Communications
DOI: 10.1002/anie.200804231
Nanoparticle Sensors
Europium-Based Fluorescence Nanoparticle Sensor for Rapid and
Ultrasensitive Detection of an Anthrax Biomarker**
Kelong Ai, Baohua Zhang, and Lehui Lu*
During the past decade the threat of biological attack with
B. anthracis spores, a potential biological warfare agent, has
been of particular concern throughout the word.[1,2] Inhala-
tion of more than 104 B. anthracis spores can lead to death
unless medical attention is received within 24–48 h.[3] There-
fore, the rapid and ultrasensitive detection of B. anthracis
spores prior to infection is critical for the prevention and
control of anthrax disease or bioterrorism.
with rapid response, high sensitivity, and improved selectivity
is of great importance.
Herein, we present the first example of a europium-based
fluorescence nanoparticle sensor for rapid and ultrasensitive
detection of B. anthracis spores in aqueous solution. By
grafting the sensing moiety (EuIII complex) onto the surface
of organic-dye-doped silica nanoparticles, a nanoparticle
sensor with optimal geometry is produced, which sequesters
the reference dye in the core while offering the greatest
possible surface area for sensing of B. anthracis spores.
Several features of such a sensor make it particularly
attractive for the detection of B. anthracis spores: 1) the
stabilization through covalent bonding of both the reference
dye and the sensing moiety minimizes their leaching thus
giving a stable sensor with low background signal and
enabling quantitative chemical sensing down to the single
nanoparticle level; 2) incorporation of the reference dye as a
non-interfering internal calibration makes it possible for use
as a ratiometric sensor; 3) this architecture can improve the
thermal stability and mechanical properties of the sensing
moiety, enabling practical applications, and allowing the
sensing moiety to be separated from the analyte solution.
B. anthracis spores can be identified through the detection
of calcium dipicolinate (CaDPA), a unique biomarker for
bacillus spores, which accounts for about 10% of the spore
dry weight and is not found in other common spores, such as
pollen or mold.[1b,f] With this novel sensor, a procedure taking
only 2 min is capable of achieving a limit of detection (LOD)
of 0.2 nm CaDPA, which is approximately six orders-of-
magnitude lower than an infectious dosage of the spores and
is two orders-of-magnitude better than those reported using
TbCl3 as a sensor reagent.[1b,c]
Our strategy for the fabrication of a europium-based
fluorescence nanoparticles sensor is outlined in Scheme 1. In
the first step, uniform fluorescein isothiocyanate (FITC) dye-
doped silica nanoparticles are prepared using a reverse
microemulsion method and subsequently modified with 3-
aminopropyltriethoxysilane (APTES).[8] With this synthesis
approach, FITC with high quantum yield (F = 0.93), as a
reference dye, is protected from external influences such as
solvents, providing a stable reference signal. Next, ethyl-
enediamine tetraacetic acid dianhydride (EDTAD) is synthe-
sized according to the procedure published by Paik et al.[9]
and then covalently grafted onto the surface of the FITC-
doped silica nanoparticles by the reaction of the amino group
present on the APTES molecules with the anhydride group of
EDTAD molecules. The resulting EDTA ligand is then
readily converted into a [Eu(EDTA)(H2O)3] complex on
reaction with EuCl3 (Sensor 1). Upon exposure of Sensor 1 to
CaDPA, water molecules are excluded from the EuIII
Lanthanide-based sensors have gained a great deal of
attention in recent years owing to their unique spectroscopic
characteristics, including long fluorescence lifetime, large
Stoke shift, and sharp line-like emission bands.[4,5] These
properties are particularly attractive because they enable
temporal and spectral discrimination against background
fluorescence often associated with commonly used fluoro-
phores in chemical biology, leading to excellent detection
sensitivity. To date, terbium has been considered as the best
lanthanide for the detection of B. anthracis spores because of
its bright fluorescence, long fluorescence lifetime, and high
enhancement ratio.[1b,c,f,5b,6] To our knowledge, the use of
europium(III) for fluorescent sensing of B. anthracis spores
has not been achieved. This is probably based on the general
belief that excited states of EuIII can couple more efficiently
with the high-frequency OH oscillators of water molecules
than TbIII, resulting in enhancement of nonradiative quench-
ing of the EuIII emission and hence lower detection sensitiv-
ity.[7] Nevertheless, EuIII offers several advantages over TbIII in
terms of larger Stoke shift, a red emission, and exclusion of
second-order scattering interference in the maximum fluo-
rescence intensity. These advantages apply particularly to the
case of a nanoparticle sensor, which is very desirable for
applications in complex environments, such as those found in
spores. More importantly, false-positive results arising from
the nonselective binding of aromatic compounds to TbIII can
be decreased with the europium-based sensor, as discussed
below. Hence, the quest to prepare europium-based sensors
[*] K. L. Ai, B. H. Zhang, Prof. L. H. Lu
State Key Laboratory of Electroanalytical Chemistry Changchun
Institute of Applied Chemistry
Chinese Academy of Sciences
5625 Renmin Street, Changchun 130022 (P.R. China)
Fax: (+86)431-8526-2406
E-mail: lehuilu@ciac.jl.cn
[**] Financial support by the “Hundred Talents Project” (Initialization
Support) of the Chinese Academy of Sciences, the National Natural
Science Foundation of China (No. 20873138; No. 20845001), and
the Program for Excellent Doctoral Thesis of Chinese Academy of
Sciences is gratefully acknowledged.
Supporting information for this article is available on the WWW
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Angew. Chem. Int. Ed. 2009, 48, 304 –308