Tetracycline generated red luminescence based on a novel

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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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Journal of Agricultural and Food Chemistry

1

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

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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

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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

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* Corresponding Author

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Tel.: 86-20-39310258, Fax: 86-20-39310187. E-mail: [email protected]; 1

ACS Paragon Plus Environment


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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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46

INTRODUCTION

47

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,

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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

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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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112

10℃/min (Bruker, Karlsruhe, Germany). All error bars represent standard deviations from

113

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

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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.

125

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

132

acetone. Then the samples were vacuum-dried at 65℃ for 7 hours.43,44

133

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-,

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NO2-, NO3-, CO32-, HCO3-, Ac-, PO43-, HPO42-, MnO42-, Cr2O72-, SO42-, SO32-, HS-, F-, Cl-),

147

respectively.

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Detection of Tc in real samples. For Tc detection in real sample, milk samples were

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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

157

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.

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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

176

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

179

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



200

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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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222

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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244

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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266

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



Page 14 of 33

288

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


Page 15 of 33


310

(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



Page 16 of 33

332

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


Page 17 of 33


354

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



Page 18 of 33

376

Research Fund of Henan Provincial Education Department (17A150016) and Natural Science

377

Foundation of Henan (162300410200).

378 379

REFERENCES

380

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2017, 81, 156-163.

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(53) Liu, Y.; Ouyang, Q.; Li, H.H.; Chen, M.; Zhang, Z.Z.; Chen, Q.S. A turn-on

532

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535

(54) Hu, W.W.; Chen, Q.S.; Li, H.H.; Ouyang, Q.; Zhao, J.W. Fabricating a novel label-free

536

aptasensor for acetamiprid by fluorescence resonance energy transfer between NH2-NaYF4:

537

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538

(55) Chauvin, A.S.; Comby, S.; Song, B.; Vandevyver, C.D.B.; Thomas, F.; Bunzli, J.C.G. A

539

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540

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541

(56) Zhou, Z.; Wang,Q.M.; Zhang, C.C.; Gao, J.W. Molecular imaging of biothiols and in

542

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543

Trans. 2016, 45, 7435-7442.

544 545 546 547 548 549 550 551 25



Page 26 of 33

552

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


Page 27 of 33


574 575 576 577

578 579

Scheme 1

580 581 582 583

27



Page 28 of 33

584 585

Figure 1

586 587

588 589

Figure 2

590 591 592 593 594 28


Page 29 of 33


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



Page 30 of 33

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


Page 31 of 33


613 614 615 616 617

618 619

Figure 7

620 621 622 623 624 625 626 627 628 629 630 31



Page 32 of 33

631 632 633

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

32


Page 33 of 33


645

33


Tetracycline generated red luminescence based on a novel

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