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Agricultural and Environmental Chemistry
Tetracycline generated red luminescence based on a novel lanthanide functionalized layered double hydroxide nanoplatform Zhan Zhou, Xiangqian Li, Jinwei Gao, Yiping Tang, and Qianming Wang J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 26 Mar 2019 Downloaded from http://pubs.acs.org on March 27, 2019
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Tetracycline Generated Red Luminescence Based on a Novel
2
Lanthanide Functionalized Layered Double Hydroxide
3
Nanoplatform
4 5
Zhan Zhou a, Xiangqian Li b, Jinwei Gao c, Yiping Tang d, Qianming Wang b*
6 7
a. College of Chemistry and Chemical Engineering, Henan Key Laboratory of
8
Function-Oriented Porous Materials, Luoyang Normal University, Luoyang 471934, PR
9
China
10
b. Key Laboratory of Theoretical Chemistry of Environment, Ministry of Education, School
11
of Chemistry & Environment, South China Normal University, Guangzhou 510006, China
12
c. Guangdong Provincial Engineering Technology Research Center For Transparent
13 14 15
Conductive Materials, South China Normal University, Guangzhou 510006, China d. College of Material Science and Engineering, Zhejiang University of Technology, Hangzhou, Zhejiang, 310014, China
16 17 18 19 20 21 22
* Corresponding Author
23
Tel.: 86-20-39310258, Fax: 86-20-39310187. E-mail:
[email protected]; 1
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ABSTRACT : The considerable interests of lanthanide complexes in optics have been
25
well known for a long period of time. But such molecular-based edifices have been excluded
26
from practical application because of poor thermal or photo-stabilities. Here a novel europium
27
embedded layered double hydroxide (Mg-Al LDH-Eu) has been established and such
28
inorganic-organic framework demonstrates improved thermal performance due to hydrolysis
29
and poly-condensation of trimethoxysilyl- unit. In addition, the incorporation of functional
30
building block such as ethylenediamine triacetic acid can significantly minimize the negative
31
effects of hydroxyl groups. In the presence of tetracycline (Tc), the nanoprobe exhibits an
32
“off-on” change in aqueous solution and the red luminescence can be excited in the visible
33
light range (405 nm). It provides a very sensitive signal response to Tc with an excellent
34
linear relation in the range of 0.1 µM to 5.0 µM and the detection limit of this probe is
35
measured to be 7.6 nM. This nanoplatform exhibits low cytotoxicity during in vitro
36
experiments and can be employed for the detection of tetracycline in 293T cells.
37 38
KEYWORDS: Layered double hydroxide, Lanthanide, Tetracycline, Fluorescence,
39
Nanoprobe, Stability
40 41 42 43 44 45 2
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INTRODUCTION
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Tetracycline (Tc), one of the frequently-used antibiotics, may serve as effective drugs in
48
human therapy or animal medicine for curing diseases and treating infections.1-3 But the
49
uncontrollable usage of tetracycline would lead to a series of problems, such as bacterial
50
resistances, ear injury, kidney damage, liver damage and gastrointestinal reactions.4-7
51
Therefore, the development of high selectivity and sensitivity probes for monitoring Tc
52
residues in environment and bio-medical analysis has caused considerable interests in
53
scientific fields. Over the past decade, several analytical tools for the detection of Tc have
54
been provided, such as high-performance liquid chromatography (HPLC), capillary
55
electrophoresis (CE), mass spectrum (MS), fluorescence, electrochemical sensor and so on.8-12
56
It has been explored that fluorescent probe possessed a few advantages because of its high
57
sensitivity, ease of operation, fast-response, cost-effectiveness and real-time sensing. To date,
58
numerous smart materials including gold nanoclusters, quantum dots and modified
59
fluorescent dye were employed for Tc detection.13-18 However, such fluorophores would be
60
easily influenced by external factors. Low thermo- or photo-stability and short-lived excited
61
states would severely restrict their practical applications.
62
Recently, lanthanide complexes are investigated in the range of chemical sensing in virtue
63
of their special luminescence characteristics, including extraordinary color purity, high
64
quantum efficiency, long lived excited states, large Stokes shifts and narrow emission
65
bands.19-27 The sharp difference between lanthanide species (excited states from microsecond
66
to millisecond) and conventional fluorophores (excited state in the nanosecond scale) will
67
make time-resolved acquisition of emission curves and the influence of background signals 3
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would be negligible. However, the rational design of molecular-based lanthanide indicators
69
still has much difficulty in real mediums due to the competitive coordination of polar solvents
70
or water molecules. Consequently, quite a few new strategies have been developed for the
71
integration of lanthanide complex systems into a wide range of solid supports.28-33 It is known
72
that layered double hydroxide (LDH), a typical 2D inorganic layered solid host, is composed
73
of positively charged layers and interlayer balancing anionic species and water molecules.34-41
74
Its well-defined inorganic layered structure will certainly improve the chemical stability of
75
luminescent centers and the natural basicity of LDH can effectively protect the lanthanide
76
emissions. It will be the first case study for the coordination entrapment of europium complex
77
into LDH matrix and its sensing performance has been explored.
78
In this study, a novel lanthanide functionalized layered double hydroxide based
79
fluorescent nanoprobe (Mg-Al LDH-Eu) with “turned on” effect has been afforded. Its
80
detection behavior for Tc has been completely carried out in aqueous solution (Scheme 1).
81
Firstly, Mg-Al LDH was prepared according to the co-precipitation method. Subsequently,
82
N-(trimethoxysilylpropyl) ethylenediamine triacetic acid (EDTA) and europium (III) ions
83
were incorporated to achieve Mg-Al LDH-Eu. In the presence of Tc, the emission intensity at
84
618 nm was substantially improved and displayed very striking red luminescence. As for the
85
case of Tc, the emission intensities followed the linear equation Y = 11.8 X + 4.05 (R2 =
86
0.999) with the various concentrations of Tc (from 0.1 to 5 µM). The detection limit was
87
determined
88
complex-encapsulated layered double hydroxide will contribute to the development of new
89
intelligent optical sensors.
to
be
7.6
nM.
This
effective
strategy
for
assembling
lanthanide
4
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EXPERIMENTAL SECTION
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Chemicals and materials. N-(trimethoxysilylpropyl) ethylenediamine triacetic acid
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sodium (EDTA, 40 wt.%), Tetracyclinehydrate (Tc), L-Alanine, (Ala, 98%), L(+)-Arginine
93
(Arg, 99%), L-Asparagine (Asn, 99%), L-Glutamine (Gln, 99%), L-Aspartic Acid (Asp, 98%),
94
Glycine (Gly, 99%), L-Leucine (Leu, 99%), DL-Serine (Ser, 98%), L-Tryptophan (Trp, 99%),
95
L-Histidine (His, 99%), L-Lysine (Lys, 98.5%), L-cysteine (Cys, 99%), Homocysteine (Hcy,
96
95%) and Glutathione (GSH, 98%) were purchased from Aladdin Reagent Company.
97
Analytical grade Mg(NO3)2·6H2O, Al(NO3)3·9H2O, NH3·H2O and other reagents were
98
purchased from Guangzhou Chemical Reagent Factory and used without further purification.
99
Ultrapure water was used throughout this research.
100
Characterizations. Fluorescence and excitation spectra were measured using a Hitachi
101
F-7000 fluorescence spectrophotometer with a 150 W xenon lamp as a light source (Hitachi,
102
Tokyo, Japan). Time-gated emission spectra were measured on a Edinburgh FLS980
103
luminescence spectrometer with the settings of delay time, 0.2 ms; gate time,0.4 ms; cycle
104
time, 20 ms; excitation slit, 5 nm; and emission slit, 5 nm. Infrared spectra were recorded by a
105
Nicolet Magna 550 FT-IR instrument (resolution: 1 cm-1; range 4000-750 cm-1) with the KBr
106
pellet technique (Nicolet Magna, Illinois, USA). TEM images were obtained with a JEOL
107
JEM-2100HR transmission electron microscope (Hitachi, Tokyo, Japan). SEM images were
108
measured with a Zeiss Ultra 55 scanning electronic microscope (Zeiss, Oberkochen,
109
Germany). X-ray diffraction measurements were carried out on powder samples through a
110
Bruker D8 diffractometer using Cu-Kα1 radiation (λ = 1.54 Å) (Netzsch, Selb, Germany).
111
Thermogravimetric analysis was explored by a STA409PC system under air at a rate of 5
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10℃/min (Bruker, Karlsruhe, Germany). All error bars represent standard deviations from
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three repeated experiments. High performance liquid chromatography (HPLC) analysis was
114
carried out by using a LC2010A HPLC system (Shimadzu, Japan). C18 column (250×4.6 mm)
115
and a UV detector were used.
116
Synthesis of Mg-Al LDH. Mg-Al LDH were prepared according to the co-precipitation
117
method with a slight modified described in the literature.42 In brief, solution A:
118
Mg(NO3)2·6H2O (0.769 g, 3 mmol) and Al(NO3)3·9H2O (0.563 g, 1.5 mmol) were dissolved
119
in 40 mL of ultrapure water. Solution B: NaOH (0.4 g, 10 mmol) was dissolved in 40 mL of
120
ultrapure water. Solution A and solution B were simultaneously added to a 500 mL
121
round-bottomed flask and mixed for 1 min using a rotor speed of 3000 rpm. The mixture was
122
placed in a 100 mL Teflon-lined stainless-steel autoclave and heated at 100℃ for 24 hours.
123
The obtained milky white sample (Mg-Al LDH) was recovered by centrifugation, washed
124
with ultrapure water and ethanol until the pH = 7, and vacuum-dried at 65℃ for 7 hours.
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Synthesis of Mg-Al LDH-COOH. In order to obtain the polydentate ligands to chelate
126
europium (III) ions in Mg-Al LDH surface, 0.1 g of Mg-Al LDH was added into a 250 mL
127
round bottom flask with 100 mL of ethanol and dispersed through ultrasonication at 0℃ for
128
30 min. 0.25 mL portion of a 40 wt% hexane solution of N-(trimethoxysilylpropyl)
129
ethylenediamine triacetic (EDTA) acid sodium was then added and stirred for 12 hours at 65℃
130
for silanization. Then, 100 mL of methanol was added to dilute the unreacted silane solution.
131
The product was obtained by filtration and washed sequentially with methanol, water, and
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acetone. Then the samples were vacuum-dried at 65℃ for 7 hours.43,44
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Synthesis of Mg-Al LDH-Eu. The obtained carboxyl groups modified Mg-Al 6
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LDH-COOH (100 mg) was added in a round bottom flask with 20 mL bicarbonate buffer (10
135
mM, pH = 9.6). Then, Eu(NO3)3·6H2O (50 mg) was added to the mixture with stirring for 2
136
hours. After completely reaction, Mg-Al LDH-Eu was collected by centrifugation, washed
137
with ultrapure water for three times to remove the excessive europium ions and vacuum-dried
138
at 65℃ for 7 hours.
139
Detection of Tc in aqueous solution. To verify the feasibility of the Mg-Al LDH-Eu as
140
the biosensor for Tc, 1 mL of Mg-Al LDH-Eu aqueous solution (0.1 mg/mL) was injected to
141
a spectrophotometer quartz cuvette. Then various concentrations of Tc (0-7 µM) were added,
142
the fluorescence emission spectra were recorded under excitation at 405 nm. For comparison,
143
control experiments were conducted by replacing Tc with other interfering analytes (0.1 mM),
144
including amino acids (GSH, Cys, Hcy, Ala, Arg, Asn, Gln, Asp, Gly, Leu, Ser, Trp, His,
145
Lys), common cation ions (Cu2+, Fe2+, Fe3+, Zn2+, Cd2+, Hg2+) and common anions (ClO-,
146
NO2-, NO3-, CO32-, HCO3-, Ac-, PO43-, HPO42-, MnO42-, Cr2O72-, SO42-, SO32-, HS-, F-, Cl-),
147
respectively.
148
Detection of Tc in real samples. For Tc detection in real sample, milk samples were
149
collected from the local supermarket. The various volumes of the standard Tc were added into
150
the corresponding vial to obtain the different final concentrations (1.0, 2.0 and 3.0 µM).
151
Finally, the probe was added into the above solution and subjected to measure the
152
concentration of Tc, respectively. The fluorescence emission spectra were recorded under
153
excitation at 405 nm and calculated the concentration of Tc using the linear equation,
154
respectively. As for HPLC analysis, the pretreatment process for the raw milk was performed
155
according to the literature.45 The HPLC operation parameters were given as follows: mobile 7
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phase: 0.01 M oxalic acid/acetonitrile/methanol (80:15:5, v/v/v) at 1 mL/min; the column
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temperature was kept at 40 oC; UV detection wavelength was fixed at 350 nm; an aliquot of
158
60 μL was injected. Tetracycline standard solutions of 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 µM were
159
prepared and determined by HPLC for the establishment of a standard curve.
160
Cell culture and viability assay. 293T cell was cultured in Dulbecco’s modified medium
161
(DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics penicillin (PS)
162
and maintained at 37℃ under an atmosphere of 5%CO2. To investigate the cytotoxicity of
163
Mg-Al LDH-Eu, the 293T cells (106 cells/well) were dispersed within replicated 12-well
164
microtiter plates and incubated for 24 h at 37℃ under an atmosphere of 5% CO2. After
165
removal of the medium, cells were cultured with fresh medium containing various
166
concentrations of Tc(0, 2, 7 M) for another 24 h. The cytotoxicity of Mg-Al LDH-Eu (0.1
167
mg/mL) was evaluated by MTT assay based on ISO 1099.
168
Cell imaging. Cells were added on polylysine-coated cell culture glass slides inside the 30
169
mm glass culture dishes. The cells were washed with DMED medium and incubated in the
170
fresh medium containing Mg-Al LDH-Eu (0.1 mg/mL) at 37℃ for 2 h. Cells were washed
171
with medium again and further cultured for another 0.5 h with the various concentrations of
172
Tc (0, 2, and 7 M). The cells were imaged on a Leica confocal laser scanning microscope
173
(TCS SP5 CLSM) equipped with a UV laser (405 nm).
174 175
RESULTS AND DISCUSSION
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The inorganic layered compounds possess excellent surface features to be modified for
177
the construction of host-guest interactions and its microstructure has been explored. The size 8
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and morphology of the as-prepared Mg-Al LDH and Mg-Al LDH-Eu were examined by
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transmission electron microscopy (TEM) and scanning electron microscope (SEM). Mg-Al
180
LDH gave rise to a moderately narrow particle size distribution with a polydispersity index
181
(PDI) of 0.162, suggesting Mg-Al LDH was homogenously dispersed in aqueous solution.
182
The average particle size of Mg-Al LDH was 126.8 nm (Figure S1). TEM images (Figure 1A,
183
B) showed that Mg-Al LDH was randomly dispersed and possessed plate-like morphology. It
184
has been observed that Mg-Al LDH was composed of numerous sheets from side views and
185
the lateral dimension of the nanosheets was determined to be about 100 nm (Figure 1C,D),
186
which was consistent with the particle size distribution given by DLS (Figure S1). The lattice
187
fringe spacing of about 0.15 nm was attributed to the (110) plane of an Mg-Al LDH phase
188
(Figure 1B, inset).46 SEM images indicated that the Mg-Al LDH samples possessed uniform
189
plate-like morphology and the surface was smooth (Figure 1C,D). Elemental analysis of
190
Mg-Al LDH by using energy-dispersive X-ray spectroscopy (EDS) indicated that it’s
191
composed of C, N, O, Mg and Al elements (Figure 2A). After the surface was functionalized
192
with EDTA group and the encapsulation of europium ions, the microstructures of Mg-Al
193
LDH-Eu with round shapes and average particle size of 100 nm were identified (Figures S1,
194
S2). A lattice spacing of 0.15 nm with (110) facet was also observed and the result was very
195
similar to bare Mg-Al LDH. Several lamellar structures were observed in perpendicular
196
directions. Although the grafting reaction induced slight changes, the influence on the internal
197
structure of the free Mg-Al LDH due to the surface modification with lanthanide complexes
198
would be limited. Elemental analysis of Mg-Al LDH-Eu by using energy-dispersive X-ray
199
spectroscopy (EDS) indicated that it’s successful assembly and chelation of europium ions 9
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(Figure 2B).
201
FT-IR spectra of Mg-Al LDH, Mg-Al LDH-COOH, Mg-Al LDH-Eu were carried out for
202
the investigation of the modification and coordination processes. As provided in Figure 3, a
203
broad band in Mg-Al LDH centered at 3405 cm-1 was observed, which was attributed to the
204
stretching vibration of the hydroxyl group from both the interlayer water and hydroxide layers.
205
The band at 1350 cm-1 was ascribed to the stretching vibration of nitrate anions.47 In the curve
206
of Mg-Al LDH-COOH, the emerging intense absorption bands at about 2959, 2920 and 2849
207
cm-1 were assigned to the stretching vibrations of methylene groups from EDTA units.48 The
208
band at 1034 cm-1 was ascribed to the stretching vibration of Si-O bond.49 In addition, two
209
typical bands at 1680 and 1580 cm-1 were corresponding to the stretching vibrations of
210
carboxylate units. These results revealed that EDTA has been successfully grafted to the
211
Mg-Al LDH surface by Mg(Al)-O-Si bonds and the surface contained quantities of functional
212
carboxyl groups. Notably, after the introduction of europium ions, the peaks in Mg-Al
213
LDH-COOH at 1680 and 1580 cm-1 were shifted to 1665 and 1535 cm-1 in the curve of
214
Mg-Al LDH-Eu, respectively. It was clear that the carboxyl groups were involved in the
215
chelation reaction with europium ions.
216
The powder X-ray diffraction (XRD) patterns of the synthesized Mg-Al LDH, Mg-Al
217
LDH-COOH and Mg-Al LDH-Eu were given in Figure 4. Mg-Al LDH prepared by the
218
conventional co-precipitation method showed the typical LDH diffraction peaks. The
219
diffraction peaks indexed to the (003), (006), (009) and (110) planes supported the presence
220
of Mg-Al LDH as the matrix.42,50 The sharpness and symmetry of these peaks indicated that
221
the prepared LDH was in a highly crystalline phase. Upon grafting EDTA groups and 10
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chelated europium ions, the obtained Mg-Al LDH-Eu displayed almost the same reflections
223
as that of Mg-Al LDH, showing that the silanization and the incorporation of lanthanide
224
complexes could hardly affect the crystalline integrity of Mg-Al LDH. So far as Mg-Al
225
LDH-Eu was concerned, the diffraction peaks were nearly identical with the Mg-Al LDH and
226
slight changes of the peak intensities were monitored. Hence, the surface functionalized with
227
EDTA groups and the incorporation of europium ions would lead to negligible changes of the
228
structural regularity of original layered double hydroxide.
229
The thermogravimetric (TGA) analysis of Mg-Al LDH-Eu and EDTA-Eu complex was
230
performed to evaluate their thermal stability. As presented in Figure 5, the first weight loss of
231
10% in the range of 30-180℃ was primarily caused by the adsorbed solvent species and
232
coordinated water molecules from EDTA-Eu complex. The results revealed that the
233
EDTA-Eu complex had decomposed at around 340℃, while the weight of the Mg-Al
234
LDH-Eu nanocomposites decreased much slower in the temperature range from 30-340℃.
235
The formation of strong hydrogen bonds between the carbonyl oxygen atoms of coordinated
236
carboxylate groups and the LDH layers can be closely related to such results. Based on the
237
TGA curves of EDTA-Eu complex and Mg-Al LDH-Eu, it could be said that the Mg-Al LDH
238
would substantially improve the thermal stability of EDTA-Eu complex.
239
pH value is a key factor and can possibly exert influence on the formation of resultant
240
materials. The effects of pH on the microstructure of Mg-Al LDH-Eu were investigated and
241
the diffraction peaks remained almost unchanged between pH = 5 and pH = 11 (Figure S3).
242
When the pH value was less than 5.0 (pH = 4.0), the acidic environment would dissolve the
243
hydroxide precipitates and its crystalline structure would be affected. Since the layered double 11
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hydroxides were considered to be alkaline supports, the increasing pH values would be
245
favorable for maintaining the stability. But the higher alkaline solution (pH = 12) would also
246
induce leaching problem of europium ions and such lanthanide element with positive charges
247
might be involved in the coordination with hydroxide. It was found that europium ions were
248
absent in the corresponding EDS spectrum (Figure S4). Temperature is also very important
249
for its practical uses. Based on XRD evolution curves, it has been found that its crystalline
250
structure was kept relatively stable until the heating treatment was elevated to 100 oC (Figure
251
S5) and higher temperature (150 oC) would cause the dissociation of the internal structure.
252
Fluorescent sensing allowed the control of chemical process and the host-guest
253
interactions would result in a detectable optical response. The capability of Mg-Al LDH-Eu to
254
recognize Tc was examined by introducing Tc to the Mg-Al LDH-Eu aqueous solution. As is
255
described in Figure 6, in the presence of various concentrations of Tc (0-7 µM), the Mg-Al
256
LDH-Eu solution exhibited narrow emission bands that were assigned to the deactivation of
257
the Eu (III) excited states (5D0-7F0, 5D0-7F1, 5D0-7F2, 5D0-7F3 and 5D0-7F4) under 405 nm
258
excitation (excitation spectrum was shown in Figure S6). In particular, the fluorescence
259
changes could be perceived clearly by naked-eye under the UV-light excitation at 365 nm
260
(Figure 6, inset photo). The correlation between the emission ratio intensity F/F0 (F0
261
represents the fluorescence intensity of Mg-Al LDH-Eu at 618 nm without Tc, F represents
262
the fluorescence intensity of Mg-Al LDH-Eu at 618 nm at various concentrations of Tc) and
263
the concentration of Tc followed the linear equation Y = 11.8 X + 4.05 (R2 = 0.999) and the
264
calibration curve was achieved (Figure 6, inset). The detection of limit (DL) was calculated to
265
be 7.6 nM through the equation DL = 3 × SD/slope. SD refers to the standard deviation of the 12
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blank solutions and the value of denominator is corresponding to the slope of the calibration
267
curve.
268
As for clarification of detection process, it is closely related to electronic configurations of
269
lanthanide ions. These elements possess f-f forbidden transitions and the extinction
270
coefficients are quite small. The nature of lanthanide luminescence is completely different
271
from molecular signal of organic compounds. Direct excitation of lanthanide ion will be very
272
inefficient and generally a sensitizing chromophore would be incorporated to realize the
273
antenna effect via intramolecular energy transfer.51 In this contribution, the organic ligand
274
N-(trimethoxysilylpropyl) ethylenediamine triacetic acid was selected for the coordination
275
with europium ions. However, such EDTA type ligand generated very weak absorbance in
276
ultra-violet region and no effective lanthanide emission was monitored based on Mg-Al
277
LDH-Eu (Fig. 6, starting line). Tetracycline owns several proton-donating groups and
278
carbonyl units which would show strong affinity to the binding reaction with europium ions.
279
Moreover, it effectively harvested ultra-violet energy and the peak wavelength was located at
280
360 nm (Fig. S7). Because of complexation with europium ions, tetracycline would form
281
stable chelates which demonstrated broad-band excitation (Fig. S6) and the sharp emission
282
peaks derived from 5D0-7FJ transitions.
283
The interference study of the probe is a highly significant factor for the evaluation of
284
fluorescence sensor performance. To evaluate the selectivity of the developed nanomaterial, a
285
series of interferential amino acids (GSH, Cys, Hcy, Ala, Arg, Asn, Gln, Asp, Gly, Leu, Ser,
286
Trp, His, Lys), common cations ions (Cu2+, Fe2+, Fe3+, Zn2+, Cd2+, Hg2+) and anions (ClO-,
287
NO2-, NO3-, CO32-, HCO3-, AcO-, PO43-, HPO42-, MnO42-, Cr2O72-, SO42-, SO32-, HS-, F-, Cl-) 13
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were examined. Within expectation, no specific optical responses were achieved in the
289
presence of various species (Figure S8). Additionally, several analogues including
290
chloramphenicol, oxytetracycline, gentamycin, amoxicillin, hemoglobin, human albumin,
291
sparfloxacin, marbofloxacin and glucose were measured and no specific “turned-on” effects
292
were observed (Fig. S9). Sarafloxacin could lead to a slight change at a concentration of 20
293
μM. But its improvement ratio was much less than that of tetracycline. These results revealed
294
that nanoprobe owned excellent selectivity towards Tc.
295
In recent years, the new system concerning the detection of tetracycline has also been
296
developed.52 In this work, the layered double hydroxides have been employed in terms of
297
their special layered frameworks, large surface area and extended internal structures. These
298
excellent adsorption capabilities would be very favorable for improving the resultant
299
sensitivity and detection potentials. In addition, LDHs were derived from very cheap
300
precursors (magnesium and aluminum salts) and regarded as cost-effective. Moreover, LDHs
301
possessed very low toxicity and in this study, it has been found in the following study that
302
Mg-Al LDH-Eu gave rise to live cell-staining features in the presence of tetracycline. As for
303
the choice of lanthanides, a few literatures described the preparation of rare earth doped
304
upconversion nanoparticles and their sensing abilities were extensively evaluated.53,54
305
Different from the published work, our study was focused on the down-conversion mode of
306
lanthanide luminescence. Furthermore, the emissive lanthanide ion (Eu3+) was not located
307
within the inorganic crystal lattice such as NaYF4 and we established coordination structure in
308
this hybrid system. It is accepted that lanthanide luminescence would be affected by high
309
frequency vibrations of hydroxyl groups.51 Here we used the powerful organic ligand 14
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(ethylenediamine triacetic acid units) with multiple carboxyl groups and nitrogen atoms to
311
firmly bind with europium ions and the negative influence of water molecules would be
312
suppressed. More importantly, the low thermal stability of lanthanide complex structure could
313
be reinforced (Fig. 5) by the incorporation of it into solid matrice such as LDH and the
314
inorganic-organic hybrid material was achieved.
315
As for the stability of original material (Mg-Al LDH-Eu), we measured its XRD curve
316
when it was synthesized initially in the middle of last December, 2018. In the middle of
317
January, 2019, its XRD pattern was measured for the second time. Recently, we prepared the
318
material again and it was also explored by XRD analysis. The collected results exhibited that
319
the crystalline structures maintained relatively stable after almost two months (Fig. S10).
320
During the photoluminescence studies in the presence of Tc on different days, the
321
enhancement ratio of peak intensity at 618 nm was almost the same (Fig. S11). It revealed
322
Mg-Al LDH-Eu possessed enough stability for the monitoring of tetracycline.
323
In order to investigate the application of nanoprobe, it was employed to determine Tc in
324
milk samples. Different amounts of Tc were spiked into the samples, and the measured
325
concentrations were determined in real environments. The obtained results were provided in
326
Table S1. The recovery of the spiked sample was ranged from 97.0% to 100.8% with relative
327
standard deviation (RSD) less than 1.08%. For the sake of elucidating the reliability of the
328
proposed method, we have employed high performance liquid chromatography (HPLC)
329
analysis for the comparison purpose. Three milk samples spiked with different concentrations
330
of tetracycline were measured by HPLC. Based on Table S2, it can be found that the
331
recoveries varied from 95 to 99.5 % and the results were in agreement with the 15
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above-mentioned technique. It indicated that fluorescent method in this contribution would be
333
reproducible and accurate for the detection of tetracycline and such nanoprobe can
334
quantitatively detect Tc in practical samples with good performance.
335
With the aim of expanding its applicability, a fat-containing concentrated milk sample
336
(Nestle, Carnation Evaporated Milk) was selected. It has been well-documented that the
337
special property of trivalent lanthanide ions with long excited states would be valuable in
338
practical fields. The employment of time-resolved spectra will effectively decrease the
339
background signals and auto-fluorescence effects. The red emission was turned on in the
340
presence of different concentrations of Tc in a well-resolved manner and the spectra were
341
recorded over a period of 0.2 ms by using of long lifetime of lanthanide elements (Fig. S12).
342
Based on the reported literatures concerning several different lanthanide appended
343
nanostructures, generally they had negligible toxicity during in vitro studies.55,56 To explore
344
the cytotoxicity of Mg-Al LDH-Eu in live cells, methyl thiazolyl tetrazolium (MTT) assay
345
was used (Fig. S13). Following incubation with Mg-Al LDH-Eu (0.2 mg/mL) for 24 hours,
346
the cellular viabilities of 293 T cells were well above 90 %, indicating that the achieved
347
material possessed very low cytotoxicity. After the cells were incubated with Mg-Al LDH-Eu
348
for 2 h, confocal microscopic images exhibited no emissions and such phenomenon was
349
consistent with the photoluminescence results (Fig. 6). Interestingly, the red luminescence
350
was switched on upon exposure of cells to 2 μM of Tc and the red signal was improved in the
351
presence of 7 μM of Tc, suggesting that tetracycline can be monitored inside living cells (Fig.
352
7).
353
The detection of Tc would be very important and beneficial for many applications. Herein, 16
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we have designed and developed a novel lanthanide functionalized layered double hydroxide
355
(LDH) fluorescent nanoprobe for light-up and detection of Tc. The probe could quantitatively
356
determine the concentrations of Tc with excellent selectivity, high sensitivity, as well as low
357
limit of detection (7.6 nM). The nanoprobe has been successfully applied to the determination
358
of Tc in milk sample and also in 293 T cells. This research will enable the utilization of
359
unique luminescence properties of lanthanides in the development of new intelligent optical
360
sensors.
361 362
ASSOCIATED CONTENT
363
Supporting information available
364
Size distribution, SEM and TEM images of Mg-Al LDH and Mg-Al LDH-Eu; XRD patterns
365
and EDS survey at different conditions; Excitation spectrum of nanoprobe and UV-visible
366
spectrum of Tc; Emission responses in the presence of various interfering species; Stability
367
experiments; Time-gated spectra of Mg-Al LDH-Eu in fat-containing milk sample upon the
368
addition of various concentrations of Tc; Viability of 293T cells were treated with various
369
concentrations of Mg-Al LDH-Eu; Recoveries of Tc in milk samples detected by the
370
proposed approach and HPLC method.
371 372
Funding
373
J. W. thanks grants from National Natural Science Foundation of China (NSFC)-Guangdong
374
Joint funding support (No. U1801256) and Innovation team project by the Department of
375
Education of Guangdong Province (2016KCXTD009). Z. Zhou is grateful to the Scientific 17
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Research Fund of Henan Provincial Education Department (17A150016) and Natural Science
377
Foundation of Henan (162300410200).
378 379
REFERENCES
380
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538
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Trans. 2016, 45, 7435-7442.
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Figure captions
553
Scheme 1 Schematic illustration of preparation and sensing processes of Mg-Al LDH-Eu for
554
Tc (Step I: Co-precipitation reaction among Mg(NO3)2·6H2O, Al(NO3)3·9H2O and NaOH;
555
Step II: Hydrothermal treatment for 24 h and Mg-Al LDH was afforded; Step III: Silanization
556
reaction with N-(trimethoxysilylpropyl) ethylenediamine triacetic (EDTA) acid sodium; Step
557
IV: Encapsulation of europium ions and Mg-Al LDH-Eu was obtained; Step V: An “off-on”
558
recognition process in the presence of tetracycline ).
559
Figure 1 TEM image of Mg-Al LDH (A), Lattice fringe and TEM image of Mg-Al LDH (B),
560
SEM images of as-prepared Mg-Al LDH (C and D).
561
Figure 2 EDS survey of Mg-Al LDH (A) and Mg-Al LDH-Eu (B).
562
Figure 3 FT-IR spectra of Mg-Al LDH (a), Mg-Al LDH-COOH (b) and Mg-Al LDH-Eu (c).
563
Figure 4 XRD patterns of Mg-Al LDH, Mg-Al LDH-COOH and Mg-Al LDH-Eu.
564
Figure 5 TG curves of the prepared Mg-Al LDH-Eu and EDTA-Eu complex.
565
Figure 6 Fluorescence titration spectra of Mg-Al LDH-Eu aqueous solution (0.1 mg/mL)
566
upon addition of different concentrations of Tc (0-7 µM). (Inset: (left) photographs of Mg-Al
567
LDH-Eu (0.1 mg/mL) alone and in the presence of 7 µM Tc exposed to a UV lamp at 365 nm,
568
(right) linear relationship between the fluorescence intensity ratio (F/F0) and the
569
concentrations of Tc (0.1-5 µM), (λex = 405 nm; λem = 618 nm)).
570
Figure 7 Confocal microscopy images of Mg-Al LDH-Eu (0.1 mg/mL) in 293T cells. The
571
cells were incubated with only Mg-Al LDH-Eu (0.1 mg/mL) for 2 h as control (A). The cells
572
were further incubated with 2 µM (B), or 7 µM (C)Tc for 1 h.
573 26
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574 575 576 577
578 579
Scheme 1
580 581 582 583
27
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584 585
Figure 1
586 587
588 589
Figure 2
590 591 592 593 594 28
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595
a
-
1350 (NO3 )
Transmittance
b
3405 (O-H) 2963, 2913, 2847 (-CH2-) 1034 (Si-O)
-
1680 (COO , asymmetric)
c
-
1580 (COO , symmetric)
-
1665 (COO , asymmetric) -
1535 (COO , symmetric)
4000
3500
3000
2000
Wavenumber cm
596 597
2500
1500
1000
-1
Figure 3
598 599
Intensity / a.u.
Mg-Al LDH-Eu
Mg-Al LDH-COOH
(003)
(006)
Mg-Al LDH (009)
10
30
40
50
60
70
2 / degree
600 601
20
(110)
Figure 4
602 603
29
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604 605
1.0
EDTA-Eu complex Mg-Al LDH-Eu
Mass / %
0.8 0.6 0.4 0.2 0.0
400
600
800
Temperature / C
606 607
200
Figure 5
608 609
1000 Y = 11.8 X + 4.05 2 R = 0.999
800
40 20
600 400
0
7 M
0
1
2
3
4
5
Tc concentrations / M
Tc 0 M
200 0 550
600
650
700
Wavelength / nm
610 611
F/F0
Relative intensity / a.u.
60
Figure 6
612 30
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613 614 615 616 617
618 619
Figure 7
620 621 622 623 624 625 626 627 628 629 630 31
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Table of Contents Graphic
634
635 636 637
A novel europium embedded layered double hydroxide (Mg-Al LDH-Eu) offers an alternative
638
way to newly-developed analytical approaches. It displays rapid and highly selective
639
detection to tetracyclines (Tc) in water and milk. This nanoplatform exhibits low cytotoxicity
640
during in vitro experiments and can be employed for the detection of tetracycline in 293T
641
cells.
642 643 644
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