Molecular imaging of biothiols and: In vitro diagnostics based on an organic chromophore bearing a terbium hybrid probe

Article abstract of DOI:10.1039/c6dt00156d

In this research, a novel terbium-based luminescent hybrid inorganic/organic probe was designed and synthesized. Mesoporous silica nanospheres dispersed in water were used as the appropriate host for the covalently linked lanthanide-containing organic structures. The lanthanide structure was linked to a sulfonate ester unit, which, in the presence of biothiols, was cleaved to result in terbium emission. The hybrid probe exhibited the capabilities of quantitative determination and detection limits for biothiols were presented (36.8 nM for Cys, 32.5 nM for GSH, and 34.7 nM for Hcy). Evaluation of luminescence changes in cell culture demonstrated that this smart probe is cell membrane permeable and selectively lights up in the presence of cysteine and glutathione in human embryonic kidney cells and human lung adenocarcinoma cells. This variation in the presence of biothiols can be controlled by the treatment with N-methylmaleimide. The narrow line-like bands and long-lived excited states of this terbium luminescent sensor allows the discrimination of scattering signals and interfering fluorescence derived from biological tissues.

Full text of DOI:10.1039/c6dt00156d

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Molecular imaging of biothiols and in vitro diagnostics based on an
organic chromophore bearing terbium hybrid probe
b
a,b,c*
d
e
Zhan Zhou, Qianming Wang,
, Cheng Cheng Zhang , Jinwei Gao
a. Key Laboratory of Theoretical Chemistry of Environment, Ministry of Education, School
of Chemistry & Environment, South China Normal University, Guangzhou 510006, China
b. School of Chemistry & Environment, South China Normal University, Guangzhou 510006,
China
c. Guangzhou Key Laboratory of Materials for Energy Conversion and Storage Guangzhou
510006, P. R. China
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d. Departments of Physiology and Developmental Biology, University of Texas, Southwestern
Medical Center, Dallas, TX 75390-9133, USA
e. Institute for Advanced Materials, Academy of Advanced Optoelectronics, South China
Normal University, Guangzhou 510006, P. R. China
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* To whom the correspondence should be addressed. Eꢀmail: qmwang@scnu.edu.cn
Tel: 86ꢀ20ꢀ39310258; Fax: 86ꢀ20ꢀ39310187
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Abstract
For this research, A novel terbiumꢀbased luminescent hybrid inorganic/organic probe was
designed and synthesized. Mesoporous silica nanospheres dispersed in water were used as the
suitable host for the covalently linked lanthanideꢀcontaining organic structures. The
lanthanide structure was linked to a sulfonate ester unit, which, in the presence of biothiols,
was cleaved to result in terbium emission. The hybrid probe exhibited the capabilities of
quantitative determination and detection limits for biothiols were presented (36.8 nM for Cys,
32.5 nM for GSH, and 34.7 nM for Hcy). Evaluation of luminescence changes in cell culture
demonstrated that this smart probe is cell membrane permeable and selectively lights up in
the presence of cysteine and glutathione in human embryonic kidney cells and human lung
adenocarcinoma cells. This variation in the presence of biothiols can be controlled by the
treatment with Nꢀmethylmaleimide. The narrow lineꢀlike bands and longꢀlived excited states
of this terbium luminescent sensor permits discrimination from scattering signals and
interfering fluorescence derived from biological tissues.
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KEYWORDS: Terbium, Nanostructures, Probe, Bioimaging
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INTRODUCTION
Rare earth elements have long been applied in the field of optical materials due to their
narrow and characteristic emission signals derived from forbidden f-f electronic transitions. In
order to achieve an efficient luminescent framework, the organic chromophore must absorb
ultraviolet irradiation effectively and must efficiently transfer this energy to lanthanide centers.
Therefore, numerous aromatic acids, βꢀdiketones, and macrocylic ligands have been
developed that form stable complexes with lanthanide ions and shield these cations from
deꢀactivation[1ꢀ4]. These light conversion molecular complexes have proven difficult to use
in optical devices because of their poor thermal stabilities, photoꢀdegradation, and low
mechanical resistance. To avoid these issues, lanthanide organic/inorganic hybrid materials
have been prepared [5ꢀ8].
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Generally, direct doping with organometallic complexes results in problems such as
clustering of emissive species and leaching of luminescent components due to weak
interactions. Covalent anchoring of the organic functionalities to silica matrix enables
preparation of high doping concentrations and homogenous dispersions [9ꢀ11]. These
organically modified hybrid xerogels based on lanthanide emissions will be not only of
fundamental interests, but also have superior applications in various fields like organic light
emitting diodes, amplifiers or sensors [12,13]. As far as the inorganic hosts were concerned,
hybrid networks composed of surfactantꢀtemplated porous organosilicas possess high surface
area, large pore volume, and uniformly distributed pore size making them attractive matrices
for the encapsulation of bulky organic moieties [14,15]. These embedded structures are
readily constructed and have potential as luminescent probes [16ꢀ18].
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In this work, a novel terbiumꢀbased luminescent hybrid inorganic/organic probe was
designed and synthesized (Fig. 1). The achieved ligand L played dual roles. It can coordinate
to lanthanide ions effectively and undergo solꢀgel reactions based on polymerizable silylated
3
groups (ꢀSi(OEt) ). In view of the molecular structure, it is accepted that two nitroꢀ groups in
DNBS have strong electronꢀwithdrawing features and enormously disturb the electron
distributions of pyridine derivative units in the fluorophore [19]. The photoinduced electron
transfer affects the complexation with lanthanide ions and the hybrid nanosphere is weakly
green fluorescent. But it has high affinity for thioꢀcontaining amino acids such as glutathione
(GSH), cysteines (Cys) and Homocysteine (Hcy). The cleavage reaction of sulfonate ester by
biothiols changes the electronic structure of the chromophore and in turn enhances the narrow
green emissions. Although optical probes, including organic dyes, metal complexes, silver
particles, and quantum dots, have been developed for the measurement of biothiols [20ꢀ25],
the utilization of lanthanide luminescence as signaling moieties will gain advantages over the
analogous sensing systems. The lanthanide lineꢀlike emission peaks (around 10 nm) could
achieve desired signal to noise ratio compared to conventional fluorophores (emission bands
covering more than 100 nm). Its longꢀlived excited states will avoid the side effects of
autoꢀfluorescence and light scattering of biological tissues. Additionally, the metal emission
might be highly sensitive to delicate changes in the coordination environments. More
importantly, in this work, the selection of organoalkylsilanes as precursors to anchor the
lanthanide organic complex inside the mesoporous framework will give rise to improved
chemical or thermal stabilities. Here the first case of terbiumꢀcontaining hybrid
inorganicꢀorganic thiols sensor has been described. The green luminescence of the probe
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enables the quantitative assay of thiols in the nanomolar range with high selectivity.
Validation of the probe in two live cell systems (HeLa and A549) demonstrates the
potentialities of this sensor in medical diagnostics.
EXPERIMENTAL SECTION
4 7
Materials. Tb O (99.9%) was purchased from Shanghai Yuelong. Terbium hydrochlorate
4 7
was prepared by dissolving Tb O in concentrated hydrochloric acid. Tetraethyl orthosilicate
(TEOS, AR grade), cetyltrimethylammonium bromide (CTAB, AR grade),
3ꢀ(triethoxysilyl)propyl isocyanate (TEPIC, 96%), 4ꢀhydroxypyridineꢀ2,6ꢀdicarboxylic acid
(97%), tetramethylammonium hydroxide (TMAOH), tetramethylammonium chloride and
NꢀBocꢀethylenediamine (98%) were purchased from J&K Scientific. Nꢀmethylmaleimide,
glutathione (GSH), and amino acids, including cysteines (Cys), homocysteine (Hcy), alanine
(Ala), arginine (Arg), aspartic acid (Asp), glutamic acid (Glu), glycine (Gly), histidine (His),
leucine (Leu), lysine (Lys), methionine (Met), proline (Pro), serine (Ser), threonine (Thr),
tryptophan (Trp), tyrosine (Tyr), and valine (Val), were purchased from SigmaꢀAldrich.
CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) was purchased from
Promega. All the other reagents were purchased from Guangzhou Chemical Reagent Factory
and used without further purification.
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Synthesis of ligand (L). The synthesis of L is shown schematically in Figure S1, and the
experimental details are as follows.
Synthesis of compound 3. Chelidamic acid hydrate (compound 1) was reacted with
methanol to form dimethyl 4ꢀhydroxypyridineꢀ2,6ꢀdicarboxylate (compound 2) based on
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concentrated H
2
SO
4
. Compound 3 was obtained by the treatment of compound 2 with excess
1
amounts of NꢀBocꢀethylenediamine. Compound 3 (yield: 72%) H NMR (500 MHz, CDCl
3
,
ppm): 9.26 (s, 2H), 8.23 (s, 2H), 5.18 (s, 2H), 3.58 (m, 4H), 3.45 (m, 4H), 1.36 (s, 18H). MS
(m/z): 468.6 [M + H].
2 2
Synthesis of compound 4. Compound 3 (467 mg, 1.0 mmol) in dry CH Cl (25 mL) and
NEt (300 ꢁL) were mixed, and the solution was stirred in an iceꢀwater bath for 15 min.
3
2,4ꢀDinitrobenzenesulfonyl chloride (532 mg, 2.0 mmol) was added, and the mixture was
stirred for 3 h at room temperature. The solvent was evaporated, and the crude product was
purified over a silica gel column (petroleum ether/ethyl acetate; 4:1, v/v) to obtain compound
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4 as a yellow solid (yield: 52%). Compound 4 H NMR (500 MHz, CDCl
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, ppm): 8.91 (s, 2H),
8.71 (d, J = 2.0 Hz, 1H), 8.58 (dd, J = 2.0 Hz, 2.5 Hz, 1H), 8.35 (d, J = 8.5 Hz, 1H), 8.15 (s,
2H), 5.25 (s, 2H), 3.62 (m, 4H), 3.46 (m, 4H), 1.38 (s, 18H). MS (m/z): 720.2 [M + Na].
2 2 3
Synthesis of ligand (L). Compound 4 was stirred with CH Cl /CF COOH (3:1, v/v) for 3
h at room temperature. The solvent was evaporated, yielding compound 5. Compound 5
(385.5 mg, 0.75 mmol) was dissolved in 20 mL of anhydrous tetrahydrofuran under a steady
flow of nitrogen, and TEPIC (592.8 mg, 2.4 mmol) was added with stirring. The mixture was
refluxed under nitrogen for 6 h. The reaction was monitored by thin layer chromatography.
Solvent was removed, and the product was purified on silica gel to give the ligand (L, yield:
1
57%). H NMR (500 MHz, CDCl
3
, ppm; Fig. S2): 8.93 (s, 2H), 8.71 (s, 1H), 8.62 (dd, J = 2.0
Hz, 2.5 Hz, 1H), 8.39 (d, J = 8.5 Hz, 1H), 8.18 (s, 2H), 5.78 (s, 2H), 5.36 (s, 2H), 3.82 (q, J =
8.5 Hz, 12H), 3.61 (m, 4H), 3.35 (m, 4H), 3.18 (t, J = 10.0 Hz, 4H), 1.62 (q, J = 8.5 Hz, 4H)
1.21 (t, J = 10.0 Hz, 18H), 0.65 (t, J = 10.0 Hz, 4H). MS (m/z): 992.5 [M + H]. Elemental
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Analysis: Anal. Calcd for [C H N O SSi ]: C, 44.79; H, 6.20; N, 12.71; S, 3.23. Found: C,
37 61 9 17 2
44.65; H, 6.13; N, 12.68; S, 3.30.
Synthesis of the lanthanide complex (TbL). To prepare the terbium complex, L (198.4 mg,
0.2 mmol) was dissolved in 10 mL ethanol in a 25ꢀmL round bottom flask, and 5 mL of a 0.04
3
M solution of TbCl and ammonia (400 ꢁL) were added with stirring. The mixture was
o
refluxed for 3 h at 90 C. The crude product was washed with ethanol and with water and then
dried in vacuo overnight. The resulting precipitate was collected to give the desired terbium
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complex (yield: 82%). Elemental Analysis: Anal. Calcd for TbL·3H O
61 9 2 2
[C37H N O17SSi Tb·3H O]: C, 36.88; H, 5.60; N, 10.46; S, 2.66. Found: C, 36.72; H, 5.72; N,
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10.49; S, 2.58.
Synthesis of mesoporous silica nanoparticles (MSNs) and the hybrid material
(H-MSN-Tb). Briefly, TEOS (0.1 ml) was mixed with 0.1 g CTAB, 0.6 g urea, and 30 ml
o
water in an autoclave. The autoclave was sealed and hydrothermally heated to 180 C. After
24 h, the autoclave was taken out of the oven and rapidly cooled under tap water. The
o
precipitate was centrifuged and calcined at 550 C for 5 h to remove the surfactant and yield
the desired MSNs. To a suspension of MSNs (200 mg) in ethanol, 1 mL L in ethanol (0.5 M)
and 500 ꢁL of aqueous ammonia (30%) were added, and the suspension was stirred for 3 h.
3
TbCl solution (1 mL, 0.5 M) was added. After 12 h, the precipitate was centrifuged and
redispersed in ethanol and water at least three times to remove unreacted L and lanthanide
ions. The precipitate was dried in vacuo to give HꢀMSNꢀTb.
Cell culture and MTS assay. HeLa cells (human embryonic kidney cells) were cultured in
Dulbecco’s modified medium (DMEM) supplemented with 10% fetal bovine serum (FBS)
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and 1% penicillin. Cells were maintained at 37 °C in 5% CO . A549 cells (human lung
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adenocarcinoma cells) were cultured in Kaighn’s Modification of Ham’s Fꢀ12 supplemented
2
with 10% FBS and 1% penicillin and maintained at 37 °C in 5% CO . To investigate
cytotoxicity, cells were cultured in wells of 96ꢀwell plates for one day. Medium was then
replaced with medium containing HꢀMSNꢀTb (0, 5, 10, 20, 50, and 100 ꢁg/mL), and cells
were cultured for another 24 h. An aliquot of 20 ꢁL MTT solution was added to each well.
After incubated with 4 h, the absorbance at 490 nm was measured using a Polarstar
microplate reader.
Cell imaging. For the experiments, cells were washed with medium, then incubated in
medium containing HꢀMSNꢀTb (100 ꢁg/ml) for 2 hours. After washing twice with PBS, the
cells were observed using a fluorescent microscope (Olympus BX50, Japan). For control
experiments, cells were pretreated with Nꢀmethylmaleimide (100 ꢁM) for 1 h and then
incubated with HꢀMSNꢀTb (50 ꢁg/mL) for 2 h. When indicated, cells were incubated with 50
ꢁM Cys or 50 ꢁM GSH.
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H-MSN-Tb characterization. HꢀNMR spectra were recorded at 293 K using a Bruker
Avance spectrometer (500 MHz) with TMS as an internal standard; all chemical shifts are
reported in the standard notation of parts per million. Elemental analysis was carried out by a
Vario EL cube machine (Elementar, Germany). Fluorescence emission and excitation spectra
were measured using an Edinburgh FLS920 spectrometer. The absolute quantum yields were
collected at room temperature through an integration sphere (Edinburgh FLS 920
spectrometer) method. FTꢀIR spectra were measured using a Shimadzu Prestigeꢀ21. LCꢀMS
spectra were measured with a Thermo LCꢀMS equipment (LCQꢀ Advantage). Transmission
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electron microscopy (TEM) was performed on a JEOL JEMꢀ2100HR. Scanning electron
microscopy (SEM) was measured on a Zeiss Ultra 55. The adsorption desorption isotherms of
nitrogen were measured at 77 K using the ASAP2020M system. Thermogravimetric analyses
o
(TGA) were carried out on a STA409PC system under air at a rate of 10 C/min. HPLC
analyses were performed on an Inertsil ODSꢀSP (5 ꢁM, 4.6mm×250mm) column using a
Shimadzu HPLC system that consists of two LCꢀ20AT pumps and a SPDꢀ20A UVꢀvis
detector at 254 nm with acetonitrile/water (V/V = 4/1) containing 0.5 % trifluoroacetic acid as
eluents at a flow rate of 0.8 mL/min. 10 ꢁL was used for the injection volume.
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RESULTS AND DISCUSSION
Synthesis of terbium inorganic/organic mesoporous hybrid nanosphere probe.
The terbiumꢀbased luminescent hybrid inorganic/organic probe is shown in Figure 1.
Mesoporous silica nanospheres (MSN) dispersed in water were used as the host for the
covalently linked lanthanideꢀcontaining organic structures. The MSN was derivatized with
ligand (L), which chelates the lanthanide and contains a moiety reactive with thiols, and
complexed with terbium to yield the hybrid probe HꢀMSNꢀTb. The ligand was synthesized by
conversion of chelidamic acid hydrate 1 to the ester 2 (Fig. S1). Compound 2 was reacted
with NꢀBocꢀethylenediamine to produce amide 3. An excellent electron acceptor,
2,4ꢀdinitrobenzenesulfonyl (DNBS), was introduced into the organic framework to yield
compound 4. Following deprotection, 3ꢀ(triethoxysilyl)propyl isocyanate was grafted to the
chromphore through amide linkages. This multifunctional precursor (L) was used to
coordinate with terbium ions during a solꢀgel process in order to prepare the mesoporous
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hybrid material (Fig. 1).
Characterization of the probe.
The organic chromophore and the formation of the complex within the siloxane host were
investigated by roomꢀtemperature infrared spectroscopy. The intense bands observed between
ꢀ1
2800ꢀ3000 cm were assigned to asymmetric and symmetric stretching vibrations of multiple
CH units in the ligand (Fig. 2a) and indicate that the 3ꢀ(triethoxysilyl)propyl isocyanate was
2
ꢀ
1
successfully introduced into the organic framework. A broad signal detected at 1081 cm was
attributed to SiꢀO stretching vibration modes from the silica matrix (Fig. 2b). The successful
ꢀ1
grafting reaction was confirmed by two sharp peaks located at 1665 and 1554 cm that
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correspond to C=O and NꢀH vibrations, respectively, due to urea units in the ligand. The C=O
ꢀ
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vibration mode was shifted to lower frequency (ꢂυ ca. 15 cm ) in the terbium complex than
in the uncomplexed ligand, suggesting that all carbonyl moieties participate into the
coordination with terbium ions.
Adsorptionꢀdesorption isotherms and pore size distribution curves of the uncomplexed
and terbium containing hybrid material are shown in Fig. S3. Type IV isotherms were
observed for both sample, indicating the existence of mesoporous ordered structures. The
BrunauerꢀEmmettꢀTeller (BET) specific surface area and the pore volume measured for the
2
−1
−3 −1
terbiumꢀcontaining material (529.6 m g , 0.61 cm g , respectively) varied significantly
2
−1
−3 −1
from those of the uncomplexed sample (861.1 m g , 0.95 cm g , respectively), suggesting
that the terbiumꢀcontaining organic structures were incorporated into the pores or the
channels.
The thermogravimetric analysis (TGA) was carried out to determine thermal stabilities of
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the organometallic complex and the mesoporous hybrid network (Fig. S4). At temperatures
o
lower than 100 C, weight loss was mainly due to desorption of water molecules. The second
o
weight loss region between 200 and 500 C is likely due to the decomposition of loaded
organic structures. The pure metal complex was thermally unstable compared with that in the
highly ordered mesostructured silica, indicating that the integration of the complex into the
o
inorganic backbone improved durability. Above 500 C, the weight loss is assumed to be due
to decomposition of silica and the further condensation of silanols.
Representative SEM and TEM images of the probe are shown in Fig. 3. TEM micrograph
displayed that the materials consist of wellꢀpreserved and uniform nanospheres. The narrow
size distribution of the obtained granules was between 80 and 100 nm. Numerous
wellꢀordered mesopores were found within the spherically shaped nanoparticles. Especially
porous and hollow structures were identified in regular spheres.
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Emission spectra in the presence of thiol and non-thiol biomolecules.
HꢀMSNꢀTb exhibited a typical metal centered luminescence in HEPES buffer. Excitation of
the organic chromophore at 284 nm (Fig. S5) resulted in a relatively weak green emission
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(Fig. S6). The narrow peaks were assigned to the electronic transitions from D level to the
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J
F levels (J = 6, 5, 4, and 3 at 490, 545, 583, and 621 nm, respectively). Upon addition of Cys,
a dramatic emission intensity increase was observed depending on the time duration. The
luminescence enhancement has reached a plateau within 15 min (Fig. S7). The relationship
between pH value and the emission intensity of HꢀMSNꢀTb with or without the biothiol
treatment was shown in Fig. S8. When pH is above 5.0, the emission intensity of HꢀMSNꢀTb
was increased in the presence of Cys. In contrast, its fluorescence maintained relatively stable
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in the absence of Cys at different pH environments. Therefore, HꢀMSNꢀTb can be potentially
used for the monitoring of thiols within physiological pH conditions.
The detection limit of HꢀMSNꢀTb for thiols was investigated using fluorescence titration
spectra. As shown in Fig. 4, the luminescence of HꢀMSNꢀTb was dramatically enhanced with
the addition of Cys. When the peak intensity at 545 nm was plotted against Cys concentration
from 0 to 22 ꢁM, the curve demonstrated an excellent linearity (correlation coefficient = 0.98).
The plateau in the presence of Cys has been reached at the concentration of 22 µM. The
detection of limit (36.8 nM) was calculated by the equation DL = 3×SD/S with a
signalꢀtoꢀnoise ratio of 3 (DL means detection limit; S refers to the slope of the calibration
curve; SD corresponds to standard deviation which was obtained by ten times parallel
experiments of the blank sample). In addition, the quantum yields in the absence of Cys and
in the presence of Cys were measured to be 0.35 % and 11.4 % respectively. Significant
increase in luminescence intensity was also observed upon the addition of GSH to HꢀMSNꢀTb
(Fig. 5). A similar linear regression fitting equation has been generated with a 20ꢀfold increase
at the end of the titration (detection limit = 32.5 nM). Under the same conditions, the hybrid
probe also demonstrated the analogous responses to Hcy, and the detection limit is 34.7 nM
(Fig. S9). Therefore, the target probe can be used for quantitative determination of biothiols
including GSH, Cys, and Hcy.
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The selectivity was investigated by analysis of fluorescence responses in the presence of
various amino acids. In the presence of up to 100 ꢁM of other fifteen analytes (Ala, Arg, Asp,
Glu, Gly, His, Len, Lys, Met, Pro, Ser, Thr, Trp, Tyr, and Val) there was little change in
ꢀ
ꢀ
ꢀ
ꢀ
2ꢀ
2ꢀ
4 3
HꢀMSNꢀTb emission (Fig. S10). Furthermore, common anions (F , Cl , Br , I , SO , CO , and
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+
2+
2+
2+
3+
3+
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NO ) or cations (K , Mg , Ca , Zn , Al , and Fe ) did not induce the emission intensity
3
changes of the hybrid probe. However, when Cys was added into the above solution, it gave rise to
a drastic enhancement effect which was similar to the addition of Cys alone (Fig. S11). Those
results indicated that the hybrid probe can be used to selective detection of biothiols, and the
recognize process was not influenced by the common competitive ions.
The above results suggest that lanthanideꢀbased inorganicꢀorganic mesoporous probe
interacts specifically with biothiols, which induce cleavage of sulfonate ester. In the absence
of the biothiol, the double nitro groups with potential electronꢀaccepting features in ligand L
pull electrons from pyridine ring to DNBS unit quenching its singlet emission and
suppressing energy migration to terbium ions. In the presence of biothiols, the sulfonate ester
bonds are cleaved and DNBS moiety is removed. The electronic structure of pyridine ring has
been reꢀarranged and its emission is restored. So it could transfer ultraꢀviolet energy to the
central ions. Thus, only in the presence of biothiols was the terbium green emission at 545 nm
detected. Although it was not convenient to apply routine molecular structure
characterizations to study the hybrid inorganicꢀorganic material (HꢀMSNꢀTb) due to its
solubility, we have found the useful information in the functional ligand L and its cleavage
molecule L’ (Fig. S12). Mass spectra results firmly supported the cleavage reaction. The mass
signal assigned to the molecular weight of the cleaved compound (L’) was detected in the
presence of Cys (Fig. S13). In addition, we also utilized HPLC to explore the reaction process
of L with Cys. As shown in the following Fig. 6, ligand L gave a typical chromatographic
band at 9.05 min. After the addition of Cys, this peak was reduced dramatically. A new peak
at 6.92 min was observed and this product with the retention time of 6.92 min has been
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confirmed to be ligand L’ by mass result.
Use of H-MSN-Tb as a thiol sensor in cells.
Nanoscale hybrid sensors clearly have potential for use as diagnostic tools [26].
Thiolꢀcontaining amino acids like Cys and GSH play crucial roles in controlling regular
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1
cellular functions, and levels of these compounds are indicative of numerous diseases. To
determine whether our probe is useful in living cells, we performed experiment in two types
of immortalized cells, HeLa and A549 cells. Probes used in vivo cannot be toxic to cells.
Cytotoxicity of the HꢀMSNꢀTb probe was determined using an MTS assay. Both HeLa and
A549 cells were 95% viable at a concentration of 20 ꢁg/mL HꢀMSNꢀTb (Fig. 7). Under
practical live cell imaging conditions of about 50 ꢁg/mL probe, HꢀMNSꢀTb also had very low
cytotoxicity. These negligible toxicities suggest that these nanospheres are biocompatible and
have potential for visualization of targets in cells.
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Cell imaging under a fluorescence microscope revealed that both HeLa and A549 cells
were stained intracellularly by HꢀMSNꢀTb resulting in an intense green emission, indicating
that the hybrid material penetrated cell membranes and was activated by thioꢀreactive
components within cells (Figs. 8A and 9A). When cells were pretreated with the biothiol
scavenger Nꢀmethylmaleimide prior to addition of HꢀMSNꢀTb, the green luminescence was
completely quenched (Figs. 8B and 9B). The significant fluorescence decrease was related to
the removal of Cys and GSH by Nꢀmethylmaleimide. When GSH or Cys was added to cells
pretreated with Nꢀmetylmaleimide, luminescence was observed again upon incubation with
HꢀMSNꢀTb (Figs. 8C, D and 9C, D). These results confirmed that the nanosphereꢀbased
lanthanide hybrid probe has potential as for use as a biosensor in living organisms.
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CONCLUSIONS
A general strategy for the design and synthesis of novel lanthanide turnꢀon fluorescence
probes has been described. The hybrid inorganicꢀorganic network combines terbium
coordinated pyridine derivative moieties as the fluorophore and 2,4ꢀdinitrobenzenesulfonyl
ester group as the recognition unit. The cleavage reaction releases the original terbium
lineꢀlike green emissions and this sensor demonstrates rapid detection, excellent selectivity
and high sensitivity for Cys and GSH in buffer solution. The low cytotoxicity displayed that
the nanoꢀsized luminescent probe has great potentials in visualizing biothiols efficiently in
two live cell lines (including HeLa and A549). This switchꢀon type hybrid nanoꢀsensor that
enables clear observation of terbium emission changes will contribute to the new
development of highly efficient smart probes in thiols sensing field.
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ACKNOWLEDGMENTS
Q. M. appreciates National Natural Science Foundation of China (No. 21371063 and
No.21328503), excellent university young scholar fund of Guangdong province (Yq2013053),
Science and Technology Project in Guangzhou (2014J4100054), Natural Science Foundation
of Guangdong (2015A030313378) and Guangdong Science and Technology plan
(2013B010403025). J. W. thanks supports from National Natural Science Foundation of
China (No.51571094) and Guangdong province fund (2014B09091505).
REFERENCES
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Figure captions
Figure 1. Schematic illustration of responsive MSNꢀbased sensor HꢀMSNꢀTb.
Figure 2. IR spectra of ligand L (solid line) and HꢀMSNꢀTb (dotted line).
Figure 3. TEM (top) and SEM (bottom) images of HꢀMSNꢀTb.
Figure 4. Emission spectra of HꢀMSNꢀTb (50 ꢁg/mL) upon addition of Cys (from 0 to 22 ꢁM)
in 30 mM HEPES buffer, pH 7.0 (λ = 284 nm). Inset: Relative intensity of HꢀMSNꢀTb at 545
ex
nm as a function of Cys concentration from 2 to 22 ꢁM. Detection limit: 36.8 nM.
Figure 5. Emission spectra of HꢀMSNꢀTb (50 ꢁg/mL) upon addition of GSH (from 0 to 20
ꢁM) in 30 mM HEPES buffer, pH 7.0 (λex= 284 nm). Inset: Relative intensity of HꢀMSNꢀTb
at 545 nm as a function of GSH concentration from 2 to 20 ꢁM. Detection limit: 32.5 nM.
Figure 6. Typical HPLC of chromatogram of ligand L (20 µM, a) and ligand L (20 µM) incubated
with Cys (40 µM) for 30 min (b). Peaks in the chromatograms were detected by monitoring the
absorption at 254 nm. The mobile phase was 4/1 acetonitrile/water containing 0.5 %
trifluoroacetic acid at a flow rate of 0.8 mL/min.
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Figure 7. Viability of HeLa cells (red bar) and A549 cells (black bar) after 24 h in the
presence of indicated concentrations of HꢀMSNꢀTb.
Figure 8. (A) Fluorescence image of HeLa cells incubated with 50 ꢁg/mL HꢀMSNꢀTb for 2 h.
(B) Fluorescence image of HeLa cells pretreated with 100 ꢁM Nꢀmethylmaleimide for 1 h,
then incubated with 50 ꢁg/mL HꢀMSNꢀTb for 2 h. (C) Fluorescence image of HeLa cells
pretreated with 100 ꢁM Nꢀmethylmaleimide for 1 h, then incubated with 50 ꢁM Cys and 50
ꢁg/mL HꢀMSNꢀTb for 2 h. (D) Fluorescence image of HeLa cells pretreated with 100 ꢁM
Nꢀmethylmaleimide for 1 h, then incubated with 50 ꢁM GSH and 50 ꢁg/mL HꢀMSNꢀTb for 2
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h.
Figure 9. (A) Fluorescence microscope images of A549 cells incubated with 50 ꢁg/mL
HꢀMSNꢀTb for 2 h. (B) Fluorescence images of A549 cells pretreated with 100 ꢁM
Nꢀmethylmaleimide for 1h, then incubated with 50 ꢁg/mL HꢀMSNꢀTb for 2h. (C)
Fluorescence microscope images of A549 cells pretreated with 100 ꢁM Nꢀmethylmaleimide
for 1 h, and then incubated with 50 ꢁM Cys and 50 ꢁg/mL HꢀMSNꢀTb for 2 h. (D)
Fluorescence microscope images of A549 cells pretreated with 100 ꢁM Nꢀmethylmaleimide
for 1 h, and then incubated with 50 ꢁM GSH and 50 ꢁg/mL HꢀMSNꢀTb for 2 h.
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