Terbium Functionalized Micelle Nanoprobe for Ratiometric Fluorescence Detection of Anthrax Spore Biomarker

Article abstract of DOI:10.1021/acs.analchem.8b00050

Rapid, sensitive, and selective quantitative detection of pyridine dicarboxylic acid (DPA) as biomarker of anthrax spores is in great demand since anthrax spores are highly lethal to human beings and animals and also potential biological warfare agents. H

Full text of DOI:10.1021/acs.analchem.8b00050

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Article
A novel terbium functionalized micelle nanoprobe for
ratiometric fluorescence detection of anthrax spore biomarker
Ke Luan, Ruiqian Meng, Changfu Shan, Jing Cao, Jianguo Jia, Wei-Sheng Liu, and Yu Tang
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00050 • Publication Date (Web): 01 Feb 2018
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A novel terbium functionalized micelle
nanoprobe for ratiometric fluorescence
detection of anthrax spore biomarker
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Ke Luan, Ruiqian Meng, Changfu Shan, Jing Cao, Jianguo Jia, Weisheng Liu and Yu
Tang*
State Key Laboratory of Applied Organic Chemistry, Key Laboratory of Nonferrous
Metal Chemistry and Resources Utilization of Gansu Province, College of Chemistry and
Chemical Engineering, Lanzhou University, Lanzhou 730000, P. R. China
*Eꢀmail: tangyu@lzu.edu.cn. Fax: +86 931ꢀ891ꢀ2582.
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ABSTRACT: Rapid, sensitive and selective quantitative detection of pyridine
dicarboxylic acid (DPA) as biomarker of anthrax spores is in great demand since
anthrax spores are lethal to human beings and animals highly and also the potential
biological warfare agents. Herein, we prepared a ratiometric fluorescence lanthanide
functionalized micelle nanoprobe by “oneꢀpot” selfꢀassembly, with an amphiphilic ligand
containing βꢀdiketone derivative which can “immobilize” terbium ions through the
coordination interaction and a fluorophore as fluorescence reference (FR). The detection
strategy was ascribed to Tb3+ ions in lanthanide functionalized micelle, which can be
sensitized to emit the intrinsic luminescence upon addition of DPA due to the presence of
energy transfer when DPA chromophore coordinated with Tb3+ ion. And the fluorescence
intensity of FR remained essentially constant, leading to ratiometric fluorescence
response toward DPA. The results demonstrate that the terbium functionalized micelle
was able to sensitively detect DPA with a linear relation in the range of 0 ꢁM to 7.0 ꢁM
in aqueous solution, which also showed remarkable selectivity to DPA over other
aromatic ligands. Our work paves a new way in the design of ratiometric fluorescence
lanthanide functionalized micelle nanoprobes which can be promising for selective and
sensitive detection of bacterial spores or biomolecules.
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INTRODUCTION
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Bacillus anthrax, a rodꢀshaped, Gramꢀpositive, and sporeꢀforming bacterium, which
can cause anthrax, further resulting in deadly infection against living organism.1 During
dormant period, the bacterial spores can survive under harsh environment such as high
temperature, freeze, ultraviolet radiation, desiccation, strong acid and strong base.2
Bacillus anthrax spores can transform into an active form when the surrounding
environmental conditions are favorable. The inhalation of 104 B. anthrax spores is deadly,
unless effective treatment is received within 24 ~ 48 h.3ꢀ5 Accordingly the Bacillus
anthrax spore as the potential biological warfare agent has attracted considerable
attention around the world.6,7 The rapid and sensitive detection of Bacillus anthrax is
crucial to minimize the probability of anthrax infection and prevent bioterrorism. During
the past decades, a number of sensitive, accurate and selective detection methods
including biological methods, highꢀpressure liquid chromatography, electrochemical
detection and optical methods have been developed for detecting anthrax spores.8,9
Thereinto, optical methods such as polymerase chain reaction (PCR),3 surface enhanced
Raman spectroscopy (SERS), immunoassays and luminescence techniques for detecting
anthrax spores have attracted plenty of interest.10ꢀ13 The method of PCR assay is to
amplify the bacterial DNA by chain polymerization. But it requires expensive reagents,
complicated pretreatment and long test time.3 The luminescence methods are primary
based on detecting dipicolinic acid (DPA), which accounts for about 5ꢀ15% of the dry
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weight of anthrax spores and is always used as a biomarker for anthrax spores.14,15
Compared to other assays, the advantages of luminescence method show a low detection
limit (LOD) for rapid, sensitive and selective response to anthrax spores.
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The luminescence of lanthanide ions own intrinsic narrow fꢀf emission bands, long
lifetimes and large Stokes shifts, which can keep off the interference from emission
overlap.16 However, fꢀf transition of single lanthanide ions is the Laporte forbidden, so it
is significant to introduce a chromophore called "antenna" which can sensitize the
lanthanide ions through the coordination interaction and energy transfer to the lanthanide
ions.17,18 Interestingly, DPA as the chromophore molecule can coordinate with lanthanide
ions and achieve transfer energy to lanthanide ions, thus the sensitized lanthanide ions
can exhibit characteristic luminescence emission through antenna effect.19ꢀ21 On account
of the particular intrinsic characteristics of lanthanide ions, different platforms containing
lanthanide ions have been developed for anthrax spore biomarker detection such as
carbon materials,9,22ꢀ24 silica nanoparticles,15 metalꢀorganic frameworks,25 and solid
films.26 To our knowledge, most of lanthanideꢀbased sensors used to determine the
concentration of DPA are according to the single change of lanthanide characteristic
fluorescence intensity, and show limit of detections (LODs) varying from subnaomolar to
a few tens of nanomolar of DPA.15,27ꢀ30 However, the fluorescence intensity based on
lanthanide ions always suffers from the disturbances of instrumental factors or
microenvironments. To overcome these drawbacks, ratiometric fluorescence probes are
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highly attractive because they are endowed with a selfꢀcalibration ability, which can
reduce the aforesaid interferences.31ꢀ33
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Selfꢀassembled supramolecular materials derived from amphiphilic small molecule
monomers have drawn more and more attention for their remarkable salient features.34ꢀ37
The amphiphilic monomers are composed of hydrophilic part and hydrophobic part. By
adjusting the structural parameters of organic structural unit, various topologies such as
micelles, liposomes, and vesicles can be fabricated in aqueous solution by
supramolecular selfꢀassembly.38,39 Recently, the selfꢀassembly of metalloꢀamphiphiles
has received considerable attention. These assemblies have the characteristics of organic
selfꢀassembled supermolecules, which show more sensitive to external stimuli, good
biocompatibility and low cytotoxicity compared with small organic molecules. Moreover,
they also display different features including in the luminescence,40 magnetism,41 and
catalytic properties.42 Some fluorescence nanoprobes based on selfꢀassembly of
metalloꢀamphipathic molecules have also been developed for detecting
biomolecules.43ꢀ44ꢀ46
Herein, we have manufactured a ratiometric fluorescence lanthanide functionalized
micelle nanoprobe that was made up of the fluorescence interiorꢀreference and
amphiphilic Tb3+ complexes as a “turn on” fluorescence probe by “oneꢀpot” method in
aqueous solution for the selective and sensitive detection of DPA (Scheme 1). The
lanthanide functionalized micelle was constructed on the account of following
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considerations: (1) Amphipathic ligand L was composed of alkyl chain moiety as
hydrophobic part and hyamine moiety as hydrophilic part, which contained βꢀdiketone
derivative to “immobilize” lanthanide ions in the micelle through the coordination
between the βꢀdiketone and lanthanide ions. (2) Tb3+ ion was selected to coordinate with
the ligand L for its characteristic emission properties, such as large Stokes shift,
resistance to photobleaching, narrow emission bands and long luminescence lifetime. Its
intrinsic luminescence was weak when the micelle was dispersed in aqueous solution as a
result of the Laporte forbidden fꢀf transitions and the nonꢀradiative transition of Tb3+ ion
excited states to the vibrational relaxation of water molecules. Only when the lowest
triplet energy level of the chromophore molecule is appropriate to the lowest triplet
energy level of Tb3+ ions, Tb3+ ions can be sensitized and excited to emit intrinsic
fluorescence. (3) A fluorophore as fluorescence reference (FR) which emits blue
fluorescence (440 nm) at 275 nm excitation was added into the micelle. Through the
above design thought, the ratiometric fluorescence terbium functionalized micelle was
prepared by “oneꢀpot” method in aqueous solution. The terbium functionalized micelle
rapidly exhibited characteristic fluorescence of Tb3+ ions when adding DPA into the
micelle solution, while the fluorescence of FR had negligible change. As far as we know,
it is for the first time to achieve the detection of the anthrax spore biomarker DPA by
ratiometric fluorescence terbium functionalized micelle as nanoprobe. And this design
strategy can also be applied to other lanthanide micelle nanoprobes used for the detection
of the biomarkers or biomolecules with high selectivity and sensitivity.
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Scheme 1. Selfꢀassembly of terbium functionalized micelle in H2O and the response
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property for DPA.
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EXPERIMENTAL
Instruments and Reagent
The steadyꢀstate corrected luminescence spectra in water solution were performed on
an Edinburgh Instruments FSL920 fluorescence spectrometer, with a 450 W Xe arc lamp
as the steadyꢀstate excitation source. The NMR spectra were obtained on 400 MHz
spectrometer with TMS as inner standard (Bruker). The UVꢀvis absorbance spectrum was
determined on a Cary 5000 spectrophotometer (Agilent). Fourier transform infrared
(FTIR) spectroscopy was obtained using FTꢀIR spectrometer ranging from 400 cmꢀ1 to
4000 cmꢀ1 by potassium bromide pellet on Vertex 70 (Burker). The surface tension (γ)
was determined on Surface Tension Meter (DCAT21, Dataphysics Company, Germany)
by Du Noüy ring. The morphologies of micelle were obtained on transmission electron
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microscopy (TecanaiTM G2 F30; FEI Company, USA) working at 300 kV. The ζ potential
and the dynamic light scattering (DLS) of micelles were measured on a Zetasizer (Nano
ZS, Malvern, Worcestershire, UK). Thermogravimetric analyses (TGA) were measured
using a Netzsch STA 449 F3 Jupiter up to 800 °C at a heating rate of 10 °C·minꢀ1 under a
nitrogen atmosphere. All materials and reagents were of analytical grade and were
purchased from J＆K Chemical Ltd. These reagents were used without any purification.
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Synthesis of the Ligand L
The synthetic procedure of the amphiphilic ligand L is shown in Scheme 2. And the
detailed synthetic route and the characterization data of the products are listed as below.
Scheme 2. Synthetic procedure of the amphiphilic ligand L.
Synthesis of 1-(4-dodecyloxy-phenyl)-ethanone: 4ꢀHydroxyacetophenone (4.08 g, 30
mmol) and potassium carbonate (24.8 g, 180 mmol) were dissolved in dimethyl
formamide (90 mL) at room temperature, the resulting mixture was heated to 60 °C and
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stirred for 1 h. Then 1ꢀbromooctane (8.7 mL, 36 mmol) was added dropwise to the
solution and stirred for an additional 12 h at 80 °C under N2 atmosphere. After cooling,
200 mL of icy water was added to the reaction solution and the mixture was subjected to
extraction with ethyl acetate (3×50 mL), collected organic layers, washed with brine (100
mL), dried over anhydrous MgSO4 and concentrated under reduced pressure to give a
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faint yellow solid (8.1g). H NMR (400 MHz, CDCl3, ppm): δ 7.90 (d, J = 4.0 Hz, 2 H),
6.90 (d, J = 4.0 Hz, 2 H), 2.52 (s, 3 H), 1.75 ꢀ 1.70 (m, 2 H), 1.40 ꢀ 1.24 (m, 18 H), 0.86 (t,
J = 7.2 Hz, 3 H). 13C NMR (400 MHz, CDCl3, ppm): δ 14.19, 22.77, 26.04, 26.36, 29.17,
29.43, 29.64, 29.72, 31.98, 68.30, 114.16, 130.10, 130.62, 163.18, 196.78. ESI MS (m/z):
305.3 [M + H]+.
Synthesis of 3-(4-dodecyloxy-phenyl)-3-oxo-propionic acid methyl ester: Sodium
hydride (0.36 g, 15 mmol) and dimethyl carbonate (0.9 g, 10 mmol) were dissolved in
anhydrous methylbenzene (30 mL) and heated to 110 °C. Then
1ꢀ(4ꢀdodecyloxyꢀphenyl)ꢀethanone (1.82 g, 6 mmol) was added dropwise to the reaction
mixture over half an hour and stirred at 110 °C for 2 h. When the reaction cooled to room
temperature, glacial acetic acid (1.5 mL) was slowly added and a heavy, pasty solid
appeared. Subsequently icy water was added until the solid was dissolved completely.
The mixture was extracted with ethyl acetate (3 × 20 mL), organic layers were combined
and washed with brine, dried over anhydrous MgSO4, concentrated under reduced
pressure and the residue purified by column chromatography over silica gel with
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petroleum ether / ethyl acetate = 10 / 1 as the eluents to give a yellow solid (1.28 g). H
NMR (400 MHz, CDCl3, ppm): δ 7.90 (d, J = 8.2 Hz, 2 H), 6.92 (d, J = 8.2 Hz, 2 H),
4.02 (t, J = 6.4 Hz, 2 H), 3.94 (s, 2 H), 3.73 (s, 2 H), 1.73 ꢀ 1.68 (m, 2 H), 1.40 ꢀ 1.24 (m,
18 H), 0.87 (t, J = 7.2 Hz, 3 H). 13C NMR (400 MHz, CDCl3, ppm): δ 14.22, 22.78, 26.04,
29.14, 29.44, 29.63, 29.67, 29.72, 29.75, 32.00, 45.62, 52.55, 68.45, 114.47, 128.83,
131.00, 163.79, 168.32, 190.90. ESI MS (m/z): 363.30 [M + H]+.
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Synthesis
of
N-(2-amino-ethyl)-3-(4-dodecyloxy-phenyl)-3-oxo-propionamide:
3ꢀ(4ꢀDodecyloxyꢀphenyl)ꢀ3ꢀoxoꢀpropionic acid methyl ester (1.81 g, 5 mmol), ethane
diamine (0.48 g, 8mmol), and DMAP (0.2 g, 1.5 mmol) were dissolved in 10 mL of
ethanol, and then the reaction solution was stirred overnight at 78 °C. The
Nꢀ(2ꢀaminoꢀethyl)ꢀ3ꢀ(4ꢀdodecyloxyꢀphenyl)ꢀ3ꢀoxoꢀpropionamide was consumed as
indicated by TLC. The resulting mixture was concentrated under reduced pressure and
the residue purified by column chromatography over silica gel with dichloromethane /
methanol = 50 / 1 as the eluents to give a reddish brown oil (1.07 g). 1H NMR (400 MHz,
CDCl3, ppm): δ 7.32 (d, J = 8.8Hz, 2 H), 6.86 (d, J =8.8Hz, 2 H), 3.94 (t, J = 6.4Hz, 2 H),
3.65 (s, 2 H), 3.15 (t, J = 6.4Hz, 2 H), 2.36 (t, J = 6.4Hz, 2 H), 2.17 (s, 6H), 1.79 ꢀ 1.72
13
(m, 2 H), 1.40 ꢀ 1.24 (m, 18 H), 0.86 (t, J = 7.2Hz, 3 H). C NMR (400 MHz, CDCl3,
ppm): δ 14.16, 22.73, 26.11, 29.11, 29.27, 29.40, 29.70, 31.97, 42.68, 45.49, 50.15, 59.68,
68.06, 114.25, 128.39, 129.20, 164.66, 170.64, 190.76. ESI MS (m/z): 419.31 [M + H]+.
Synthesis
of
the
ligand
L:
To
a
solution
of
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Nꢀ(2ꢀaminoꢀethyl)ꢀ3ꢀ(4ꢀdodecyloxyꢀphenyl)ꢀ3ꢀoxoꢀpropionamide (0.418 g, 1 mmol) in
acetone (4 mL), iodomethane (0.282 g, 5 mmol) in acetone (2 mL) was added dropwise
to the solution at 0 °C. Then the reaction solution was stirred for two days at room
temperature. When reaction completion, the ethyl ether (25 mL) was added, and the
resulting precipitate was collected by filtration, washed with ethyl ether several times,
and dried under reduced pressure to give a faint yellow solid (0.28 g). 1H NMR (400
MHz, DMSOꢀd6, ppm): δ 7.32 (d, J =8.8Hz, 2H), 6.97 (d, J =8.8Hz, 2H), 3.97 (t, 6.4Hz,
2H), 3.55 (s, 2H), 3.46 (t, J = 6.4Hz, 2H), 3.37 (t, J = 6.4Hz, 2H), 3.15 (s, 3H), 2.98 (s,
9H), 1.72 ꢀ 1.64 (m, 2H), 1.40 ꢀ 1.24 (m, 18H), 0.82 (t, J = 7.2Hz, 3H). 13C NMR (400
MHz, CDCl3, ppm): δ 14.35, 22.56, 26.10, 29.17, 29.26, 29.48, 31.78, 38.63, 50.45,
53.32, 65.10, 68.30, 86.71, 115.23, 127.45, 129.76, 160.35, 163.96, 169.80, 193.75. ESI
MS (m/z): 447.44 [M + H]+.
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Preparation of terbium functionalized micelle
A solution of terbium(III) nitrate hexahydrate (45 mg, 0.1 mmol) in deionized water (3
mL) was added dropwise to a solution of the ligand L (57.3 mg, 0.1 mmol) in deionized
water (5 mL). Then, 150 ꢁL of sodium 6ꢀ(dimethylamino)naphthaleneꢀ2ꢀsulfonate (FR,
10 mM, dissolved in acetone) was added to the mixture and stirred for 24 h at room
temperature. Then the reaction mixture was dialyzed with a cellulose ester dialysis
membrane (500 MWCO) for 2 days to completely remove “free” Tb3+ ions, ligand L and
FR. The resulting material was dried by lyophilization to obtain micelle powder, which
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was dispersed in HEPES buffer solution (10 mM, pH = 7.0) to prepare 1 mg·mLꢀ1 stock
solution of micelles for further characterization and use.
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RESULTS and DISCUSSION
Synthesis, morphology and characterization of the terbium functionalized micelle
The sensitive and selective nanoprobe for DPA detection was based on a ratiometric
lanthanide functionalized micelle, which was constructed with ligand L, Tb3+ ions and FR
by “oneꢀpot” method as shown in Scheme 1. The ligand L showed aggregation properties
in aqueous solution due to its peculiar structure: a hydrophilic hyamine and a
hydrophobic dodecyl chain. In order to investigate the selfꢀassembly properties of micelle,
critical micellar concentration (CMC) of micelle was determined by measuring the
surface tension (γ) in HEPES buffer solution (10 mM, pH = 7.0) with different
concentrations of Tb3+ꢀL.47,48 With increasing concentration of Tb3+ꢀL in aqueous
solution, the surface tension rapidly decline and remained steadily, which implied that the
hydrophobicity of the dodecyl chain in the ligand L is enough to form micelles. The
CMC was determined to be 36.4 ꢁM from the two linear segments in the curve of γ
versus concentration of the micelle solution (Figure 1a). And the successfully prepared
terbium functionalized micelle was studied by transmission electron microscopy (TEM)
and dynamic light scattering (DLS) in aqueous solution. The morphology of micelle was
homogeneous spherical nanoparticles with an average diameter of 135 nm (Figure 1c, d),
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which are features of micelles. When FR was added into the terbium functionalized
micelle to construct a ratiometric fluorescence lanthanide functionalized nanoprobe, the
DLS experiment showed the average diameter increased from 269.5 nm (Tb3+ꢀL micelle,
without loading FR) to 271.4 nm (terbium functionalized micelle, with loading FR)
(Figure S1b and Figure 1b), indicated that the load of FR had negligible effect on the
particle size of terbium functionalized micelle. And the average diameter of terbium
functionalized micelle determined by DLS was larger than that measured through TEM
resulting from the swelling of micelles in aqueous solution.49,50
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The coordination interaction between Tb3+ and the ligand L was studied by UVꢀvis
absorption spectroscopy and Fourier Transform Infrared (FTIR) spectroscopy (Figure S2).
The UVꢀvis absorption spectrum of ligand L showed absorption peak at 283 nm, which
can be attributed to the πꢀπ* transition of βꢀdiketone group.51 In contrast, the absorption
peak of Tb3+ꢀL was stronger and slightly redꢀshifted than the absorption peak of L,
indicated that Tb3+ ions coordinated with ligand L (Figure S2a). When βꢀdiketone group
in the ligand L coordinates with Tb3+ ion, the degree of electronic delocalization is
increased, and the degree of conjugation enhances, resulting in a larger activity range of π
electron in the lanthanide complex. Thus, the energy for energy level transition of the
associated conjugated molecular orbital is reduced, leading to the redꢀshifting of the
absorption peak of the lanthanide complex.52 FTIR spectrum of ligand L showed the
ν(CH2) bands at 2922 cmꢀ1 and 2850 cmꢀ1, indicated the presence of hydrophobic dodecyl
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chain. Besides, the sharp absorb band at 1645 cmꢀ1 and 1611 cmꢀ1 was ascribed to
stretching vibration of carbonyl. The covalent conjugation can be confirmed by a slightly
blue shift of FTIR absorption band of Tb3+ꢀL compared to the ligand L (Figure S2b).
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Figure 1. (a) Plot of surface tension as a function of the concentration of the solution of
micelle in HEPES buffer (10 mM, pH = 7.0). (b) Particle distribution of terbium
functionalized micelle in HEPES buffer (10 mM, pH = 7.0) solution measured by DLS.
TEM image of terbium functionalized micelle (c) the grid was untreated and (d) the grid
was postꢀstained with uranyl acetate.
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The binding of Tb3+ ions to ligand L was also identified by ζ potential (Table S1). The
ζ potential of Tb3+ꢀL micelle (ζ = 72.2 mV) showed a significant increase in comparison
to the ζ potential of ligand L micelle (ζ = 39.2 mV), which indicated that Tb3+ ions
incorporated in micelle surfaces. In addition, it can be seen that Tb3+ꢀL micelle formed
nanoparticle with an average diameter of 269.5 nm is larger than the nanoparticle formed
with ligand L (an average diameter, 193.6 nm) (Figure S1a). Through the above
characterization, the coordinated interaction between Tb3+ ions and ligand L can be
demonstrated. The Tb3+ content (29.5 %) in micelle was obtained by thermogravimetric
analysis (TGA) (Figure S3). It can be calculated that the complex ratio of ligand L to
Tb3+ is approximately 1 : 2.
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Optical responses of micelle as nanoprobe for DPA detection
The ability of terbium functionalized micelle for ratiometric detection of DPA was then
investigated. Firstly, the stability of fluorophore as the fluorescent reference was
measured by the detection of the fluorescence of FR at different pH in aqueous solution.
The fluorescence of fluorophore exhibited ignorable change within a large pH range
(5.0ꢀ10.0), which reveals that the pH of solution has a negligible influence on the
fluorescence of FR (Figure S4). The results indicated that the micelles have reliable
capacity as the ratiometric fluorescence nanoprobe for detecting bioꢀrelevant molecules.
Then the photophysical characteristics of Tb3+ꢀL micelles were measured in HEPES
buffer (10 mM，pH = 7.0). The micelle only showed blue luminescence (the fluorescence
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of FR, 440 nm) in aqueous solution under UV irradiation (275 nm). Interestingly, upon
addition of 5 ꢁM DPA, the aqueous of terbium functionalized micelle showed the
intrinsic absorption peak of DPA and gave a significant bright green luminescence
(Figure S5). In the emission spectrum, the emission bands were observed at 495 nm
(5D4→ 7F6), 545 nm (5D4→ 7F5), 586 nm (5D4 → 7F4), and 621 nm (5D4 → 7F3) under 275
nm excitation (Figure S6),53,54 which were intrinsic bands of Tb3+ ions. As neither of the
micelle nor DPA alone showed any green luminescence under these conditions, Tb3+ ions
were sensitized and emitted characteristic luminescence, which was attributed that the
coordinated H2O is replaced by DPA from the lanthanide center and DPA as the “antenna”
molecule transferring energy to Tb3+ ions. The binding of DPA to the micelles was also
confirmed by ζ potential changes. The ζ value of the micelles showed pronounced
decrease from 20.17 mV to 0.48 mV after addition of 100 ꢁM DPA (Table S1). Although
coordination between DPA and Tb3+ ions reduced the surface charge of the micelle, the
obtained micelles remained monodisperse (Figure S7) and the particle diameter has no
significant change (Figure S8). The terbium functionalized micelle can be used for
precise ratiometric fluorescence detection of DPA concentration due to the emission
intensity of Tb3+ ions increased significantly while the reference emission intensity at 440
nm hardly changed.
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The optical sensing property of micelle for DPA was investigated further in HEPES
buffer solution (10 mM, pH = 7.0) at room temperature. With addition of DPA, the
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terbium functionalized micelle emitted stronger and stronger green fluorescence with
negligible change in the fluorescence intensity of FR and the fluorescence enhancement
of Tb3+ ions in the micelle reached a plateau in seconds. Since the characteristic
fluorescence intensity of Tb3+ ions at 545 nm was highest increase upon addition of DPA,
the fluorescence intensity change at 545 nm was set to quantitatively detect DPA (Figure
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S6). It was also investigated that the effect of pH on the fluorescence intensity ratio (I545
/
I440) of the micelles when DPA was added (Figure S9). The I545 / I440 was found to vary
slightly from pH = 5.0 to 9.0, and decreased sharply at higher pH (pH = 10.0) on account
of the formation of Tb3+ hydroxide and precipitated. The result indicated that the micelle
nanoprobe can be used to detect DPA at normal pH range. Then the sensitivity of
lanthanide functionalized micelle to detect DPA was investigated through measuring the
fluorescence changes when different concentration of DPA was added into the aqueous
solution of lanthanide functionalized micelle. As the Figure 2a shows, the fluorescent
intensity at 545 nm gradually increases with the DPA concentration of 0 ꢁM to 18 ꢁM,
and the fluorescent intensity at 440 nm was almost invariant. The intensity of
fluorescence emission at 545 nm was observably enhanced about 140 fold when the
concentration of DPA was increased to 18 ꢁM (Figure S10). The fluorescent intensity
ratio of the peak at 545 nm and 440 nm showed linear variation with R2 = 0.999 in the
range of 0 ꢁM to 7 ꢁM DPA (Figure 2b). Moreover, the detection limit of the lanthanide
functionalized micelle for DPA was calculated to be about 54 nM from the calibration
curve using the method of signalꢀtoꢀnoise ratio (S / N) of 3, which is lower than the
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detection limit (87 nM, R2 = 0.998) by measuring “turnꢀon” fluorescence at 545 nm
(Figure S10), which is sufficient for the detection of anthrax spore stock suspension.55 It
was obviously demonstrated that ratiometric fluorescence detection own superiority over
singleꢀfluorescence detection. The luminescence of the aqueous of lanthanide
functionalized micelle changed from blue to green emission upon adding 60 ꢁM DPA
(the infectious dose in anthrax spores), which can be distinctly visible under 254 nm
UVꢀlamp illumination (Figure 2a inset).22 Interestingly, the fluorescence intensity of Tb3+
linearly changes with the increase of DPA concentration, so it can be easily changed the
detection range by adjusting concentration of micelles. The fluorescence titration curve
showed the micelle and DPA concentrations combined with a 1 : 1.25 binding model
(Figure S11) and stability constant (KS) was 2.17 × 1010 L molꢀ1 (Figure S12).56 The
ratiometric sensing capability of the micelle can be used for rapidly and quantitatively
analyzing the concentration of DPA without being influenced by external condition
arising from instrumental or environmental factors.
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Figure 2. (a) Fluorescent spectra changes of micelle (50 ꢁg·mLꢀ1) upon addition of
different concentrations of DPA (0ꢀ18 ꢁM) in HEPES buffer (10 mM, pH = 7.0) at room
temperature, slit: 5 nm / 3 nm. (Inset: photographs of the fluorescence of micelle before
(left) and after (right) adding 60 ꢁM DPA under a UV lamp at 254 nm) (b) Linear
relationship of the fluorescence intensity (I545 / I440) against the concentration of DPA
from 0 to 7 ꢁM. The data were reported as the mean ± standard deviation of triplicate
experiments and were fitted by the equation inserted.
High selectivity is very crucial to a probe, thus the selectivity of lanthanide
functionalized micelle for DPA was evaluated by addition of various other aromatic
ligands or amino acid. As showed in Figure 3, upon addition of benzoic acid (BA),
oꢀphthalic acid (oꢀPA), mꢀphthalic acid (mꢀPA), pꢀphthalic acid (pꢀPA), glutamine (Glu),
glycine (Gly), methionine (Met) and phenylalanine (Phe), the fluorescence intensity of
the lanthanide functionalized micelle (50 mg·mLꢀ1) didn’t show noticeable change.
Moreover, competition experiments showed that the luminescent response of lanthanide
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functionalized micelle to DPA was not interfered by all of these aromatic ligands and
amino acid. The high selectivity of lanthanide functionalized micelle for DPA can be
attributed that the carboxylate groups and nitrogen atom in the pyridine ring of DPA can
provide strong interactions with Tb3+ ions. In the opposite, other aromatic ligands may
not sufficiently chelate with Tb3+ ions. Further, the lowest triplet level of the DPA (3ππ*)
is more suitable for transferring energy from the ligand to the metal ion, so that Tb3+ ions
can be sensitized and emit intrinsic fluorescence. The coordination interaction between
Tb3+ and DPA was demonstrated on the basis of the details in the support information.
The experiment results indicate that almost two H2O molecules coordinate with Tb3+ ion
in the micelle. And the number of H2O molecules has negligible change upon addition of
DPA into the aqueous of terbium functionalized micelle. And we deduce that the DPA
may replace the coordinated anion and enter in the coordination sphere of Tb3+ through
metalꢀligand chelate coordination and ionꢀpairing mutual effect.57
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Figure 3. Fluorescence responses of terbium functionalized micelle toward different
potentially interfering aromatic ligands or amino acids. (a) Fluorescent spectra of micelle
in the presence of 5 ꢁM DPA, BA, oꢀPA, mꢀPA, pꢀPA, Glu, Gly, Met or Phe, slit: 5 nm / 5
nm. (Inset: photographs of the fluorescence of micelle upon addition of different analytes
under a UV lamp at 254 nm). (b) The blue bars represent addition of 5 ꢁM analytes to a
50 mg·mLꢀ1 solution of micelle (pH = 7.0, HEPES buffer solution): (1) blank, (2) BA, (3)
oꢀPA, (4) mꢀPA, (5) pꢀPA, (6) Glu, (7) Gly, (8) Met, (9) Phe. The green bars represent
subsequent addition of 5 ꢁM DPA to the mixture. The excitation wavelength was set to
275 nm.
Overall, these results clearly proved that lanthanide functionalized micelle own
excellent selectivity and antiꢀinterference ability for effective recognition of anthrax
spores biomarker. Furthermore, when DPA was introduced into the aqueous of lanthanide
functionalized micelle, the micelle showed a rapid fluorescence response, and the
luminescence of Tb3+ ions reached a plateau within seconds. As compared to previously
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lanthanide(III)ꢀbased probe (Table S2), the terbium functionalized micelle was
constructed through selfꢀassembly, with excellent biocompatibility and broad detection
range. Thus, the lanthanide functionalized micelle nanoprobe was allowed for rapid
detection of anthrax spore biomarker.
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Application of terbium functionalized micelle
To investigate the application performance of the terbium functionalized micelle
nanoprobe, the silica gel plate was impregnated with the micelle nanoprobe as test paper
for detection of DPA. As shown in Figure 4a, the silica gel plate impregnated with DPA
solution (60 ꢁM, 0.5 mL) emitted green fluorescence under the irradiation of a UV lamp
(254 nm). In the opposite, the silica gel plate impregnated with other analyte solution (60
ꢁM, 0.5 mL) only emitted the blue fluorescence of FR. Then, we used the solution of
DPA as ink, and the preꢀprocessed silica gel sheet as paper. After writing by DPA, the
portion of silica gel plate written by DPA showed green fluorescence under a UV lamp at
254 nm (Figure 4b), and the plate had no color change under visible light. Therefore, the
device pretreated with terbium functionalized micelle has potential capacity as test paper
for detecting anthrax spore biomarker, and may have application in the security.
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Figure 4. (a) The photographs of the fluorescence of silica gel plate impregnated with
different analytes (60 ꢁM, 0.5 mL) under a UV lamp at 254 nm. (b) The color changes of
silica gel plate under visible light (left) and under UV lamp at 254 nm (right) after writing
by DPA solution (1 mM·Lꢀ1).
CONCLUSIONS
In summary, we have successfully developed a lanthanide functionalized micelle
nanoprobe for ratiometric fluorescence detection of the anthrax spore biomarker, DPA.
The lanthanide functionalized micelle exhibited excellent selectivity and sensitivity to
DPA (detection limit 54 nM), which can be attributed to the emitted fluorescence of Tb3+
ions sensitized by DPA. When different concentration of DPA was added into the
aqueous of lanthanide functionalized micelle, the luminescence of Tb3+ ion in the micelle
enhanced linearly and the fluorescence of FR remained constant. Therefore, a lanthanide
functionalized micelle nanoprobe for ratiometric fluorescence detection of DPA was
constructed, which can response to DPA within seconds, and the detection range can be
easily changed by adjusting the concentration of the micelle. The lanthanide
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functionalized selfꢀassembled micelle would provide a new strategy for designing
ratiometric fluorescence nanoprobe to sensitively and rapidly detect bacterial spore
biomarker, biomolecules or other applications.
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ASSOCIATED CONTENTS
Supporting Information
Synthesis of FR and Tb3+ꢀL micelle, DLS and TEM measurement, spectrometric
properties of micelle, calculating stability constant, luminescence decay curves, 1H NMR,
13C NMR, MS. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Email: tangyu@lzu.edu.cn. Fax: +86 931ꢀ891ꢀ2582. Tel: +86 931ꢀ891ꢀ2552.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGEMENT
This study has been financially supported by the National Natural Science Foundation
of China (Projects 21471071, 21431002) and Fundamental Research Funds for the
Central Universities Grants (Project lzujbkyꢀ2017ꢀOt05).
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REFERENCES
6
7
(1) Goel, A. K. World J. Clin. Cases 2015, 3, 20ꢀ33.
8
9
(2) Bhardwaj, N.; Bhardwaj, S.; Mehta, J.; Kim, K. H.; Deep, A. Biosens. Bioelectron. 2016, 86, 799ꢀ804.
(3) King, D.; Luna, V.; Cannons, A.; Cattani, J.; Amuso, P. J. Clin. Microbiol. 2003, 41, 3454ꢀ3455.
(4) Zhang, B. H.; Wang, H. S.; Lu, L. H.; Ai, K. L.; Zhang, G.; Cheng, X. L. Adv. Funct. Mater. 2008, 18,
2348ꢀ2355.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(5) Huan, T. N.; Ganesh, T.; Han, S. H.; Yoon, M. Y.; Chung, H. Biosens. Bioelectron. 2011, 26, 4227ꢀ4230.
(6) Enserink, M. Science 2001, 294, 490ꢀ491.
(7) Zhang, X. Y.; Zhao, J.; Whitney, A. V.; Elam, J. W.; Van Duyne, R. P. J. Am. Chem. Soc. 2006, 128,
10304ꢀ10309.
(8) Paulus, H. Anal. Biochem. 1981, 114, 407ꢀ410.
(9) Tan, C. L.; Wang, Q. M.; Zhang, C. C. Chem. Commun. 2011, 47, 12521ꢀ12523.
(10) Hurtle, W.; Bode, E.; Kulesh, D. A.; Kaplan, R. S.; Garrison, J.; Bridge, D.; House, M.; Frye, M. S.;
Loveless, B.; Norwood, D. J. Clin. Microbiol. 2004, 42, 179ꢀ185.
(11) Zhang, X. Y.; Young, M. A.; Lyandres, O.; Van Duyne, R. P. J. Am. Chem. Soc. 2005, 127, 4484ꢀ4489.
(12) Li, Q.; Sun, K.; Chang, K. W.; Yu, J. B.; Chiu, D. T.; Wu, C. F.; Qin, W. P. Anal. Chem. 2013, 85,
9087ꢀ9091.
(13) Hang, J.; Sundaram, A. K.; Zhu, P. X.; Shelton, D. R.; Karns, J. S.; Martin, P. A. W.; Li, S. H.; Amstutz,
P.; Tang, C. M. J. Microbiol. Meth. 2008, 73, 242ꢀ246.
(14) Goodacre, R.; Shann, B.; Gilbert, R. J.; Timmins, E. M.; McGovern, A. C.; Alsberg, B. K.; Kell, D. B.;
Logan, N. A. Anal. Chem. 2000, 72, 119ꢀ127.
(15) Ai, K. L.; Zhang, B. H.; Lu, L. H. Angew. Chem., Int. Ed. 2009, 48, 304ꢀ308.
(16) Tsukube, H.; Shinoda, S. Chem. Rev. 2002, 102, 2389ꢀ2403.
(17) Kupcho, K. R.; Stafslien, D. K.; DeRosier, T.; Hallis, T. M.; Ozers, M. S.; Vogel, K. W. J. Am. Chem.
Soc. 2007, 129, 13372ꢀ+.
(18) Hanaoka, K.; Kikuchi, K.; Kobayashi, S.; Nagano, T. J. Am. Chem. Soc. 2007, 129, 13502ꢀ13509.
(19) Hindle, A. A.; Hall, E. A. H. Analyst 1999, 124, 1599ꢀ1604.
(20) Curiel, D.; Sanchez, G.; MasꢀMontoya, M.; Tarraga, A.; Molina, P. Analyst 2012, 137, 5499ꢀ5501.
(21) Binnemans, K. Chem. Rev. 2009, 109, 4283ꢀ4374.(22) Chen, H.; Xie, Y.; Kirillov, A. M.; Liu, L.; Yu,
M.; Liu, W.; Tang, Y. Chem. Commun. 2015, 51, 5036ꢀ5039.
(23) Sun, Y. P.; Zhou, B.; Lin, Y.; Wang, W.; Fernando, K. A. S.; Pathak, P.; Meziani, M. J.; Harruff, B. A.;
Wang, X.; Wang, H. F.; Luo, P. J. G.; Yang, H.; Kose, M. E.; Chen, B. L.; Veca, L. M.; Xie, S. Y. J. Am.
Chem. Soc. 2006, 128, 7756ꢀ7757.
(24) Feng, L.; Liu, Y. W.; Tang, X. Y.; Piao, Y. M.; Chen, S. F.; Deng, S. L.; Xie, S. Y.; Wang, Y. H.; Zheng,
L. S. Chem. Mater. 2013, 25, 4487ꢀ4496.
(25) Zhang, Y.; Li, B.; Ma, H.; Zhang, L.; Zheng, Y. Biosens. Bioelectron. 2016, 85, 287ꢀ293.
(26) Yilmaz, M. D.; Hsu, S. H.; Reinhoudt, D. N.; Velders, A. H.; Huskens, J. Angew. Chem., Int. Ed. 2010,
49, 5938ꢀ5941.
(27) Oh, W. K.; Jeong, Y. S.; Song, J.; Jang, J. Biosens. Bioelectron. 2011, 29, 172ꢀ177.
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2
3
4
5
(28) Xu, H.; Rao, X.; Gao, J.; Yu, J.; Wang, Z.; Dou, Z.; Cui, Y.; Yang, Y.; Chen, B.; Qian, G. Chem.
Commun. 2012, 48, 7377ꢀ7379.
(29) Tan, H.; Ma, C.; Chen, L.; Xu, F.; Chen, S.; Wang, L. Sens. Actuators B 2014, 190, 621ꢀ626.
(30) Wang, Y.; Li, Y.; Qi, W.; Song, Y. Chem. Commun. 2015, 51, 11022ꢀ11025.
(31) Ma, B. L.; Zeng, F.; Zheng, F. Y.; Wu, S. Z. Analyst 2011, 136, 3649ꢀ3655.
(32) Chen, Y.; Zhu, C.; Yang, Z.; Chen, J.; He, Y.; Jiao, Y.; He, W.; Qiu, L.; Cen, J.; Guo, Z. Angew. Chem.,
Int. Ed. 2013, 52, 1688ꢀ1691.
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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35
36
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(33) Forzani, E. S.; Lu, D. L.; Leright, M. J.; Aguilar, A. D.; Tsow, F.; Iglesias, R. A.; Zhang, Q.; Lu, J.; Li,
J. H.; Tao, N. J. J. Am. Chem. Soc. 2009, 131, 1390ꢀ1391.
(34) Ma, X.; Zhao, Y. L. Chem. Rev. 2015, 115, 7794ꢀ7839.
(35) Peng, H. Q.; Niu, L. Y.; Chen, Y. Z.; Wu, L. Z.; Tung, C. H.; Yang, Q. Z. Chem. Rev. 2015, 115,
7502ꢀ7542.
(36) Ramanathan, M.; Shrestha, L. K.; Mori, T.; Ji, Q. M.; Hill, J. P.; Ariga, K. Phys. Chem. Chem. Phys.
2013, 15, 10580ꢀ10611.
(37) Zheng, L. Z.; Li, J. H. J. Phys. Chem. B 2005, 109, 1108ꢀ1112.
(38) Zhu, J. H.; Yu, C.; Chen, Y.; Shin, J.; Cao, Q. Y.; Kim, J. S. Chem. Commun. 2017, 53, 4342ꢀ4345.
(39) Zhou, Z.; Song, J.; Tian, R.; Yang, Z.; Yu, G.; Lin, L.; Zhang, G.; Fan, W.; Zhang, F.; Niu, G.; Nie, L.;
Chen, X. Angew. Chem., Int. Ed. 2017, 56, 6492ꢀ6496.
(40) Liu, J.; Morikawa, M. A.; Lei, H.; Ishiba, K.; Kimizuka, N. Langmuir 2016, 32, 10597ꢀ10603.
(41) Polarz, S.; Landsmann, S.; Klaiber, A. Angew. Chem., Int. Ed. 2014, 53, 946ꢀ954.
(42) Lanigan, N.; Wang, X. Chem. Commun. 2013, 49, 8133ꢀ8144.
(43) Lei, H.; Liu, J.; Yan, J.; Lu, S.; Fang, Y. ACS Appl. Mater. Interfaces 2014, 6, 13642ꢀ13647.
(44) Liu, J.; Morikawa, M.ꢀa.; Kimizuka, N. J. Am. Chem. Soc. 2011, 133, 17370ꢀ17374.
(45) Lei, H.; Liu, J.; Yan, J.; Quan, J.; Fang, Y. Langmuir 2016, 32, 10350ꢀ10357.
(46) Wei, X.; Dong, R.; Wang, D.; Zhao, T.; Gao, Y.; Duffy, P.; Zhu, X.; Wang, W. Chem. ꢀ Eur. J. 2015, 21,
11427ꢀ11434.
(47) Yao, Y.; Xue, M.; Chen, J.; Zhang, M.; Huang, F. J. Am. Chem. Soc. 2012, 134, 15712ꢀ15715.
(48) Shau, S. M.; Chang, C. C.; Lo, C. H.; Chen, Y. C.; Juang, T. Y.; Dai, S. A.; Lee, R. H.; Jeng, R. J. ACS
Appl. Mater. Interfaces 2012, 4, 1897ꢀ1908.
(49) Luo, X.; Chen, M.; Zhang, Y.; Chen, Z.; Li, X. Chem. ꢀ Eur. J. 2016, 98, 9ꢀ19.
(50) Cao, Y.; Liu, M.; Zhang, K.; Zu, G.; Kuang, Y.; Tong, X.; Xiong, D.; Pei, R. Biomacromolecules 2017,
18, 150ꢀ158.
(51) Abbas, Z.; Dasari, S.; Patra, A. K. RSC Adv. 2017, 7, 44272ꢀ44281.
(52) Lincheneau, C.; Leonard, J. P.; McCabe, T.; Gunnlaugsson, T. Chem. Commun. 2011, 47, 7119ꢀ7121.
(53) Li, G.; Zhang, Y.; Geng, D.; Shang, M.; Peng, C.; Cheng, Z.; Lin, J. ACS Appl. Mater. Interfaces 2012,
4, 296ꢀ305.
(54) de Faria, E. H.; Nassar, E. J.; Ciuffi, K. J.; Vicente, M. A.; Trujillano, R.; Rives, V.; Calefi, P. S. ACS
ACS Appl. Mater. Interfaces 2011, 3, 1311ꢀ1318.
(55) Cable, M. L.; Kirby, J. P.; Levine, D. J.; Manary, M. J.; Gray, H. B.; Ponce, A. J. Am. Chem. Soc.
2009, 131, 9562ꢀ9570.
(56) Liu, G.; Feng, D.ꢀQ.; Mu, X.; Zheng, W.; Chen, T.; Qi, L.; Li, D. J. Mater. Chem. B 2013, 1, 2128.
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(57) Salvia, M. V.; Ramadori, F.; Springhetti, S.; DiezꢀCastellnou, M.; Perrone, B.; Rastrelli, F.; Mancin, F.
J. Am. Chem. Soc. 2015, 137, 886ꢀ892.
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