BODIPY modified silica coated magnetite nanoparticles as fluorescent hybrid material for Cu (II) detection in aqueous medium

Article abstract of DOI:10.1016/j.dyepig.2013.07.031

A novel ratiometric fluorescent sensor for copper II based on silica coated magnetite hybrid fluorescent material functionalized with boron-dipyrromethene containing a di (2-picolyl) amine was prepared and characterized. The binding abilities of the functionalized silica coated magnetite nanoparticles towards different metal ions have been examined by fluorescence spectroscopy, and displays high selectivity and sensitivity for Copper II among the all tested metals in Ethanol/water: 8:2.

Full text of DOI:10.1016/j.dyepig.2013.07.031

Accepted Manuscript
BODIPY modified Silica coated magnetite nanoparticles as fluorescent hybrid material
for Cu (II) detection in aqueous medium
Jalal Isaad, Ahmida El Achari
PII:
S0143-7208(13)00290-8
10.1016/j.dyepig.2013.07.031
DYPI 4039
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 17 June 2013
Revised Date: 19 July 2013
Accepted Date: 25 July 2013
Please cite this article as: Isaad J, El Achari A, BODIPY modified Silica coated magnetite nanoparticles
as fluorescent hybrid material for Cu (II) detection in aqueous medium, Dyes and Pigments (2013), doi:
10.1016/j.dyepig.2013.07.031.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
BODIPY modified Silica coated magnetite nanoparticles as
fluorescent hybrid material for Cu (II) detection in aqueous
medium
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Jalal Isaad*a,b and Ahmida El Acharia,b
aUniv Lille Nord de France, F-59655, Villeneuve d’Ascq Cedex, France
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b ENSAIT/GEMTEX, Roubaix, France. Tel: +33 3 20 25 86 87;
E-mail: jalal.isaad@ensait.fr
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Abstract: A novel ratiometric fluorescent sensor for copper II based on silica coated
magnetite hybrid fluorescent material functionalized with boron-dipyrromethene containing a
di (2-picolyl) amine was prepared and characterized. The binding abilities of the
functionalized silica coated magnetite nanoparticles towards different metal ions have been
examined by fluorescence spectroscopy, and displays high selectivity and sensitivity for
Copper II among the all tested metals in Ethanol/water: 8:2.
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Keywords: Silica coated magnetite; nanomaterial; Hybrid Material; BODIPY, Copper II,
Water
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Introduction
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There are a number of ingeniously designed supramolecular systems for the selective
chemosensing of chemically and biologically important ionic species.[1,2] Special attention has
been focused on the design of fluorescent chemosensors for Cu2+ due to its essential yet toxic
nature.[3] in addition to the Fe2+ and Zn2+ ions, Cu2+ is the third in abundance in human body
and plays an important roles in various physiological processes.[4,5] Besides the involvement
in various biological functions[6], Cu2+ plays pivotal role in haemoglobin synthesis, bone
development and nerve function regulation[7]. Also, Cu2+ can cause serious threats especially
when its level exceeds the cellular needs and displace other metal ions acting as cofactor in
various enzyme catalyzed reactions.[5] It is also implicated in Alzheimer’s disease,[8] lipid
metabolism,[9,10] and inflammatory disorders.[11] This has led to a strong interest in the
development of selective Cu2+ probes for biological and environmental applications.
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In this connection, many fluorescent chemosensors for Cu (II)-selective detection were
reported and have been used with some success in biological applications.[12-20] However;
some of them have shortcomings for practical application such as cross-sensitivities toward
other metal cations, low water solubility, a narrow pH span, slow response, a low
fluorescence quantum yield in aqueous media, and cytotoxicity of ligand. Thus, there is still
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strong demand for the development of efficient Cu2䟌-selective fluorescent sensor, especially
ratiometric probes that can be acted in eco-friendly aqueous medium.
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Recently, and in order to reduce all these limitations, we developed a several
chemosensors based on water soluble dyes with a specific functionality to detect different ions
[21-27]
such as cyanides
and mercury [28] in aqueous medium. On the other hand, new azathia-
crown ether possessing a dansyl fluorophore moiety functionalized Silica nanoparticles as
hybrid material was prepared in our laboratory for the mercury detection.[29] This sensor
shows selective detection of Hg (II) and can be used for Hg (II) chemosensing in
physiological environment and water quality monitoring. However, the prepared nanosilica
type materials suffer from some difficulties in handling and filtration from the reaction
mixtures. Therefore, our attention was focused on the use of other inorganic material for the
development of hybrid materials as chemosensors for copper. In this sense, magnetites have
been widely used as an inorganic support in different fields such as biology and medicine.[30–
36]
Magnetite (Fe3O4), a common magnetic iron oxide, has a cubic inverse spinel structure
with oxygen forming a FCC closed packing and Fe cations occupying the interstitial
tetrahedral and octahedral sites.[37] The electrons can hop between Fe2+ and Fe3+ ions in
octahedral sites even at room temperature, rendering magnetite an important half-metallic
material.[38] Due to the anisotropic dipolar attraction, unmodified magnetic nanoparticles of
iron oxides tend to aggregate into large clusters and thus lose the specific properties
associated with single-domain. In addition, the iron oxide particles reactivity has been shown
to increase greatly as their dimensions are reduced and they may undergo rapid
biodegradation when exposed directly to biological systems.[39-40]. In order to prevent these
limitations, suitable coating is essential.[41] In this context, silica coating represents a very
interesting pathway for different reasons such as (1) the possibility of grafting with a variety
of functional groups and (2) high surface area make the silica valuable as support for various
hybrid materials.
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Silica coating of magnetic nanoparticles has become a promising and important
approach in the development of magnetic nanoparticles for both fundamental study and
technological research. The formation of a silica coating on the surface of iron oxide
nanoparticles could help prevent their aggregation in liquid and improve their chemical
stability, and give them a surface based on silica with various kinds of silanol and siloxanes
which facilitate the covalent grafting of an adequate organic functionality on the surface of
the resulting magnetite/silica nanoparticles. Therefore, the final hybrid material with a silica
layer provides chemically stable magnetic nanoparticles with a surface chemically friendly to
biological systems.
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Herein, we report the synthesis of BODIPY fluorescent chemosensor with Bis-pyridin-
2-ylmethyl-amine as ligand. The final BODIPY fluorophore was grafted on the silica surface
of the magnetite/silica nanoparticles to afford a hybrid material as reported in figure 1, with
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high selectivity toward Cu2䟌 over all the tested metal ions and ~10-fold fluorescence ratio
change after Cu2䟌 binding upon excitation at 535 nm in H2O/EtOH mixture.
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Figure 1
Experimental section
Materials and Instrumentation
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All chemicals were reagent grade (Aldrich Chemical Co.) and were used as purchased
without further purification. Thin layer chromatography (TLC) analysis was performed using
Fluka aluminium foils coated with 25 mm particle size silica gel matrix F254. TLC
development involved either UV (254 and 366 nm) or visible light inspection, followed by
either treatment with an acid solution of p-anisaldehyde or a basic solution of KMnO4 and
heating. Flash column chromatography was performed on Merck silica gel 60 (particle size
0.040 - 0.063 nm, 230 – 400 mesh ASTM) according to the procedure of Still.[42] Uv-vis
spectra were recorded on a Cary-4000 Varian spectrophotometer, using either 0.1 or 1 cm
quartz cuvettes. Infra-red spectra were recorded in KBr disk on a Perkin Elmer-Spectrum BX
FTIR system. Absorptions are quoted in wavenumbers (cm-1). 1H and 13C NMR spectra were
recorded at 200 MHz 1H (50.0 MHz 13C) on a Varian Gemini spectrometer. Spin resonances
are reported as chemical shifts (d) in parts per million (ppm) and referenced to the residual
peak as an internal standard of the solvent employed, as follow: CDCl3 7.27 ppm (1H NMR),
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77 ppm (13C NMR, central band), DMSOd6 2.50 ppm (1H NMR, central band), 39.5 ppm (13C
NMR, central band). Spin multiplicity is showed by s=singlet, d=doublet, t=triplet, q=quartet,
m=multiplet, br=broad. Coupling constants J are reported in Hertz. Mass spectra were
recorded on a ThermoScientific LCQ-Fleet mass spectrometer under electrospray ionization
(ESI, +c or −c technique). High Resolution Mass Spectra (HRMS) were recorded on an
LTPOrbitrap mass spectrometer from Thermo Electron Corporation under ESI (+c) technique.
Mass spectrometric analysis is quoted in the m/z form. Elemental analyses were recorded on a
Perkin Elmer 240 C Elemental Analyzer. Thermogravimetric analysis (TGA) of the samples
was performed using a Perkin Elmer DiamondThermogravimeter. Dried samples was placed
in the TGA furnace and heated at a rate of 10 °C/inform room temperature to 700 °C in static
air. The fluorescence spectra were measured by Hitachi F-4500 fluorescence
spectrophotometer with a 1 cm quartz cell. Doubly distilled deionized water (DIW) was used
for all experiments,
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4-[1,3] Dioxolan-2-yl-benzaldehyde (2)
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To a solution of Benzene-1,4-dicarbaldehyde 1 (1g, 7.46 mmol) in toluene (20 mL),
ethylene glycol (0.50 g, 8.26 mmol), catalytic quantity of TsOH were added and the resulting
mixture was refluxed for 3h. After, the solvent was removed under the reduce pressure and
the crude product purified by flah chromatography (AcOEt/PE: 5/3) to give 2 in 72 % as
yield. 1H NMR (CDCl3, 200 MHz): δ= 9.83 (1s, 1H, CHO), 7.66-7.43 (m, 4H, ArH), 6.15 (s,
1H, CH), 3.89 (t, 4H) ppm. 13C NMR (CDCl3, 50 MHz): δ= 190.2, 144.2, 135.4, 129.6, 128.6,
114.7, 72.3 ppm. MS (ESI): m/z = 179.28 [M + 1]+. C10H10O3 (178.06): calcul C, 67,41; H,
5,66, found C, 67,54; H, 5,75
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4-[1,3] Dioxolan-2-yl, 5-(phenyl) dipyrromethene (3).
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Pyrrole (8,7 mL, 0,125 mol) and compound 2 (0,89 g, 5,0 mmol) were dissolved in 20
mL of CH2Cl2 purged with azote in a 100 mL flask under azote atmosphere. 1 drop of TFA
was added and the mixture was stirred at room temperature for 3 hrs. Then, the solvent was
evaporated and the crude reaction mixture was purified by flash chromatography using a pure
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ethyl acetate as eluent to obtain the compound 3 in 64% as yield. H NMR (200 MHz,
CDCl3): δ= 7.11-6.92 (m, 4H, ArH), 6.31-6.21 (m, 3H), 5.82-5.75 (m, 4 H), 5.31 (s, 1 H), 3.9
(t, 4H) ppm. 13CNMR (50 MHz, CDCl3) δ= 137.2, 134.5, 130.6, 129.4, 127.9, 116.8, 114.2,
109.1, 107.6, 71.8 ppm. MS (ESI): m/z = 295.36 [M + 1]+. C18H18N2O2 (294.14): calcul C,
73.45; H, 6.16; N, 9.52, found C, 73.54; H, 6.27; N, 9.67.
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1,1’-dichloro-5-(4-[1,3]Dioxolan-2-yl phenyl) dipyrromethane (4)
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Compound 3 (1.00 g, 3.41 mmol) was dissolved in 25 mL of dry THF and the
resulting mixture was purged with azote and cooled to -78 °C. Then, a suspension of of N-
chlorosuccinimide (0.90 g, 6.82 mmol) in 5 mL of THF was added and the reaction mixture
was stirred at -78 °C for 2 hrs and placed in the freezer overnight at -20 °C. To the cold
reaction mixture, 30 mL of water was added and extracted with chloroform (3x30 mL), the
organic layer was dried over anhydrous Na2SO4, the solvent was removed under reduced
pressure and the residue was purified by flash chromatography using a DCM / ethyl acetate
mixture (1:1) as eluent to yield the compound 4 in 51% as yield. 1H NMR (200 MHz, CDCl3):
δ= 7.92 (s, 2H, NH), 7.13 (d, J = 7.7 Hz, 2H,), 6.85 (d, J = 7.7 Hz, 2H,), 6.16 (s, 1H), 6.05
(dd, J = 4.2 Hz, 2H,), 5.87 (dd, J = 4.2 Hz, 2H), 5.26 (s, 1H) ppm. 13C NMR (50MHz,
CDCl3): δ= 136.3, 134.4, 131.3, 129.6, 127.6, 114.5, 119.7, 108.3, 107.8, 72.6, 45.3 ppm. MS
(ESI): m/z = 363.34 [M + 1]+. C18H16Cl2N2O2 (362.06): calcul C, 59,52; H, 4,44; Cl, 19,52;
N, 7,71, found C, 59,64; H, 4,53; N, 7,84.
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4,4-Difluoro-8-(4-[1,3]Dioxolan-2-ylphenyl)-3,5-dichloro-4-bora-3a,4a-diaza-s-indacene
(5)
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To a solution of compound 4 (1.00 g, 2.75 mmol), a suspension of p-chloranil (0.67 g,
2.75 mmol) in CH2Cl2 was added and the reaction mixture was stirred for 1 hr. The solvent
was evaporated under reduced pressure and the crude was purified and dissolved in 15 mL of
toluene and the solution was purged with azote. To this solution, triethylamine (2.72 g, 27
mmol) was added and the resulting mixture was heated at 70 °C for 30 min. After, BF3-OEt2
(5.83 g, 41.12 mmol) was added and the reaction solution was heated at reflux for 2 h. To the
cooled reaction mixture, NaOH 1N solution was added and the layers were separated. The
water layer was brought to pH =5-6 by addition of 1N HCl solution and extracted with
CH2Cl2 (3x50 mL), the organic solutions was concentrated under reduce pressure and the
residue was purified by flash chromatography using CH2Cl2/AcOEt: 2/1 as eluant to afford
compound 5 in 56% as yield. 1H NMR (200 MHz, CDCl3): δ= 7.26 (d, J = 8.2 Hz, 2H,). 7.07
(d, J = 8.2 Hz, 2H,), 6.54 (d, J =4.8 Hz, 2H), 6. 22 (d, J = 4.8 Hz, 2H), 6.13 (s, 1H), 3.95 (t,
4H) ppm. 13C NMR (50 MHz, CDCl3): δ= 145.3, 136.8, 134.6, 132.8, 127.3, 127.8, 126.5,
125.6, 122.2, 114.4, 113.8, 72.7 ppm. MS (ESI): m/z = 409.29 [M + 1]+. C18H13BCl2F2N2O2
(408.05): calcul C, 52,86; H, 3,20; N, 6,85 found C, 52,96; H, 3,33; N, 6,98
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.4,4-Difluoro-8-(4--[1,3]Dioxolan-2-yl,phenyl)-3,methoxy,5-chloro-4-bora-3a,4a-diaza-s-
indacene (6)
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Compound 5 (1.00 g, 2.46 mmol) was dissolved in 20 mL of absolute methanol under
azote. Then, freshly prepared sodium methoxide (1 equiv) in methanol solution was added
dropwise within 10 min with stirring and the resulting mixture was stirred at room
temperature for 30 min. The reaction was quenched by the addition of 20 mL of water and
extracted with chloroform 3x30 mL. The organic layer was dried over Na2SO4, concentrated
and purified through flash chromatography using AcOEt/PE: 3:7 as eluent to obtain
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compound 6 in 47% as yield. H NMR (200 MHz, CDCl3): δ= = 7.24 (d, J = 7.5 Hz, 2H,).
7.09 (d, J = 7.5 Hz, 2H,), 6.94 (d, 1H, J = 4.8 Hz); 6.63 (d, 1H, J = 3.1 Hz), 6.54 (d, 1H, J =
3.1 Hz); 6.32 (d, 1H, J = 3.1 Hz), 6.11 (s, 1H), 4.11 (s, 3H, OCH3), 3.94 (t, 4H) ppm. 13C
NMR (50MHz, CDCl3): δ= 149.4, 137.9, 137.1, 135.8, 134.5, 133.2, 127.7, 126.3, 119.4,
116.2, 114.8, 108.8, 73.0, 57.2 ppm. MS (ESI): m/z = 405.37 [M + 1]+. C19H16BClF2N2O3
(404.09): calcul C, 56,40; H, 3,99; N, 6,92 found C, 56,52; H, 4,14; N, 7,11.
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3- (bis - pyridin – 2 – ylmethyl - amine), 4,4 – Difluoro – 8 - (4 - [1,3]Dioxolan-2-
yl,phenyl)-3a,methoxy, 5a-chloro-4-bora-3a,4a-diaza-s-indacene (8)
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To a solution of the compound 6 (1.00 g, 2.47 mmol) in 20 mL of MeCN, 7 (0.60 g,
2.98 mmol) and 0.4 mL triethylamine was then added and the reaction mixture was refluxed
for 16 h. Then, the reaction mixture was cooled to room temperature and 30 mL of water was
added and the mixture was extracted with DCM 3x30 mL. The combined organic layers were
dried over Na2SO4, the solvent was evaporated, and the residue was purified through flash
chromatography using AcOEt as eluent to afford compound 7 in 37% as yield. 1H NMR (200
MHz, CDCl3): δ= 8.54 (d, J=8.3 Hz, 2H), 7.88 (dd, 2H), 7.48-7.11 (m, 8H), 6.74 (d, 1H, J =
6.6 Hz), 6.36 (d, 1H, J = 4.2 Hz), 6.29 (d, 1H, J = 6.4 Hz), 5.60 (d, 1H, J = 4.2 Hz), 6.12 (s,
1H), 4.78 (s, 4H), 4.09 (s, 3H, OCH3), 3.71 (d, 4H) ppm. 13C NMR (50MHz, CDCl3): δ=
159.4, 150.1, 149.2, 136.8, 135.4, 135.0, 134.3, 133.7, 132.1, 128.7, 126.4, 123.4, 120.8,
119.1, 117.2, 114.5, 108.7, 72.5, 62.6, 48.2 ppm. MS (ESI): m/z = 568.45 [M + 1]+.
C31H28BF2N5O3 (567.23): calcul C, 65,62; H, 4,97; N, 12,34;found C, 65,76; H, 5,15; N,
12,42.
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Compound 9
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To a solution of 8 (1 g, 1.76 mmol) in 20 mL of acetone, 10% HCl solution (1mL) was
added and the resulting mixture was refluxed for 3 h. The reaction mixture was concentrated
at reduced pressure. The residue was extracted three times with chloroform and the extract
was washed with water. The solvent was removed after drying over anhydrous sodium sulfate.
The crude product was purified by flash chromatography (AcOEt/PE: 3/2) to give 9 in 83 %
as yield. 1H NMR (200 MHz, CDCl3): δ= 9.77 (s, 1h, CH), 8.51 (d, J=7.7 Hz, 2H), 7.82-7.21
(m, 10H), 6.71 (d, 1H, J = 5.9 Hz), 6.32 (d, 1H, J = 4.4 Hz), 6.24 (d, 1H, J = 5.9 Hz), 5.63 (d,
1H, J = 4.4 Hz), 4.71 (s, 4H), 4.11 (s, 3H, OCH3) ppm. 13C NMR (50 MHz, CDCl3): δ=
190.4, 159.1, 150.5, 149.4, 140.2, 137.3, 136.5, 135.6, 135.7, 135.2, 132.5, 129.5, 126.7,
123.6, 120.4, 119.1, 118.7, 116.5, 62.8, 48.7 ppm. MS (ESI): m/z = 523.37 [M + 1]+.
C29H24BF2N5O2 (523.20): calcul C, 66,56; H, 4,62; N, 13,38; found C, 66,64; H, 4,72; N,
13,41.
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Colloidal iron oxide nanoparticle synthesis
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The magnetite nanoparticles were prepared by the conventional co-precipitation
method with some modifications.[42] FeCl3.6H2O (2.32 g) and FeCl2.4H2O (0.86 g) were
dissolved in 20 ml of the deionized water under nitrogen gas with vigorous stirring at 85°C.
Then, ammoniac solution 25% (3 ml) was added to the solution. The colour of bulk solution
turned from orange to black immediately. The magnetite precipitates were washed twice with
deionized water and once with sodium chloride (0.02M). The final magnetite nanoparticles
were precipitated by magnetic decantation.
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Synthesis of nanostructured silica-coated magnetite
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Sodium silicate (0.95 g) was dissolved in deionized water (70 mL) and the pH value of
the solution was adjusted to 12 by HCl solution. The sodium silicate solution and Fe3O4
nanoparticles (1 g) prepared in the above were poured into a beaker equipped with a
mechanical stirrer. The mixture was ultrasonicated for 30 min. Then, the temperature of the
mixture was increased to 80 °C. HCl solution was added dropwise to adjust the pH value to
6–7. The precipitates were washed several times with deionized water by magnetic
decantation and then dispersed in 100 ml of ethanol and (3-aminopropyl) triethoxysilane (1.0
mL) was added to the reaction solution. The reaction was allowed to proceed at 60 °C for 10 h
under stirring. The core–shell magnetic nanoparticles being surface modified with amine
groups were collected by magnetic separation, washed with distilled water and ethanol, and
then dried.
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Surface Functionalization of silica-coated magnetite with the fluorophore 9
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In a round bottom flask, 300 mg of M/SiNPs-am were suspended in a solution of 25.0
ml of anhydrous toluene, and sonicated for 30 min at RT. The fluorophore 9 (0.5 g) was
hydrolyzed in 25.0 ml of anhydrous toluene for 15 min at RT then added to M/SiNPs-am
suspension and sonicated again for 30 min. The reaction mixture was refluxed for 24 h
without magnetic stirring. The functionalized SiNPs were separated and collected by
magnetic separation and washed twice with toluene/DIW and EtOH/DIW and then dried
before use.
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Results and discussions
Synthesis of the BODIPY dye 9
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3,5-dichloroBODIPY derivatives can be substituted with many different nucleophiles,
such as oxygen, nitrogen, sulfur, and carbon centered nucleophiles, which turns out to be a
very successful approach for preparing a variety of symmetric and asymmetric BODIPY
compounds with substitution patterns that are difficult to synthesize by other way.[43] Thus, we
synthesis the 3,5-dichloroBODIPY derivative 4 as intermediate for the preparation of the
fluorophore 9 as reported in the scheme 1.
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Sheme 1
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First, one aldehyde group of terephthalaldehyde 1 was protected by the treatment of 1
by one equiv of ethylene glycol with a catalytic amount of TsOH. The dipyrromethane 3 was
[44]
prepared according to a published procedure
by condensation of aldehyde 2 with neat
excess pyrrole catalyzed by trifluoroacetic acid (TFA) at room temperature and then was
chlorinated with 2 equiv of N-chlorosuccinimide (NCS) at -78 °C in tetrahydrofuran (THF)
according to the procedure described for the halogenation of pyrrole[45] to afford the
derivative 4 which was oxidized by p-chloranil followed by reaction with triethylamine and
BF3-OEt2 in refluxing toluene to afford compound 5. To synthesis the fluorophore 9, we first
prepared the mono-substituted product 6 by the treatment of 5 with sodium methoxide in the
methanol.
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The second chlorine atom of the isolated 6 was subsequently substituted by di-(2-
picolyl)-amine 7 in refluxing acetonitrile in the presence of triethylamine as a base because of
the lower reactivity of the second chlorine resulting from the electron donating effect of the
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oxygen atom, afford 8 in 37% as yield. Finally the deprotection of the aldehyde group of 8
under acidic medium affords the fluorophore 9. The structure of all derivatives was confirmed
by MS, NMR and elemental analysis
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Characterization of the functionalized magnetite/silica nanoparticles
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In order to fully characterize the inorganic core and the organic shell of the
synthesized magnetic nanoparticles for morphology, structure and magnetic properties,
different techniques such as TGA, IR and VSM were used.
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The presence of the fluorophore 9 on the surface of the silica-coated magnetite
nanoparticles was confirmed by Fourier Transform Infrared Infra-red (FTIR) spectroscopy
(Figure 2). FTIR spectra of the silica coated magnetite nanoparticles before and after reaction
with the fluorophore 9 were recorded in KBr.
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The magnetite coated silica nanoparticles (figure 2-a) have stretches at 579, 1087,
1625 and 3286 cm-1 corresponding to the Fe-O, Si-O-Si, and water (free or adsorbed water
that still remained in the sample) stretches respectively. The stretch found at 947 cm-1 is due
to the presence of Fe-O-Si bonds in the sample. For the nanocomposite material, again Fe-O
and water stretches are found at 579, 1621 and 3286 cm-1 respectively. The absorption peak of
magnetite at 575 cm-1 is also clearly visible in the sample F-M/SiNPs as shown in Figure 2 -
b. This implies that the sample F-M/SiNPs does not change chemically or physically after the
treatment with the fluorophore 9. Comparably, two new absorbent peaks appearing at 2500
and 2945 cm-1 are due to the aromatic and aliphatic stretches associated with the fluorophore
9. FTIR spectra (Figure 2) of the F-M/SiNPs showed stretches for N=C at 1690 cm-1 due to
the coupling reaction between the aldehyde group of the fluorophore 9 and the primary amine
group of the M/FSiNPs- amine (figure 1). The presence of two broad bands appearing at
1634 and 3400 cm-1 is an indication of the presence of free H–O–H molecules in the F-
M/SiNPs sample. Furthermore, two absorption peaks centered at around 805 and 1093 cm-1
can also be observed, corresponding to the stretching of Si–O–Si bond from the silica
particles.
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Figure 2
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The magnetic properties of the synthesized nanoparticles were also studied by a
vibrating-sample magnetometer VSM. Figure 3 shows the hysteresis loops of the Fe3O4, silica
coated Fe3O4 and F-M/SiNPs at room temperature. The saturation magnetization, Ms, of bulk
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magnetite (92 emu.g−1) [46] was reduced to 57 emu g−1 for magnetite nanoparticles. It is known
that the magnetization of a magnetic particle in an external field is proportional to its size
value. Therefore, a smaller saturation magnetization value for the magnetite nanoparticles
compared to the bulk material is reasonable. The saturation magnetic moments of the silica
coated Fe3O4 nanoparticles and F-M/SiNPs nanoparticles reached to 47 emu.g−1 and 44
emu.g−1, respectively. This can be explained by considering the diamagnetic contribution of
the silica shells surrounding the magnetite nanoparticles.
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Based on these low magnetization values with respect to magnetization of pure Fe3O4
nanoparticles, which was owing to decrease in the surface moments of the magnetic
nanoparticles by non-magnetic silica coating, but it is still sufficient for magnetic separation
by a conventional magnet.
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Figure 3
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The TGA analyses (figure 4) also indicated that the silica-coated Fe3O4 particles were
covered successfully by the coupling with the fluorophore 9. The weight losses of the F-
M/SiNPs, functionalized magnetite coated silica nanoparticles (curve b) below 250 °C were
resulted from the release of both the physisorbed and chemisorbed water on the surface of the
F-M/SiNPs nanoparticles. And the breakout of the structured water in F-M/SiNPs occurred
above 600 °C. The organic compounds of hybrid nanoparticles were decomposed in the
temperature range of 250–600 °C. The major weight loss is attributed to the decomposition of
fluorophore 9 which is chemically bonded onto the surface of magnetite coated silica
nanoparticles. The amount of organic compounds bound on the surface of the nanoparticles is
estimated from the percentage of weight loss from the TGA curve. TGA of the in F-M/SiNPs
indicated that the fluorophore 9 content was about 11 %. Fluorophore 9 started to decompose
at about 300 °C and was completely burned out at about 590 °C. For the unmodified
magnetite coated silica (curve a) no significant weight loss below 100 °C could be observed
and only a small change in mass percentage could be seen in the temperature range of 100–
250 °C. The weight loss recorded as 2 wt % in this temperature range mainly resulted from
the release of water molecules bonded physically and chemically in the magnetite
nanoparticles.
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Figure 4
2
䟌
Spectral characteristics of the F-M/SiNPs with and without Cu
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The titration fluorescence spectra of the F-M/SiNPs with Cu2䟌 were carried out in
Ethanol upon excitation at λex䟞541 nm.
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The F-M/SiNPs shows a fluorescence emission band with a maximum at 565 nm with
a low fluorescence quantum yield (0.007). The low Φf value for the uncomplexed F-M/SiNPs
can be attributed to an efficient quenching via an excited-state ICT process from the nitrogen
atom of the di(2-picolyl)amine moiety to the strongly electron deficient BODIPY acceptor.
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Upon the addition of Cu2䟌 ions to the ethanol solution of F-M/SiNPs, the fluorescence
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emission intensity at 565 nm decreased significantly and a new red-shifted emission band at
588 nm resulting from the formation of the F-M/SiNPs-Cu2䟌 complex. The apparition of the
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new bond at 588 nm was also accompanied by a significant increase in the fluorescence
intensity. The Фf values of the free F-M/SiNPs and F-M/SiNPs – Cu2䟌 complex were 0.007
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and 0.24 respectively. Upon binding of Cu2+, the electron-donating properties of the amine are
reduced, partially suppressing the ICT and causing an enhancement in the fluorophore’s
fluorescence intensity.
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The fluorescent intensities ratio at 588 nm and 565 nm (I588/I565) increased from
0.38 to 3.59 depending on the concentration of Cu2䟌varying from 0 to 20 ꢀM. Therefore, the
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F-M/SiNPs can serve as a ratiometric fluorescent chemosensor for Cu2䟌 ions (Figure 2).
Figure 5
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The effect of water ratio on the fluorescence measurement of the F-M/SiNPs was
investigated in the absence and the presence of Cu2+ ions in ethanol solutions in order to
optimize the conditions for practical applications of the F-M/SiNPs as Cu2+ detection in
environmental and biological samples. The fluorescence emission of F-M/SiNPs was found to
be strongly dependent on the presence of water in the aqueous ethanol solution. When the
ratio of water increase, the fluorescence intensity of the complex F-M/SiNPs–Cu2+ decrease as
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reported in the figure 6. The sensor F-M/SiNPs has no sensitivity to Cu2䟌 if the water content
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exceeded 10%. Following this results, we focused on the fluorescence behaviours of F-
M/SiNPs in response to the different metal ions in 80:20 ethanol/water solution.
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Figure 6
2
䟌
Binding stoichiometry of 1 and Cu
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Job’s method for the emission was employed to investigate the binding stoichiometry
2
of the F-M/SiNPs and Cu2䟌 ions. The total concentration of F-M/SiNPs and Cu was
䟌
2
䟌
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maintained constant at 5 ꢀM, with a continuous variation of the molar fraction of Cu
.
Figure 7 shows the fluorescence intensity variation at 588 nm as a function of the ratio of F-
M/SiNPs/Cu2䟌concentration. Figure 7 show that the complex of F-M/SiNPs / Cu2䟌 exhibited
341
a maximum fluorescence emission at 584 nm when the molecular fraction of Cu2䟌 was 0.5.
This indicated that a 1:1 stoichiometry is most possible for the binding mode of F-M/SiNPs
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2
䟌
and Cu
.
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Figure 7
The association constant Ka was evaluated graphically by the plotting 1/ꢁF against
1/[Cu2+] (Figure 8). The data were linearly fit according to the Benesi–Hilderbrand equation.
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The Ka value, obtained from the slope and intercept of the line, was found to be 2.103 M−1.
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Figure 8
Selectivity of F-M/SiNPs to Cu2䟌 over other metal ions
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In order to investigate the selectivity of the F-M/SiNPs to Cu2䟌, fluorescent spectra of
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F-M/SiNPs response to other metal ions such as ions (Hg2+, Al3+, Na+, Ag+ , Ba2+, Mg2+, Ca2+,
Cd2+, Co2+, Fe2+, Fe3+, Mn2+, Ni2+, Pb2+, Cr3+ and Zn2+) were examined. As shown in Figure 8,
titration of alkali and alkaline earth metal did not show much change to the fluorescence
intensities of F-M/SiNPs. The emission of F-M/SiNPs at 565 nm was partly quenched by
2
Fe2䟌, whereas the titration of Cu
resulted in a remarkable red-shift and a great
䟌
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enhancement of the emission at 588 nm.
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Figure 9
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However, the fluorescence intensity of F-M/SiNPs upon treatment with Cu2+ (10 ꢀM)
was measured in the presence of the different metal ions (100 ꢀM); the results are reported in
the Figure 9. The presence of background metal ions did not cause any significant quenching
ratio (F/F0) change of F-M/SiNPs. This showed that the F-M/SiNPs could selectively detect
Cu2+ in the presence of other metal ions.
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Figure 10
The detection limit calcul
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The detection limit[47] of the F-M/SiNPs as a fluorescent sensor for the analysis of
Cu2+ was determined from the plot of fluorescence intensity as a function of the Cu2+
concentration as reported in Figure 11. The detection limit DL of chemosensor F-M/SiNPs
was determined from the following equation:
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DL = K x Sb1 / S
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Where: K=3; Sb1 is the standard deviation of the blank solution and S is the slope of
the calibration curve.
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It was found that the F-M/SiNPs have a detection limit of 10-6 M, which is allowed for
the detection of micromolar concentration range of Cu2+.
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Figure 11
Conclusion
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A novel ratiometric Cu (II) fluorescent chemosensor based on silica coated magnetite
and functionalized with BODIPY containing a di (2-picolyl) amine moiety (F-M/SiNPs) was
prepared. The results of the fluorescence titrations exhibited a pronounced Cu (II) Selective
off-on type fluorescence switching upon binding over other metal in EtOH/water: 8:2. The
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binding mode of F-M/SiNPs and Cu (II) is a 1∶1 stoichiometry determined via studies of Job
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plot.
References
13

ACCEPTED MANUSCRIPT
383
384
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
Desvergne J.P, Czarnik A.W. Chemosensors of Ion and Molecule Recognition;
Kluwer: Dordrecht, 1997;
385
386
Czarnik A.W, Fluorescent Chemosensors for Ion and Molecule Recognition, Ed.
American Chemical Society: Washington, DC, 1992.
387
388
Kramer R, Fluorescent Chemosensors for Cu2+ Ions: Fast, Selective, and Highly
Sensitive. Angew. Chem., Int. Ed., 1998, 37: 772-773.
389
390
Andreini C, Banci L, Bertini I, Rosato A, Occurrence of copper proteins through the
three domains of life: a bioinformatic approach, J. Proteome Res. 2008, 7: 209-216.
391
392
Domaille DW, Que EL, Chang CJ. Synthetic fluorescent sensors for studying the cell
biology of metals. Nat Chem Biol. 2008; 4:168–175.
393
394
Solomon EI, Chen P, Metz M, Lee SK, Palmer AE, Angew. Chem. Int. Ed. 2001, 40
: 4570- 4590.
395
396
Haris E.D, Copper and Iron: A landmark connection of two essential metals. J. Trace
Elem. Exp. Med. 2001, 14: 207-210.
397
398
Barnham K.J, Masters C.L, Bush A.I, Neurodegenerative diseases and oxidative stress.
Nat. Rev. Drug Discovery 2004: 3: 205-214.
399
400
401
Gruber S.L, Tatum J.D, Engle T.E, Prusa K.J, Laudert S.B, Schroeder A.L, Platter WJ,
Effects of ractopamine supplementation and postmortem aging on longissimus muscle
palatability of beef steers differing in biological type, J. Anim. Sci. 2000, 86, 205-210.
402
403
404
405
[10] Huster D, Purnal T.D, Burkhead J.L, Ralle M, Fiehn O, Stukert F, Olson N.E. Teusper
D, Lutsenko S, High Copper Selectively Alters Lipid Metabolism and Cell Cycle
Machinery in the Mouse Model of Wilson Disease, J. Biol. Chem. 2007, 282: 8343-
8355
406
407
408
[11] Fisher A.E, Naughton D.P, Therapeutic chelators for the twenty first century: new
treatments for iron and copper mediated inflammatory and neurological disorders.
Curr. Drug. Deliv. 2005, 2: 261-268
14

ACCEPTED MANUSCRIPT
409
410
411
[12] Wu Q, Anslyn E.V. Catalytic Signal Amplification Using a Heck Reaction. An
Example in the Fluorescence Sensing of Cu(II). J. Am. Chem. Soc. 2004, 126: 14682-
14683
412
413
[13] Xu Z, Qian X, Cui J. Colorimetric and Ratiometric Fluorescent Chemosensor with a
Large Red-Shift in Emission:ꢀ Cu(II)-Only Sensing by Deprotonation of Secondary
414
415
Amines as Receptor Conjugated to Naphthalimide Fluorophore. Org. Lett. 2005, 7:
3029-3032.
416
417
418
[14] Weng Y.Q, Yue F, Zhong Y.R, Ye B.H. A Copper (II) Ion-Selective On−Off-Type
Fluoroionophore Based on Zinc Porphyrin−Dipyridylamino, Inorg. Chem. 2007, 46:
7749-7755.
419
420
[15] Royzen M, Dai Z, Canary J.W. Ratiometric displacement approach to Cu(II) sensing
by fluorescence. J. Am. Chem. Soc. 2005, 127: 1612-1613.
421
422
423
[16] Zeng L Miller, E.W, Pralle A, Isacoff E.Y, Chang C.J. A Selective Turn-On
Fluorescent Sensor for Imaging Copper in Living Cells, J. Am. Chem. Soc. 2006, 128:
10-11.
424
425
426
[17] Shao N, Zhang Y, Cheung S, Yang R, Chan W, Mo T, Li K, Liu F. Copper ion-
selective fluorescent sensor based on the inner filter effect using a spiropyran
derivative. Anal. Chem. 2005, 77: 7294-7303.
427
428
429
[18] Yang L, McRae R, Henary M.M, Patel R, Lai B, Vogt S, Fahrni C.J. Imaging of the
intracellular topography of copper with a fluorescent sensor and by synchrotron x-ray
fluorescence microscopy. Proc. Natl. Acad. Sci, 2005, 102: 11179-11184.
430
431
[19] Kramer R. Fluorescent chemosensors for Cu2+ ions: Fast, selective, and highly
sensitive. Angew. Chem. 1998, 37: 772-773.
432
433
434
[20]
Jinlong H, Bo W, Jianguo Y, Bin L, Huayu Q, Shouchun Y. A Highly Copper-
Selective Ratiometric Fluorescent Sensor Based on BODIPY, Chin. J. Chem. 2012,
30, 1857-1861.
435
436
[21] Isaad J, Perwuelz A. New color chemosensors for cyanide based on water soluble azo
dyes, Tetrahedron Lett. 2010. 51: 5810-5814.
15

ACCEPTED MANUSCRIPT
437
438
439
[22] Isaad J, El achari A. A novel glycoconjugated N-acetylamino aldehyde hydrazone azo
dye as chromogenic probe for cyanide detection in water. Anal chim acta. 2011. 694:
120-127.
440
441
442
[23] Isaad J, El Achari A. A novel cyanide chemodosimeter based on trifluoroacetamide
benzhydrol-2 as binding motif: importance of substituent positioning on intra-
molecular charge transfer. Tetrahedron. 2011. 67: 4196-4201.
443
444
445
[24]
Isaad J, El achari A. Biosourced 3-formyl chromenyl-azo dye as Michael acceptor
type of chemodosimeter for cyanide in aqueous environment. Tetrahedron. 2011. 67:
5678-5685.
446
447
448
[25] Isaad J, salaun F. Functionalized poly (vinyl alcohol) polymer as chemodosimeter
material for the colorimetric sensing of cyanide in pure water. Sens and Actualitors B.
2011, 157: 26-33.
449
450
451
[26] Isaad J, El Achari A. Colorimetric sensing of cyanide anions in aqueous media based
on functional surface modification of natural cellulose materials, Tetrahedron. 2011.
67: 4939-4947.
452
453
[27] Isaad J, El Achari A, Malek F. Bio-polymer starch thin film sensors for low
concentration detection of cyanide anions in water. Dye and Pigm, 2013, 97, 134-140.
454
455
456
[28] Isaad J, El achari A. A water soluble fluorescent BODIPY dye with azathia-crown
ether functionality for mercury chemosensing in environmental media. The Analyst.
2013, 138: 3809-3819.
457
458
459
[29] Isaad J, El achari A. Azathia crown ether possessing a dansyl fluorophore moiety
functionalized silica nanoparticles as hybrid material for mercury detection in aqueous
medium. Tetrahedron. 2013, 69: 4866-4874.
460
461
462
[30] Kondo A, Kamura H, Higashitani K, Development and application of thermo-sensitive
magnetic immunomicrospheres for antibody purification. Appl. Microbiol. Biotechnol.
1994, 41: 99-105.
463
464
465
[31] Goetz V, Remaud M,. Graves D.J, A novel magnetic silica support for use in
chromatographic and enzymatic bioprocessing. Biotechnol. Bioeng. 1991, 37: 614-
626.
16

ACCEPTED MANUSCRIPT
466
467
468
[32] Li X, Sun Z, Synthesis of magnetic polymer microspheres and application for
immobilization of proteinase of balillus sublitis. J. Appl. Polym. Sci. 1995, 58, 1991-
1997.
469
470
[33] Guesdon J.G, Avrameas S, Magnetic solid phase enzyme-immunoassay.
Immunochemistry, 1977, 14: 443-447.
471
472
[34] Sonti S.V, Bose A, Cell Separation Using Protein-A-Coated Magnetic Nanoclusters. J.
Colloid Interface Sci. 1995, 170: 575-585.
473
474
475
[35] Khng H.P, Cunliffe D, Davies S, Turner N.A, Vulfson E.N, The synthesis of sub-
micron magnetic particles and their use for preparative purification of proteins.
Biotechnol. Bioeng. 1998, 60: 419-424.
476
477
478
[36] Shinkai M, Suzuki M, Iijima S, Kobayashi T, Antibody-conjugated magnetoliposomes
for targeting cancer cells and their application in hyperthermia. Biotechnol. Appl.
Biochem. 1995, 21: 125-137.
479
480
[37] Cornell R.M, Schwertmann U, The Iron Oxides: Structure, Properties, Reactions,
Occurrence and Uses, VCH, New York, USA 1996, 28–29.
481
482
[38] Verwey E.J.W, Electronic Conduction of Magnetite (Fe3O4) and its Transition Point
at Low Temperatures; Nature. 1939, 144: 327- 328.
483
484
[39] Santra S, Tapec R, Theodoropoulou N, Dobson J, Hebard A,. Tan W.H, Synthesis and
Characterization of Silica-Coated Iron Oxide Nanoparticles in Microemulsion:ꢀ The
Effect of Nonionic Surfactants. Langmuir, 2001, 17: 2900-2906.
485
486
487
488
[40] Yang H.H, Zhang S.Q, Chen X L, Zhuang Z.X, Xu J.G, Wang X.R, Magnetite-
containing spherical silica nanoparticles for biocatalysis and bioseparations. Anal.
Chem. 2004, 76: 1316-1321.
489
490
[41] Ma Z, Guan Y, Liu H, Superparamagnetic silica nanoparticles with immobilized metal
affinity ligands for protein adsorption. J. Magn. Magn. Mater. 2006, 301: 469-477.
491
492
[42] Chang Q, Zhu LH, Yu C, Tang HQ. Synthesis and properties of magnetic and
luminescent Fe3O4/SiO2/Dye/SiO2 nanoparticles. J Lumin, 2008, 128: 1890-1895.
17

ACCEPTED MANUSCRIPT
493
494
495
496
[43] Qin W.W, Rohand T, Dehaen W, Clifford J.N, Driesen K, Beljonne D, Van Averbeke
B, Van der Auweraer M, Boens N, Boron dipyrromethene analogs with phenyl, styryl,
and ethynylphenyl substituents: synthesis, photophysics, electrochemistry, and
quantum-chemical calculations. J. Phys. Chem. A. 2007, 111: 8588-8597
497
498
499
[44] Litter B.J, Miller M.A, Hung C.H, Wagner R.W, O’Shea D.F, Boyle P.D, Lindsey J.S.
Refined Synthesis of 5-Substituted Dipyrromethanes. J. Org. Chem. 1999, 64, 1391-
1396.
500
501
[45] Gillow H.M, Burton D.E. Bromination and chlorination of pyrrole and some reactive
1-substituted pyrroles. J. Org. Chem. 1981, 46: 2221-2225.
502
503
504
[46] Zaitsev V.S, Filimonov D.S, Presnyakov I.A, Gambino R.J, Chu B, Physical and
Chemical Properties of Magnetite and Magnetite-Polymer Nanoparticles and Their
Colloidal Dispersions. J. Colloid Interface Sci. 1999, 212: 49-57.
505
506
[47] Zhu, M.; Yuan, M.; Liu, X.; Xu, J.; Lv, J.; Huang, C.; Liu, H.; Li, Y.;Wang, S.; Zhu,
D. Org. Lett. 2008, 10, 1481.
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Figure 1: synthesis of the functionalized magnetite/silica nanoparticles (F-M/SiNPs).
Reagents and conditions: (a) sodium silica, HCl (to pH 12 and then to pH=6,7); sonication,
heating at 80 °C (b) EtOH, heating at 60 °C for 10 h (c) Any toluene, Reflux, 24h.

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Scheme 1: Synthesis strategy of the fluorophore 9. Reagents and conditions: (a) ethylene
glycol, TsOH, Reflux (b) CH2Cl2, TFA, RT (c) N-chlorosuccinimide, THF, -78°C to -20°C
(d) 1- p-chloranil, CH2Cl2, RT 2- toluene, NEt3, BF3-OEt2, reflux (e) sodium methoxide,
MeOH, RT (f) MeCN, NEt3, Reflux (g) 10% HCl, reflux

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Figure 2: FT-IR spectra of the (a) magnetite coated silica (b) functionalized magnetite/silica
nanoparticles.

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Figure 3: Magnetization curve of (a) magnetite nanoparticles (b) silica coated Fe3O4 and (c)
F-M/SiNPs

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Figure 4: Thermogravimetric (TG) curves for (a) silica coated magnetite nano clusters, (b)
functionalized magnetite silica nanoparticles (F-M/SiNPs).

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Figure 5 Fluorescent emission spectra of F-M/SiNPs (0.5mg/ml) upon excitation at 541 nm
in the presence of different concentrations of Cu2䟌 (from 0 to 20 ꢀM) in EtOH.

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Figure 6: Fluorescence intensity changes of F-M/SiNPs (0.5mg/ml) as a function of water
content in aqueous ethanol solution, pH=7, at 588 nm in the absence and presence of Cu2+ (5
equiv), λex 541 nm.

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Figure 7 Job’s plot of F-M/SiNPs and Cu2䟌. The total concentrations of F-M/SiNPs and
Cu2䟌 were kept at a constant 5 ꢀM in EtOH/H2O: 80:20, pH=7. Excitation was at 541 nm and
emission intensity was measured at 588 nm.

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Figure 8: Benesi-Hildebrand plot of the chemosensor F-M/SiNPs - Cu2+ complexes in
Ethanol/water: 8:2, pH=7.

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Figure 9: Fluorescence emission spectra of F-M/SiNPs (0.5mg/ml) upon addition of various
metal ions (100 ꢀM) in EtOH/H2O: 80:20, pH=7 (λex䟞541 nm).