of H2O2 at t = 0 at different concentrations (average of ∼20 single nanoparticles). (C)
Amplitude of fluorescence recovery as a function of H2O2 concentration determined from exponential fits of the curves in
panel B. Blue bars indicate the error bar of the fit and red bars the standard deviation of fits performed on single nanoparticle
measurements. (D) Instantaneous H2O2 concentration as a function of fluorescence signal and its first temporal differential.
Reprinted from ref 77. Copyright 2009 Nature.

Figure 11. H2O2 detection in living cells using YVO4:Eu nanoparticles. (A) White-light transmission image of a vascular smooth
muscle cell loaded with nanoparticles. (B) Fluorescence image of internalized nanoparticles in the same cell. (C) Response to a
100 ng/mL PDGF stimulation. Reprinted from ref 77. Copyright 2009 Nature.

reversibility or quantitativity. In addition, the spatial image reconstruction are highly affected by the envi-
accuracy in [H2O2] measurements is only limited by ronment. Superparamagnetic iron oxide nanoparticles120
the positioning error of the nanoparticles. Using stan- shorten these relaxation times, mostly the T2-time, and
dard methods for single particle tracking, it is thus are commonly used to locally darken T2-weighted MRI
possible to detect H2O2 in living cells with an accuracy images (positive contrast agents). On the other hand, the
of 50 nm, 0.2 μM, and 30 s. presence of paramagnetic species with unpaired elec-
Contrast Agents for MRI Imaging. Magnetic resonance trons is known to notably shorten T1 and consequently
imaging (MRI) is one of the main modalities for medical increase MRI contrast leading to a brightening of T1-
imaging, notably because it is not invasive and enables weighted images (positive contrast agents).121 Gd3þ
in-depth 3-dimensional imaging. The pulse MRI tech- ions have a high magnetic moment under a magnetic
nique relies on the relaxation of water protons in a field due to their large number of unpaired electrons
strong static magnetic field after a pulse of transverse (seven) but are highly toxic. They are thus widely used
radiofrequency field. The longitudinal and transverse as MRI contrast agents in the form of Gd3þ chelates,
relaxation times T1 and T2 of the magnetization used for such as Gd-DTPA122 or Gd-DOTA, to avoid cytotoxicity.

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Figure 12. Bimodal MRI and fluorescence imaging using Gd2O3 nanoparticles. (A) Structure of the nanoparticles: The Gd2O3
core is coated by APTES containing a fluorophore and the nanoparticle is then covered by a PEG layer. (B) MRI images with (a)
standard Gd-DOTA solution, (b) 2.2 nm nanoparticles, and (c) 3.8 nm nanoparticles. (C) MRI image of a living rat 1 h after
nanoparticle injection. (D) Fluorescence image of a living rat. In both cases “B” indicates the bladder. Reprinted from ref 62.
Copyright 2007 American Chemical Society.

Recently, gadolinium-based nanoparticles have been Gadolinium chelates were also used for the coating
developed for MRI contrast enhancement62
65 and of gold nanoparticles to combine X-ray and MR imag-
multimodal imaging (MRI and fluorescence or X-ray)62,123 ing. The use of 2 nm Au@DTDTPA-Gd particles in-
or therapy by thermal neutron irradiation.124 The use of creases the contrast in both MRI and X-ray computed
nanoparticles leads to longer rotational correlation tomography (CT). Gold nanoparticles are indeed
times which increase the proton relaxivity (defined as known to be good contrast agents for CT, and this
the modification of the inverse proton relaxation time effect is enhanced by the Gd-chelate coating. These
due to the presence of the contrast agent over the nanoparticles are thus already good candidates for
contrast agent concentration) and, with an adequate medical use.
coating, to longer blood circulation times. In addition, The advantage of these hybrid nanoparticles com-
Gd3þ-ion leaching can be expected to be weaker when pared to conventional techniques is notable: they
the ions are incorporated in solid nanoparticles rather combine in a single object the chemical properties
than in a small organic molecule. Furthermore, nano- required for different imaging modalities and for
particles can be used as a platform for grafting mol- biotargeting.
ecules allowing multimodal imaging and/or specific
targeting in vivo.125 CONCLUSION
Gadolinium oxide nanoparticles (Gd2O3) were se- The possibility to use rare-earth based nanoparticles
quentially coated with aminopropyltriethoxy silane has been demonstrated for many different biological
(APTES) coupled to a fluorophore and a polyethylene- applications, covering molecular and cell biology,
glycol (PEG) layer to which biotargeting groups can be in vitro and in vivo assays. They thus are an interesting
attached (Figure 12A). These nanoparticles enhance alternative to existing methods. Their optical proper-
MRI contrast more efficiently than the Gd
DOTA ties, photostability, absence of blinking, narrow or up-
complex, commonly used in clinical MRI, for the same converted emission, large Stokes or anti-Stokes shifts,
concentration of Gd3þ ions (Figure 12B) and can also and long lifetime of excited states, and their absence of
be imaged by fluorescence.62 Notably, the effect of cytotoxicity make them suitable to replace organic
Gd3þ ions on water molecule relaxation still exists dyes or quantum dots in all their reported applications.
although they are covered by the PEG layer: This points Their potentialities in living systems have been clearly
to interesting possibilities of functionalization for bio- established and applications leading to major biologi-
targeting in vivo. Injection of Gd2O3 nanoparticles into cal breakthroughs are now appearing. The chemical
a rat allows simultaneous MR and fluorescent imaging properties of rare earth ions have thus allowed other-
(Figure 12C,D) revealing its bladder.62 Furthermore, wise inaccessible measurements, like the dynamic
GdPO4 nanoparticles coated with dextran have been detection of H2O2. In this case, rare-earth based nano-
used for tumor detection, exploiting their long blood particles are not only a convenient alternative to
circulation time and the enhanced permeability and existing methods, but have revealed new biologically
retention effect (EPR) of the tumor vasculature. Tumor important information. Finally, one of the main advan-
visualization was possible with 1/10 of the dose typi- tages of rare-earth doped nanoparticles synthesized in
cally used with Gd
DTPA.65 An appropriate functio- water is their easy functionalization compared to
nalization could enable the targeting of organ- or quantum dots. Furthermore, combined properties for
tumor-specific receptors,126 opening new perspectives multiple imaging types can be obtained with a single
for medical imaging. particle. This facile combination of different modalities

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and targeting molecules is likely to place rare-earth Properties of Luminescent Lanthanide-Doped Hybrid
based nanoparticles as a central tool for biomedical Organic
Inorganic Materials. J. Mater. Chem. 2008, 18,
23–40.
imaging. 20. Petoud, S.; Cohen, S. M.; Bunzli, J. C. G.; Raymond, K. N.
Stable Lanthanide Luminescence Agents Highly Emis-
sive in Aqueous Solution: Multidentate 2-Hydroxy-
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