Ratiometric and reusable fluorescent nanoparticles for Zn2+ and H2PO4- detection in aqueous solution and living cells

Article abstract of DOI:10.1039/c0jm01925a

In this work, three kinds of core-shell silica nanoparticle-based fluorescent materials were prepared based on a modified Stoeber-Van Blaaderen method. They were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), dynamic light scattering (DLS), FT-IR, and several other spectroscopic methods. Firstly, The silica@sensor-1 nanoparticle (SSN) showed high selectivity toward Zn²⁺, which can detect Zn²⁺ in aqueous solution and living cells. It also can be reused to detect Zn²⁺ for at least four cycles after a simple regeneration. Secondly, to create a ratiometric measurement platform, the dye-2@silica nanoparticles (DSN), a new class of core-shell fluorescent silica nanoparticles were prepared with an acenaphtho[1, 2-b]pyrrol-9-carbonitrile chromophore derivative as the inner reference. It showed negligible sensing properties towards Zn²⁺, and the fluorescent intensity was not subjected to interference induced by pH change. Thirdly, the dye-2@silica@sensor-1 nanoparticles (DSSN), with the above reference dye buried inside the silica matrix and a layer of chemosensors anchored onto the surface of silica nanoparticles were prepared. DSSN showed not only the same sensing ability as SSN, but also a clear ratiometric fluorescent signal toward Zn ²⁺ in aqueous solutions and living cells. On the other hand, H ₂PO₄^- is a well-known Zn²⁺ binder, so the [DSSN@Zn²⁺] complex was found to ratiometrically detect H ₂PO₄^-. It responded to H₂PO ₄^- at a neutral aqueous solution with a detection limit lower than 6 × 10^-6 M. Moreover, the ratio of fluorescence intensity was linearly increased in the range 6～500 μM of H ₂PO₄^-, which implies a potential application for the quantitation of H₂PO₄^- in aqueous solution. To the best of our knowledge, this is the first example of core-shell silica nanoparticle-based fluorescent materials that can be repeatedly used to ratiometrically detect Zn²⁺ and H₂PO₄ ^- in 100% neutral aqueous solutions.

Full text of DOI:10.1039/c0jm01925a

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PAPER
www.rsc.org/materials | Journal of Materials Chemistry
ꢀ
Ratiometric and reusable fluorescent nanoparticles for Zn2+ and H2PO4
detection in aqueous solution and living cells†
*
Chunsheng He, Weiping Zhu, Yufang Xu, Ye Zhong, Juan Zhou and Xuhong Qian
*
Received 17th June 2010, Accepted 7th September 2010
DOI: 10.1039/c0jm01925a
In this work, three kinds of core–shell silica nanoparticle-based fluorescent materials were prepared
€
based on a modified Stober–Van Blaaderen method. They were characterized by transmission electron
microscopy (TEM), scanning electron microscopy (SEM), dynamic light scattering (DLS), FT-IR, and
several other spectroscopic methods. Firstly, The silica@sensor-1 nanoparticle (SSN) showed high
selectivity toward Zn2+, which can detect Zn2+ in aqueous solution and living cells. It also can be reused
to detect Zn2+ for at least four cycles after a simple regeneration. Secondly, to create a ratiometric
measurement platform, the dye-2@silica nanoparticles (DSN), a new class of core–shell fluorescent
silica nanoparticles were prepared with an acenaphtho[1, 2-b]pyrrol-9-carbonitrile chromophore
derivative as the inner reference. It showed negligible sensing properties towards Zn2+, and the
fluorescent intensity was not subjected to interference induced by pH change. Thirdly, the dye-
2@silica@sensor-1 nanoparticles (DSSN), with the above reference dye buried inside the silica matrix
and a layer of chemosensors anchored onto the surface of silica nanoparticles were prepared. DSSN
showed not only the same sensing ability as SSN, but also a clear ratiometric fluorescent signal toward
Zn2+ in aqueous solutions and living cells. On the other hand, H2PO4ꢀ is a well-known Zn2+ binder, so
ꢀ
the [DSSN@Zn2+] complex was found to ratiometrically detect H2PO4ꢀ. It responded to H2PO4 at
a neutral aqueous solution with a detection limit lower than 6 ꢁ 10ꢀ6 M. Moreover, the ratio of
fluorescence intensity was linearly increased in the range 6ꢂ500 mM of H2PO4ꢀ, which implies
a potential application for the quantitation of H2PO4ꢀ in aqueous solution. To the best of our
knowledge, this is the first example of core–shell silica nanoparticle-based fluorescent materials that can
be repeatedly used to ratiometrically detect Zn2+ and H2PO4ꢀ in 100% neutral aqueous solutions.
biocompatibility, simple synthetic procedure and relative inert-
ness in many environments.8 Compared with a single dye mole-
cule, dye-doped silica nanoparticles produce a highly amplified
optical signal by bearing a large number of dye molecules in
a single silica particle, which can provide a great improvement in
sensitivity. Such a silica matrix provides a barrier keeping the dye
from its surrounding environment. Both photobleaching and
photodegradation that often affect conventional dyes can be
minimized. And the flexible silane chemistry also provides
versatile routes for the modification of the silica surface. There-
fore, fluorescent silica nanoparticles exhibit considerable
advantages over conventional molecular dyes in the design of
fluorescent labeling agents and chemosensors.9 Meanwhile,
silica-based mulitifunctional nanoparticles have demonstrated
their capability in a wide range of applications, such as early-
stage cancer diagnosis,10 drug delivery,11 pathogen detection12
and gene delivery.13
Introduction
Fluorescence chemosensors, composed of a substrate binding
unit and one or more photoactive components, can signal the
presence of selected substrates by the variation in their fluores-
cence emissions (fluorescence intensity quenching or enhance-
ment, ratiometric measurements).1 Such systems can be used to
measure analyte concentration with high selectivity, rapid
response rate and excellent spatial resolution.2 Therefore, they
have drawn considerable attention in the intracellular moni-
toring of selected species in biological applications. Ratiometric
fluorescent sensors measure the changes in the ratio of the signal
intensities, which increase the dynamic range and provide a built-
in correction for environmental effects.3
In the last few years, fluorescent nanomaterials such as
metallic nanoparticles (Au and Ag),4 quantum dots (QDs),5
lanthanide nanoparticles,6 and silica nanoparticles7 have been
used as the matrix of various chemosensors. Among such
material platforms, silica is ‘‘generally recognized as safe’’
(GRAS) by the US Food and Drug Administration (FDA), and
is an attractive support for many applications because of its good
There are two methods for the synthesis of organic-dye-
14
€
doped silica nanopraticles; one is the Stober method, and
the other is the reverse microemulsion method.15 By either
method, one can get multifunctional systems with exquisite
shape and size control. Among these architectures, some
authors have proposed multistep synthesis of core–shell
structures.16,17 In such nanostructured particle systems, silica
could be a scaffold for the template or core-shell of another
material. Around this core–shell, some functional molecules
can be deposited.18
Shanghai Key Laboratory of Chemical Biology, School of Pharmacy East
China University of Science and Technology, 130 Meilong Road, Shanghai,
200237, PR China. E-mail: wpzhu@ecust.edu.cn; xhqian@ecust.edu.cn;
Fax: (+86) 21-64252603
† Electronic Supplementary Information (ESI) available: Fig. S1–S9,
Table 1. See DOI: 10.1039/c0jm01925a/
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Recently, silica nanoparticles have been investigated as the
matrix of chemosensors, and most of them are used for the
detection of the fluorescence quenching species, such as molec-
ular oxygen19 and Cu2+,20 Pb2 + 17 ions. Reports on the elabora-
tion of optical silica nanoparticles chemically bound with
fluorophores, especially with the enhancement of the fluores-
cence intensity for Zn2+, are quite scarce.21,22 Moreover, only one
of them showed practical applications in biological systems.22 In
contrast to the common applications in homogeneous solutions,
such silica-based fluorescent sensors have the potential for
regeneration after simple post-processing, which could be used to
construct photoluminescent devices.23
Considering the important roles of zinc in various biological
processes such as gene transcription, metalloenzyme regula-
tion, neural signal transmission,24 we have focused on the
fabrication of core–shell dye-doped silica nanoparticles as
a fluorescent chemosensor for Zn2+, by covalently linking the
modified sensor 1 to the silica network. In this paper, we
prepared monodisperse silica-rich core and sensor-rich shell
nanoparticles (silica@sensor-1, SSN), as ‘‘off–on’’ fluorescent
chemosensors for Zn2+ with a high selectivity in aqueous
solution and living cells (Fig. 1-i: SSN). To create a ratiometric
measurement platform, the dye-2@silica@sensor-1 nano-
particles (DSSN) were prepared with an 8-oxo-8H-acenaph-
tho[1,2-b]pyrrol-9-carbonitrile derivative as the inner
reference-rich core and sensor 1 as a sensor-rich shell (Fig. 1-i:
DSSN). Furthermore, it is well known that phosphate ions and
their derivatives play important roles in signal transduction
and energy storage in biological systems.25 Some fluorescent
Scheme 1 Preparation, recognition (identification) and regeneration of
dye-2@sensor-1 core–shell silica nanoparticles (DSSN) for ratiometri-
ꢀ
cally fluorescence detecting Zn2+ and H2PO4
.
Experimental
General procedures
All the solvents were of analytical grades without further puri-
fication unless otherwise noted. 1H-NMR spectra were measured
on a Bruker AV-400 spectrometer with chemical shifts reported
in ppm (in CDCl3, TMS as internal standard). Electrospray
ionization (ESI) mass spectrometry was performed in a HP 1100
LC-MS spectrometer. Melting points were determined by an X-6
micro-melting point apparatus and were uncorrected. All pH
measurements were made with a Sartorius basic pH-Meter PB-
20. Fluorescence spectra were determined using a Varian Cary
Eclipse fluorescence spectrometer. Absorption spectra were
determined by a Varian Cary 100 UV-vis spectrophotometer.
Fluorescence quantum yields were determined by using quinine
sulfate in 0.05 M H2SO4 (F ¼ 0.564) as a refence.
ꢀ
and/or colorimetric sensors for H2PO4 have been reported
during the past few years.26,27 To date, there is no report about
ꢀ
the fluorescent sensor for H2PO4 in micromolar concentra-
tions in neutral aqueous solutions. To increase the detection
limit of H2PO4ꢀ, the [DSSN@Zn2+] complex was also prepared
ꢀ
as a ratiometric fluorescent sensor for H2PO4 in 100%
aqueous solution (Scheme 1).
SEM images of the particles were taken by Hitachi S-520
scanning electron microscopy to assess the particle size and shape.
To prepare the samples for SEM studies, fluorescent silica nano-
particles were dispersed in water, and the resulting suspension was
vortexed and sonicated for 2 min. A drop (1–10 mL) of the
particles suspension was then placed on a piece of a microglass
slide and dried overnight in a desiccator.
Dynamic light scattering (DLS) measurements were per-
formed by an ALV/CGS-5022F from ALV Ltd. (German).
Hydrodynamic particle diameters were obtained from cumulant
fits of the autocorrelation functions at 90ꢃ scattering angle.
TEM images of the particles were obtained with a JEM-2100
transmission electron microscope operating at 200 keV. Samples
for TEM were prepared by spreading a drop of the nanoparticle
solution in ethanol onto standard carbon coated copper grids
(200 mesh). Dimensional analysis of nanoparticles from TEM
images were performed with the Digital Micrograph software.
FT-IR spectra (4000ꢂ500 cmꢀ1) in KBr were collected on
a Nicolet NEXUS 470 FT-IR spectrometer. The fluorescent silica
nanoparticles were mixed with KBr and pressed to a thin disc for
FT-IR detection.
Fig. 1 (i) Schematic diagrams of three different core–shell architecture
fluorescent silica nanoparticles. (ii): (a, d): SEM and TEM images of
SSN; (b, e): SEM and TEM images of DSSN; (c, f): SEM and TEM
images of DSN.
Confocal Microscopy images were obtained using a Nikon
A1R spectral confocal microscope. Fluorescence imaging was
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performed with a Leica DMIRB with Xenon lamp. Blue emis-
sion was collected with a 430ꢂ510 nm window, and red emission
was collected with a 570ꢂ650 nm window.
solution) were added. The reaction mixture was stirred for another
5 h (DLS analysis yielded a diameter of 64 ꢄ 8 nm for the resulting
silica cores). Fluorescent sensor 1 (25.0 mg, 46.7 mmol), TEOS
(150 mL, 0.405 mmol), and ammonia (0.4 mL, 25% water solution)
were subsequently added, and the mixture was stirred at 313 K for
an additional 20 h. After hydrolysis, the nanoparticles were
washed extensively with 70% ethyl alcohol and water until the UV/
Vis spectrum of the filtrate showed the absence of sensor-1
Synthesis of fluoroionophore 1
Organic fluorescent sensor 1c was prepared according to the
reported procedure.28 The modified fluoroionophore 1 was
prepared as follows: compound 1c (145 mg, 0.5 mmol) and
triethoxy(3-isocyanatopropyl)silane (148 mg, 0.6 mmol) were
dissolved in anhydrous THF (10 mL) with 2–5 drops of Et3N
under nitrogen atmosphere. The yellow solution was stirred for
48 h under reflux; then the solvent was removed by rotary
evaporation and the residue was directly purified by flash column
chromatography (silica gel, CH2Cl2/CH3OH ¼ 40/1, v/v) to
provide 1 (229 mg, yield 85.2%) as the yellow oil. 1H (400 MHz,
CDCl3, 25 ꢃC): d ¼ 10.44 (–NHCO–, s, 1H), 8.82 (t, J1 ¼ J2 ¼ 2.1
Hz, 1H), 8.81 (t, J1 ¼ J2 ¼ 3.2 Hz, 1H), 8.14 (dd, J1 ¼ 9.6 Hz,
J2 ¼ 1.6 Hz, 1H), 7.51 (t, J1 ¼ J2 ¼ 2.4 Hz, 2H), 7.42 (dd, J1 ¼ 6.2
Hz, J1 ¼ 6.0 Hz, 1H), 4.25 (–COCH2–, s, 2H), 3.78 (–OCH2CH3,
m, 6H), 3.72–3.68 (–OCH2CH2O–, m, 4H), 3.62
ꢃ
absorption. And they were stored in water at 4 C for future use
(DLS analysis yielded a diameter of 80 ꢄ 7 nm for the resulting
silica@sensor-1 core–shell nanoparticles).
Preparation of the core–shell dye-2@silica nanoparticles (DSN)
For the preparation of core–shell dye-2@silica nanoparticles, the
aforementioned dye-2 precursor, usually containing around 2.35
ꢁ 10ꢀ5 M of dye-2, with TEOS (150 mL, 0.405 mmol) and
ammonia (0.9 mL, 25% water solution) were added to 25 mL of
ethanol in a 313 K thermostated vessel. The reaction mixture was
vigorously stirred for 5 h, and then a second amount of TEOS (150
mL, 0.405 mmol) and a second amount of ammonia (0.9 mL, 25%
water solution) were added. The reaction mixture was stirred for
another 5 h. After that, TEOS (150 mL, 0.405 mmol), and
ammonia (0.4 mL, 25% water solution) were subsequently added,
and the mixture was stirred at 313 K for an additional 20 h. After
hydrolysis, the nanoparticles were washed extensively with 70%
ethyl alcohol and water until the UV/Vis spectrum of the filtrate
showed the absence of dye-2 absorption, and they were stored in
water at 4 ꢃC for future use.
(-NHCH2CH2O–, t, J1
¼
J2
¼
4.8 Hz, 2H), 3.56
(-NHCH2CH2O–, t, J1 ¼ J2 ¼ 3.2 Hz, 2H), 3.31–3.26
(–CH2CH2CH2Si–, m, 2H), 1.72–1.65 (–CH2CH2CH2Si-, m,
2H), 1.19 (–OCH2CH3, t, J1 ¼ J2 ¼ 7.0 Hz, 9H), 0.67
(–CH2CH2CH2Si–, t, J1 ¼ J2 ¼ 8.2 Hz, 2H). IR(KBr): 3335,
2928, 1639, 1536, 1371, 1250, 1077, 947, 792 cmꢀ1. HRMS (EI):
[M + H+] calcd for C25H41N4O7Si, 537.2745; found, 537.2740
(100%).
Synthesis of fluoroionophore 2
Preparation of the core–shell dye-2@sensor-1 nanoparticles
(DSSN)
The precursor 2c was prepared according to the reported proce-
dure.29 The fluoroionophore 2 was prepared as follows: compound
2c (200 mg, 0.869 mmol) and 3-aminopropyltriethoxy silane (770
mg, 3.47 mmol) were dissolved in acetonitrile (25 mL) under
nitrogen atmosphere. The yellow solution was stirred for 48 h at
room temperature; then the solvent was removed by rotary evap-
oration and the residue was directly purified by flash column
chromatography (silica gel, CH2Cl2/CH3OH ¼ 80/1, v/v) to
For the preparation of nanoparticles with a compact dye-2 core,
the aforementioned dye-2 precursor, usually containing around
2.35 ꢁ 10ꢀ5 M of dye-2, with TEOS (150 mL, 0.405 mmol) and
ammonia (0.9 mL, 25% water solution) were added to 25 mL of
ethanol in a 313 K thermostated vessel. The reaction mixture was
vigorously stirred for 5 h, and then a second amount of TEOS
(150 mL, 0.405 mmol) and a second amount of ammonia (0.9 mL,
25% water solution) were added. The reaction mixture was stir-
red for another 5 h (DLS analysis yielded a diameter of 65 ꢄ 7
nm for the resulting dye-2 cores). Fluorescent sensor 1 (25.0 mg,
46.7 mmol), TEOS (150 mL, 0.405 mmol), and ammonia (0.4 mL,
25% water solution) were subsequently added, and the mixture
was stirred at 313 K for an additional 20 h. After hydrolysis, the
nanoparticles were washed extensively with 70% ethyl alcohol
and water until the UV/Vis spectrum of the filtrate showed the
absence of dye-2 and sensor-1 absorption. And they were stored
in water at 4 ꢃC for future use (DLS analysis yielded a diameter
of 82 ꢄ 5 nm for the resulting dye-2@sensor-1 core–shell nano-
particles).
ꢃ
1
provide 1 (58.9 mg, 15.1%) as the red powder. m.p. > 300 C. H
(400 MHz, CDCl3, 25 ꢃC): d ¼ 8.74 (d, J ¼ 7.6 Hz, 1H), 8.44 (d, J ¼
8.4 Hz, 1H), 8.05 (d, J ¼ 8.4 Hz, 1H), 7.82 (t, J1 ¼ J2 ¼ 7.6 Hz, 1H),
6.78 (d, J ¼ 8.8 Hz, 1H), 3.92 (–OCH2CH3, q, 6H), 3.76
(–CH2CH2CH2Si–, t, J1
(–CH2CH2CH2Si–, t, J1
¼
¼
J2
J2
¼
¼
7.2 Hz, 2H), 2.06
6.4 Hz, 2H), 0.87
(–CH2CH2CH2Si–, t, J1 ¼ J2 ¼ 7.2 Hz, 2H). IR(KBr): 3297, 2966,
2419, 1727, 1567, 1521, 1451, 1333, 1274, 1192, 1080, 951, 779 cmꢀ1.
HRMS (EI): [M + H+] calcd for C24H28N3O4Si, 450.1849; found,
450.1854 (100%).
Preparation of the core–shell silica@sensor-1 nanoparticles
(SSN)
Spectrophotometric titrations
For the preparation of core-shell silica-sensor-1 silica nano-
particles, TEOS (150 mL, 0.405 mmol) and ammonia (0.9 mL,
25% water solution) were added to 25 mL of ethanol in a 313 K
thermostated vessel. The reaction mixture was vigorously stirred
for 5 h, and then a second amount of TEOS (150 mL, 0.405
mmol) and a second amount of ammonia (0.9 mL, 25% water
The metal salts employed are Hg(ClO4)2$3H2O, Cd(ClO4)2$6H2O,
Pb(ClO4)2$3H2O, AgClO4$H2O, Zn(ClO4)2$6H2O, NaClO4,
Mg(ClO4)2,
KClO4,
Cr(ClO4)3$6H2O,
Cu(ClO4)2$6H2O and Ni(ClO4)2$6H2O, respectively. The anions
Co(ClO4)2$6H2O,
(X) used in this study are all [Na]X salts. Metal ion and anion stock
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solutions used during the spectrophotometric measurements
and titrations were prepared with Milli-Q deionized water
(R > 18 MU).
washed twice with PBS buffer. The [DSSN@Zn2+] complex
pretreated cells were then diluted with the addition of NaH2PO4
(600 mM, respectively) and incubated for another 0.5 h at 37 ꢃC.
Then, the cells were washed with PBS buffer three times for
further testing.
For the quantitative titrations, the concentration of sensor-1
subunits in the nanoparticles water suspensions was evaluated
from their absorbance using the 3 values of the corresponding
compound in aqueous solution (1: l ¼ 243 nm, 3 ¼ 1.73 ꢁ 104
Mꢀ1 cmꢀ1). The desired amount of such nanoparticle suspensions
was then transferred to a volumetric flask and an aqueous
solution was added to reach a final volume of 10 mL. Then the
adsorption and emission spectra were recorded. The excitation
wavelength was 320 nm. The same amount of fluorescent
nanoparticles were suspended in the corresponding different
concentration of metal ions or anions solution (10 mL), the
emission spectra of the fluorescent nanoparticles suspension with
different concentrations of Zn2+ (or H2PO4ꢀ) and other metal ion
Results and discussion
Design of the fluorescent core–shell silica nanoparticles
€
In 1968, Stober et al. described a pioneering synthesis method of
nearly monodisperse silica nanoparticles via the hydrolysis and
condensation of a silica alkoxide precursor (such as tetraethy-
lorthosilicate, TEOS) in alcoholic solutions in the presence of
aqueous ammonium hydroxide mixture.14 This method is
comparatively simple and both organic and inorganic dyes can
be entrapped. To address the problem of sensor leakage in the
silica backbone, Van Blaaderen et al. covalently linked fluores-
cein isothiocyanate (FITC) to 3-aminopropyltriethoxysilane and
incorporated the dye into the silica matrix to obtain robust dye-
rich silica nanoparticles.30 After that, Kopelman, Rosenzweig
and their co-workers proposed a new strategy for preparing
fluorescent chemosensors within nanoparticles, dubbed
PEBBLE sensors.19
solutions (Cd2+, Ag+, Pb2+, Hg2+, Na+, Mg2+, K+, Cr3+, Co2+,
ꢀ
Cu2+, Ni2+) [or other anions (HCO3ꢀ, Fꢀ, Brꢀ, Iꢀ, NO3
,
CH3COOꢀ, SO42ꢀ)] were measured respectively. All the
measurements were repeated three times and the general average
was obtained.
Regeneration of the SSN
Here as depicted in Scheme 1, we synthesized a type of fluo-
rescent sensor (DSSN) based on core–shell silica nanoparticles
The desired amount of fluorescent silica@sensor-1 nanoparticles
suspensions ([sensor-1] ¼ 1.2 ꢁ 10ꢀ4 M) was equilibrated with 10
mL of aqueous solutions containing 5.1 ꢁ 10ꢀ3 M Zn2+ at room
temperature, and the emission spectra of the suspension were
measured. The nanoparticles were treated with a solution of
EDTA-2Na (1.0 ꢁ 10ꢀ2 M) and then separated by centrifugation
(10000 r/min) and washed with tris-HCl (0.01 M) solution (water,
pH ¼ 7.10). The regenerated fluorescent silica nanoparticles were
suspended in the same volume (10 mL) of aqueous solutions
containing the same concentration (5.1 ꢁ 10ꢀ3 M) of zinc, and
the emission spectra were carried out again.
€
by a modified Stober-Van Blaaderen method. The sensing
systems were anchored onto the surface of the silica nano-
particles, to most extent, preserving the analytical characteristics
of chemosensors and providing the large surface area for the
sensor to interact with substrates. The reference dye 2 molecules
were buried inside the silica matrix, which protected the dye from
its surrounding environment, and the dye 2 just produced
a reference signal for ratiometric measurements. On the other
hand, metal-containing receptors could provide unsaturated
coordination sites at the metal centers, and present some
geometrical preference for anions of a given shape.31 Therefore,
the fluorescent silica nanoparticle [DSSN@Zn2+] complex also
could ratiometrically detect the anions in an aqueous solution
with a high sensitivity.
Application of fluorescent silica nanoparticles for fluorescent
images intracellular Zn2+ (H2PO4-) in HeLa cells
HeLa cells were seeded at 2 ꢁ 106 cells in a 10 cm Petri dish and
were cultured in DMEM containing 10% fetal bovine serum and
penicillin/streptomycin at 37 ꢃC in 5% CO2 and 95% air. After 24
h of cell attachment, the cells were incubated with SSN ([sensor-
Chemical synthesis
We designed sensor 1 by chemically incorporating the fluo-
roionophore to the silica network via the reaction between the
ethoxysilane groups of the fluoroionphore 1 and the silanol
groups during hydrolysis and condensation of tetraethylortho-
silicate. Synthesis of sensor 1 was depicted in Scheme 2. The
ꢃ
1] ¼ 42 mM) for 30 min at 37 C. The SSN were then removed
and the cells were washed twice with physiological saline solution
(NaCl: 0.9%, wt%). SSN pretreated cells were then diluted with
the addition of Zn(ClO4)2 (50 mM) and incubated for another 0.5
h at 37 ꢃC. Then, the cells were washed with physiological saline
solution three times for further testing.
The cells were incubated with DSSN ([sensor-1] ¼ 40 mM) for
30 min at 37 ꢃC. The DSSN were then removed and the cells were
washed twice with PBS buffer (phosphate buffered saline).
DSSN pretreated cells were then diluted with the addition of
Zn(ClO4)2 (40, 100, 700 mM, respectively) and incubated for
another 0.5 h at 37 ꢃC. Then, the cells were washed with PBS
buffer three times for further testing.
The cells were incubated with the [DSSN@Zn2+] complex
([sensor-1] ¼ 45 mM, [Zn2+] ¼ 1.5 mM) for 60 min at 37 ꢃC. The
[DSSN@Zn2+] complex was then removed and the cells were
Scheme 2 Synthesis of sensor 1.
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and TEM, the as-prepared nanoparticles were uniformly mon-
odispersed and separated from one another (Fig. 1-ii: a–f). These
three kinds of nanoparticles almost have the same size (their final
diameters of the as-prepared nanoparticles being 71 ꢄ 6 nm, 70 ꢄ
8 nm, 69 ꢄ 9 nm, respectively). Dynamic light scattering (DLS)
data, shown in Fig. 2, showed a quite narrow size distribution
centering at 80 nm. DLS hydrodynamic diameters are usually
found to be slightly greater than those measured by TEM anal-
ysis. This may be attributed to the shrinkage of the particles due
to the microscope electron beam.32 From the DLS data, it was
shown that the SSN and DSSN core–shell silica nanoparticles
had a sensor-1 rich shell of 16 and 17 nm, respectively. The core–
shell silica nanoparticles were made from the same proportions
of precursor materials. This indicated that the concentrations
of dye 2 and sensor 1 covalently bound to the silica matrix were
almost the same among SSN, DSN and DSSN.33 Moreover, the
binding of sensor 1 to the synthesized silica core was further
proved by FT-IR (see the ESI,† Fig. S1) and fluorescent spec-
troscopy (Fig. 3).
Scheme 3 Synthesis of dye 2.
compound 2-chloro-N-(quinol-8-yl)-acetamide (1b) was
prepared from 8-aminoquinoline (1a) and 2-chloroacetyl chlo-
ride in chloroform. Compound 1c was obtained with 85.2% yield
by reaction of eight equivalents of 2-(2-aminoethoxy) ethanol,
N,N-diisopropylethylamine respectively with 1b and KI (3 mol %
based on 1b) in acetonitrile. The reaction between compound 1c
and an equal equivalent of triethoxy(3-isocyanatopropyl)silane
in anhydrous tetrahydrofuran afforded the fluoroionphore 1 as
a yellow oil with a yield of 85.2%.
Dye 2 was derived from the fluorophore 2c, which was struc-
turally simple with strong long-wavelength emission and high
fluorescence quantum yield.29 As shown in Scheme 3, the starting
material acenaphthalenequinone (2a) condensed with malono-
nitrile to give the monoadduct 2b with 97.3% yield. Following
cyclization catalyzed by K2CO3 in anhydrous acetonitrile, the
precursor 2c was obtained almost quantitatively. Then, at room
temperature, the highly electron-deficient and highly reactive
precursor 2c reacted with 3-aminopropyltriethoxy silane in
acetonitrile to give the fluoroionophore 2 with 15.1% yield.
Preparation and characterization of fluorescent core–shell silica
nanoparticles
The core–shell architecture of the three different fluorescent silica
€
nanoparticles was synthesized via a modified Stober–Van Blaa-
deren method. As depicted in Fig. 1, the silica core was synthe-
sized by a pure silica precursor, tetraethoxysilane (TEOS),
catalyzed by concentrated aqueous ammonia in an ethanolic
solution. Following the synthesis of these core particles, the
sensor 1, was hydrolyzed with 0.405 mmol of TEOS to form the
sensor layer. Finally, the as-synthesized silica@sensor-1 nano-
particles (SSN) were centrifuged and resuspended repeatedly in
deionized water, 70% ethanol solution and finally deionized
water, in which they remain stable against flocculation, leaching,
and degradation for months. Meanwhile, one kind of dye-2-rich
core and silica shell nanoparticles (Fig. 1-i: DSN) were also
prepared by hydrolyzing dye 2 in a basic ethanolic solution of
TEOS to form dye-2 rich core. Then a silica shell was hydrolyzed
by pure silica precursor (TEOS). The dye-2@sensor-1 nano-
particles (DSSN) were synthesized by a similar procedure
(Scheme 1). The dye 2 was first hydrolyzed in basic ethanolic
solution of TEOS to form a dye-2-rich core, then a silica layer
was formed by the hydrolysis of a silica precursor (TEOS). After
the reference dye-2 was buried deep inside the nanoparticles, the
sensor 1 precursor was hydrolyzed with TEOS to form the sensor
layer.
Fig. 2 Dynamic light scattering (DLS) data of the core–shell architec-
ture of two different fluorescent silica nanoparticles. (a) Size distribution
of silica core and SSN nanoparticals; (b) Size distribution of dye-2 core
and DSSN nanoparticles.
Fig. 3 Emission spectra of a solution of SSN in the presence of
increasing Zn2+ concentrations (0–1.0 mM) in tris-HCl (0.01 M) solution
(water, pH ¼ 7.10). (b). Inset: Job plot for SSN in tris-HCl (0.01 M)
solution (water, pH ¼ 7.10). The total [sensor-1] + [Zn2+] ¼ 100 mM.
Conditions: [sensor-1] ¼ 3.1 ꢁ 10ꢀ5 M, lex ¼ 320 nm, lem ¼ 482 nm,
25 ꢃC.
The three different fluorescent silica nanoparticles were
investigated with scanning electron microscopy (SEM) and
transmission electron microscopy (TEM). As shown by SEM
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SSN-based zinc detection
The signal response of SSN toward Zn2+ was measured at pH
7.10 (10 mM Tris-HCl solution, water) in both emission and
absorption spectra (Fig. 3). Free SSN exhibited a weak fluores-
cence (quantum yield F0 ¼ 0.005). When Zn2+ was added to the
SSN solution, a new absorption peak at 266 nm appeared, while
the peak at 243 nm decreased, with an isosbestic point at 248 nm
(ESI,† Fig. S2). The fluorescence intensity at 482 nm is signifi-
cantly enhanced by approximately 13-fold (quantum yield, F/F0 ¼
13.4) with a 10 nm red-shift from 472 nm to 482 nm in fluorescence
emission (Fig. 3). The Job plot (Fig. 3. inset) exhibited
a maximum at 0.5 M fraction of Zn2+ in the solution of SSN, and
indicated that a 1 : 1 complex was formed between the sensor-1
anchored onto the SSN and Zn2+.
Fig. 5 Fluorescence images of SSN ([sensor-1] ¼ 4.2 ꢁ 10ꢀ5 M) induced
by intracellular Zn2+ in HeLa cells. (a) Bright-field transmission image of
HeLa Cells incubated with SSN. (b) Fluorescence image of HeLa cells
incubated with SSN. (c) Fluorescence image of HeLa cells incubated with
SSN for 30 min, washed three times, and then further incubated with 5 ꢁ
10ꢀ5 M Zn2+ for 30 min.
The selectivity of SSN towards Zn2+ was tested, as shown in
Fig. 4. The SSN showed no fluorescence intensity changes for
many metal ions such as Cd2+, Ag+, Pb2+, Hg2+, Na+, Mg2+, K+,
Cr3+, Co2+, Cu2+ or Ni2+. Under identical conditions, fluorescence
intensity increased significantly in the presence of Zn2+. The
competition experiments for SSN were also conducted (Fig. 4).
When the same amount of Zn2+ was added into the solution of
SSN in the presence of other metal ions such as Cd2+, Ag+, Pb2+,
Hg2+, Na+, Mg2+, K+, Cr3+, Ni2+, no significant variation in the
fluorescent intensity was observed by comparison with that Zn2+
only, whereas Co2+, Cu2+ quenched the fluorescence respectively.
Moreover, an obviously blue-green emission of the SSN with
Zn2+ solution can easily be observed by the naked eye (ESI,†
Fig. S3). The results were in line with the ability of reported
sensor 1c28 to selectively detect Zn2+ ions with no interferences
from other metal ions.
phospholipids.34 Therefore, the silica particles’ high affinity for
cell surfaces by adsorption, which eventually leads to endocytosis
is very convenient. We examined the application of SSN to
cultured living cells (HeLa cells) by confocal spectral microscopy
imaging system (CLSM). Incubation of HeLa cells with fluo-
rescent silica nanoparticles for 0.5 h at 37 ꢃC was followed by the
addition of Zn2+ and then was incubated for another 0.5 h. The
enhancement of fluorescence was observed (Fig. 5). The results
suggest that SSN can penetrate the cell membrane and can be
used for imaging Zn2+ in living cells.
Regeneration of SSN
The reversibility of SSN was also investigated and the results
were shown in Fig. 6. The stripping agent used in this experiment
was a solution of EDTA-2Na. Fig. 6 showed a profile of fluo-
rescence response of SSN during five sequential cycles. The
whole process consisted of (1) adding the solution of EDTA-2Na
to the [SSN@Zn2+] complex solution, (2) centrifuging the
nanoparticles solution and washing the nanoparticles with tris-
HCl (0.01 M) solution (water, pH ¼ 7.10), and 3) sensing of Zn2+
(5.1 ꢁ 10ꢀ3 M of Zn2+ in SSN solution ([sensor-1] ¼ 1.2 ꢁ 10ꢀ4
M)). As shown in Fig. 6, SSN were able to re-combine with Zn2+
for at least four cycles, and the decrease in fluorescence intensity
(I5/I1 ¼ 87.1%) may be attributed to the stripping agent’s effect
Application of SSN in cells
The taking up of molecules by cells is often facilitated by specific
binding between these species and membrane-bound receptors
(e.g. LDL or transferrin receptors). It is known that silica
particles have a great affinity for the head-groups of a variety of
on the sensing performance,35 and
a small amount of
Fig. 4 Fluorescence response of SSN in tris-HCl (0.01 M) solution
(water, pH ¼ 7.10) with different metal ions. Blue bars represent the
response of SSN in the presence of the appropriate metal ion of interest.
Red bars represent the response upon addition of Zn2+ (1.5 ꢁ 10ꢀ4 M) to
a solution of SSN and the appropriate metal ion of interest. I0 corre-
Fig. 6 Reversibility of SSN ([sensor-1] ¼ 1.2 ꢁ 10ꢀ4 M) response to Zn2+
(5.1 ꢁ 10ꢀ3 M) over five complex/stripping cycles. I0 corresponds to the
emission intensity of SSN without cation, I1ꢂI5 corresponds to the
emission of SSN with fresh zinc (II) solutions respectively. Conditions:
lex ¼ 320 nm, lem ¼ 482 nm, 25 ꢃC.
sponds to the emission intensity of SSN without cation. 1, none; 2, Zn2+
3, Na+; 4, Mg2+; 5, Pb2+; 6, Cd2+; 7, Ag+; 8, Hg2+; 9, K+; 10, Cr3+; 11, Ni2+
;
;
12, Co2+; 13, Cu2+. Conditions: [sensor-1] ¼ 3.1 ꢁ 10ꢀ5 M, lex ¼ 320 nm,
lem ¼ 482 nm, 25 ꢃC.
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agglomeration of the nanoparticles during multiple regeneration
cycles was evidenced by a TEM image of the nanoparticles after
the fifth time. However, SSN showed excellent reusability and
stability towards Zn2+ within at least four successive cycles.
DSSN-based zinc detection
As demonstrated by Kopelman and co-workers in the case of
PEBBLEs sensors,19 one of the main advantages of nanoparticle
sensors is the possibility to convert a simple on-off or off-on
chemosensor into a more valuable ratiometric one. This is done
by embedding a second, substrate-insensitive dye within the
particles which can produce a reference signal. To investigate this
possibility, we chose the 8-oxo-8H-acenaphtho[1,2-b]pyrrol-9-
carbonitrile derivative (dye 2) as the inner reference. First, we
prepared a class of dye-2 rich core and pure silica shell fluores-
cent nanoparticles (DSN) of the same diameter as the SSN
mentioned above. As the concentration of Zn2+ increased, there
was little change in fluorescence intensity of the DSN solution
(ESI,† Fig. S4). The results indicated that dye-2 is indifferent to
the substrates, and has the potential to produce a reference signal
for ratiometric fluorescence detection. On the other hand, as
shown in Fig. 7, the fluorescent intensity of DSN did not change
at a wide pH range from 3 to 11, which is different from other
fluorescent silica nanoparticles prepared by Rhodamine iso-
thiocyanate (TRITC) or fluorescein isothiocyanate (FITC).36,37
Furthermore, dye-2 was structurally simple with strong long-
wavelength emission and smaller molecules than other long-
wavelength fluorophores, which decrease the aggregation when
they were co-located into a single silica nanoparticle.33 All the
above advantages encouraged us to consider using dye-2 as an
inner reference to amply the ratio of fluorescence intensity for the
ratiometric measurements.
Fig. 8 Emission spectra of a solution of dye-2-rich core and sensor-1-
rich shell fluorescent silica nanoparticles (DSSN) in the presence of
increasing Zn2+ concentrations (0–1.2 mM) in tris-HCl (0.01 M) solution
(water, pH ¼ 7.10). Inset: Ratiometric calibration curve I482 nm/I591 nm as
a function of Zn2+ concentration (0.1ꢂ1.2 mM). Conditions: [sensor-1] ¼
3.1 ꢁ 10ꢀ5 M, lex ¼ 320 nm, lem1 ¼ 482 nm, lem2 ¼ 591 nm, 25 ꢃC.
greatest possible surface area for sensor interactions. As shown
in Fig. 8, by adding the Zn2+ (0ꢂ1.5 mM) to the solution of
DSSN, the fluorescence intensity at 482 nm (sensor-1@Zn2+) is
significantly enhanced. There is another band at 591 nm (inner
reference) which was marginally affected. The ratio of sensor-1 to
the dye-2 was estimated as 62 : 1 by utilizing the fluorescence
spectra (ESI,† Fig. S5, S6, Table S1). The inset in Fig. 8 shows
the ratiometric calibration of fluorescence intensity of I482 nm
/
I591 nm corresponding to the concentration of 0.1 ꢁ 10ꢀ3ꢂ1.2 ꢁ
10ꢀ3 M Zn2+, which was in a linear manner with a correlation
coefficient of R ¼ 0.99032. This indicated that DSSN not only
showed similar properties with SSN towards Zn2+ in aqueous
solution (ESI,† Fig. S7), but can be potentially be used to
quantitatively detect Zn2+ concentration in aqueous solutions by
measuring the ratio of fluorescence intensity at two different
wavelengths. Besides, a Job plot indicates a complex with 1 : 1
stoichiometry of the sensor-1 anchored onto DSSN and Zn2+,
Therefore, we prepared dye-2-rich core and sensor-1-rich shell
fluorescent silica nanoparticles (dye-2@silica@sensor-1, DSSN)
(Scheme 1). The reference dye-2 was buried deep inside the silica
particles and further protected by a layer of silica. This layer of
silica acted as a filter and protected the dyes from interactions
with larger molecules such as proteins or organic quenchers that
could interfere with the measurements.37 A layer of sensor-1 rich
shell was attached to the surface of as-synthesized silica nano-
particles, which protects the reference dye while providing the
with the association constant of 0.69 ꢁ 104 Mꢀ1 38
.
Application of DSSN in cells
To further demonstrate the DSSN with practical application
potential, we applied it for ratiometric fluorescence imaging of
Zn2+ in living cells. As shown in Fig. 5, the prepared fluorescent
nanoparticles could penetrate the cell membrane for imaging
Zn2+ in living cells. We changed the intracellular concentration
Zn2+ by processing a series of paralleled experiments. The DSSN
were incubated at 37 ꢃC and washed thoroughly with PBS buffer
to remove the extracellular nanoparticles. Then with the addition
of Zn2+ with different concentrations, the living cells were incu-
bated for another 0.5 h. The changes of the fluorescent intensity
were measured using an inverted fluorescence microscope.
ImageJ software gave an average emission value of the fluores-
cent photos. From the images we could see that free DSSN in
living cells showed very weak fluorescence (ESI,† Fig. S8: blue,
A2, B2, C2). With the increase of Zn2+ concentration, the fluo-
rescence from 430 nm to 510 nm increased clearly (ESI,† Fig. S8:
blue, A3, B3, C3), and the fluorescence from 570 nm to 650 nm
Fig. 7 Dependence of the fluorescence intensity of DSN on pH in tris-
HCl (0.01 M) solution (water, pH ¼ 7.10). Conditions: lex ¼ 575 nm,
lem ¼ 591 nm, 25 ꢃC.
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was shown as an inner reference signal (ESI,† Fig. S8: red, A4,
B4, C4). The ratio of fluorescence at different wavelengths (blue/
red) corresponding to different Zn2+ concentrations (ESI,†
Fig. S9) indicated that DSSN could act as reliable zinc concen-
tration indicators within living cells.22 Furthermore, with the
addition of an excess amount of EDTA in living cells which were
intracellularly stained with DSSN and Zn2+, the fluorescence was
quenched, which implied the DSSN could reversibly detect Zn2+
(ESI,† Fig. S8: blue, A5, B5, C5).
regular indicator displacement assay between the [DSSN@Zn2+]
complex and H2PO4ꢀ. Ratiometric calibration is shown in
Fig. 9a (inset). It is observed that the ratio of fluorescence
intensity of I591 nm/I482 nm corresponds to the concentration of
ꢀ
H2PO4 (50ꢂ500 mM) in a linear fashion (with a linearly
ꢀ
dependent coefficient R2 of 0.9955). If the amount of H2PO4
needed to decrease the initial fluorescence emission by 15% is
taken as a reference detection limit, an anion concentration down
to 6.0 ꢁ10ꢀ6 M was able to be measured, which is much lower
than the reported phosphate anion receptors in water under
physiological pH conditions.27 On the other hand, the signal of
ꢀ
The [DSSN@Zn2+] complex-based H2PO4 detection
ꢀ
[SSN@Zn2+] complex towards H2PO4 produced a quench at
only one wavelength (lem ¼ 482 nm) (Fig. 9b).The change of
It is reported that metal-containing receptors can provide
unsaturated coordination sites at the metal centers and present
some geometrical preference for anions of a given shape, and
metal–ligand receptors have a stronger affinity for anions in pure
water at physiological pH value.39 Based on this strategy,
a variety of zinc-based phosphate anions receptors have been
developed in the past few years.26,27 The [DSSN@Zn2+] complex’s
ability to ratiometrically detect anions and other species is of
particular interest.
fluorescence intensity of I482 nm corresponding to the concen-
ꢀ
tration of H2PO4 could not be used as a standard curve for
ꢀ
H2PO4 quantitative detection (Fig. 9b inset). Therefore, the
[DSSN@Zn2+] complex can be potentially used to quantitatively
ꢀ
and ratiometrically detect H2PO4 concentration by measuring
the fluorescence intensity at two different wavelengths compared
with the [SSN@Zn2+] complex.
It is well known that the luminescent properties exhibited by
polynuclear d10 (such as Zn2+, Cd2+) metal complexes are sensitive
to structural and/or environmental changes.40 So, the anion
selectivity of the [DSSN@Zn2+] complex was investigated among
common anions such as monovalent (H2PO4ꢀ, HCO3ꢀ, Fꢀ, Brꢀ,
Iꢀ, NO3ꢀ and CH3COOꢀ) as well as divalent (SO42ꢀ) anions in tris-
HCl (0.01 M) aqueous solution at pH 7.10 at 25 ꢃC. The fluo-
rescence spectra of the aqueous solution of the [DSSN@Zn2+]
complex upon addition of these anions were recorded. As shown
We prepared the [DSSN@Zn2+] complex ([sensor-1] ¼ 3.1 ꢁ
10ꢀ5 M) by centrifuging the nanoparticles which bound the Zn2+
from zinc-rich solution (2.0 mM) and resuspended in tris-HCl
(0.01 M) solution (water, pH ¼ 7.10) for H2PO4ꢀ detection. The
ꢀ
results were shown in Fig. 9a. The addition of H2PO4 to the
aqueous solution of the [DSSN@Zn2+] complex induced
a dramatic fluorescence quenching effect on the band 482 nm,
while the band of 591 nm was marginally affected. The decrease
in fluorescence intensity on the band 482 nm may result from the
ꢀ
in Fig. 10, the addition of H2PO4 (500 mM) to an aqueous
solution of [DSSN@Zn2+] complex ([sensor-1] ¼ 3.1 ꢁ 10ꢀ5 M)
induced a dramatic fluorescence quenching at the wavelength of
482 nm; under the same conditions, anions such as HCO3ꢀ, Fꢀ,
2ꢀ
Brꢀ, Iꢀ, NO3ꢀ, CH3COOꢀ and SO4 caused nearly no fluores-
cence intensity changes.
We further demonstrated the application of the [DSSN@Zn2+]
complex to cultured living cells by fluorescence microscopy
(Fig. 11). Incubation of HeLa cells with the [DSSN@Zn2+]
complex for 1.0 h at 37 ꢃC was followed by the addition of
ꢀ
H2PO4 and then was incubated for another 0.5 h. With the
increase of the concentrations of H2PO4ꢀ, the fluorescence from
430 nm to 510 nm was decreased (Fig. 11 blue: A2, B2). And the
Fig. 9 Fluorescence emission changes of the core–shell fluorescent silica
nanoparticles [DSSN@Zn2+] complex (a) and the [SSN@Zn2+] complex
(b) upon addition of H2PO4ꢀ (6ꢂ500 mM) in tris-HCl (0.01 M) solution
(water, pH ¼ 7.10). (a) Inset: Ratiometric calibration curve I591 nm/I482
nm of complex [DSSN@Zn2+] as a function of H2PO4ꢀ concentration. (b)
Inset: Titration curve I482 nm of complex [SSN@Zn2+] as a function of
H2PO4ꢀ concentration. Conditions: [sensor-1] ¼ 3.1 ꢁ 10ꢀ5 M, lex ¼ 320
nm, lem1 ¼ 482 nm, lem2 ¼ 591 nm, 25 ꢃC.
Fig. 10 Fluorescence response of the [DSSN@Zn2+] complex ([sensor-1] ¼
3.1 ꢁ 10ꢀ5 M) in tris-HCl (0.01 M) solution (water, pH ¼ 7.10) with
different anions. 1, H2PO4ꢀ; 2, Clꢀ; 3, Fꢀ; 4, Brꢀ; 5, Iꢀ; 6, SO42ꢀ; 7,
CH3COOꢀ; 8, NO3ꢀ. Conditions: [sensor-1] ¼ 3.1 ꢁ 10ꢀ5 M, lex ¼ 320
nm, lem1 ¼ 482 nm, lem2 ¼ 591 nm, 25 ꢃC.
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Acknowledgements
The authors are grateful for the financial support from National
Natural Science Foundation of China (Grants 90713026,
21076077), Science and Technology Foundation of Shanghai
(Grants 073919109), the Key New Drug Creation and
Manufacturing Program (Grants 2009ZX09103-102) and the
Shanghai Leading Academic Discipline Project (B507). We also
thank Dr Jinbiao Tu for the improvements of this paper.
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Conclusions
In conclusion, we have synthesized three kinds of novel core–
€
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10764 | J. Mater. Chem., 2010, 20, 10755–10764
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