Highly Sensitive and Selective Determination of Tertiary

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Highly sensitive and selective determination of tertiary butylhydroquinone in edible oils by competitive reaction induced “on-off-on” fluorescent switch Xiaoyue Yue, Wenxin Zhu, Shuyue Ma, Shaoxuan Yu, Yuhuan Zhang, Jing Wang, Yanru Wang, Daohong Zhang, and Jianlong Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05340 • Publication Date (Web): 08 Jan 2016 Downloaded from http://pubs.acs.org on January 11, 2016

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Journal of Agricultural and Food Chemistry

Highly sensitive and selective determination of tertiary butylhydroquinone in edible oils by competitive reaction induced “on-off-on” fluorescent switch Xiaoyue Yue, Wenxin Zhu, Shuyue Ma, Shaoxuan Yu, Yuhuan Zhang, Jing Wang, Yanru Wang, Daohong Zhang, Jianlong Wang*

College of Food Science and Engineering, Northwest A&F University, Yangling 712100, Shaanxi, China

Corresponding Author *Tel.: +86 29-8709-2275; Fax: +86 29-8709-2275. E-mail address: [email protected] (JL, Wang)

1

ACS Paragon Plus Environment


1

ABSTRACT As one of most common synthetic phenolic antioxidants, tertiary

2

butylhydroquinone (TBHQ) has received increasing attention due to the potential risk

3

of TBHQ for liver damage and carcinogenesis. Herein, a simple and rapid fluorescent

4

switchable methodology was developed for high selective and sensitive determination

5

of TBHQ by utilizing the competitive interaction between the photo-induced electron

6

transfer (PET) effect of carbon dots (CDs) / Fe(Ⅲ) ions and the complexation

7

reaction of TBHQ / Fe(Ⅲ) ions. This novel fluorescent switchable sensing platform

8

allows determining TBHQ in a wider range from 0.5 to 80 µg mL-1 with a low

9

detection limit of 0.01 µg mL-1. Furthermore, high specificity and good accuracy with

10

recoveries ranging from 94.29 to 105.82% in spiked edible oil samples are obtained

11

with the present method, confirming its applicability for the trace detection of TBHQ

12

in complex food matrix. Thus, the present method provides a novel and effective

13

fluorescent approach for rapid and specific screening of TBHQ in common products,

14

which is beneficial for monitoring and reducing the risk of TBHQ overuse during

15

food storage.

16

Keywords: TBHQ; Fluorescence detection; Carbon dots; PET effect; Complexation

17 18

2


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19 20

INTRODUCTION

21

Tertiary butylhydroquinone (TBHQ) is one of most common synthetic phenolic

22

antioxidants, which can prevent edible oil and lipid food putrefaction and

23

deterioration.1 Due to their excellent antioxidant property, chemical stability, low cost

24

and availability, TBHQ is very popular with food manufacturers. Although TBHQ has

25

better antioxidant property and has been permitted to be used in foods up to a

26

maximum limit of 200 mg kg-1 in some countries such as China, the United States,

27

Australia, Brazil, New Zealand and Philippines, it is banned in the European Union

28

and Japan due to their potential risks such as liver damage and carcinogenesis.

29

to ensure human health and food safety in the food industry, various analytical

30

methods have been proposed to assay TBHQ in food samples, such as

31

high-performance liquid chromatography (HPLC),5 gas chromatography–mass

32

spectrometry (GC-MS),6 and capillary electrophoresis.7 These traditional analytical

33

methods are highly accurate and widely used for TBHQ determination, however,

34

they suffer from more or less inherent drawbacks such as laborious procedures,

35

time-consuming processing and expensive instruments, which limit their practical

36

applications. Therefore, it is indispensable to develop a simple, rapid and inexpensive

37

method for the detection of TBHQ.

2-4

So,

38

Possessing the advantages of simplicity, lower-cost, high sensitivity and less

39

cell-damaging, fluorescence analytical techniques have been widely used in detection 3



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of various substances.8-11 Fluorescence carbon dots (CDs), as newly emerging

41

carbon-based nanomaterials are superior in many respects including good aqueous

42

solubility, stable photo-luminescence (PL), low toxicity, resistance to photo bleaching,

43

high fluorescent activity and excellent biocompatibility,

44

widely used in fluorescent analysis. Recently, several CDs based switchable

45

fluorescence sensors have been reported for detection of L-cysteine,

46

receptor,15 glutathione,

47

fluorescence switchable sensors can be constructed using various

48

chemical and biological functions, which mainly focus on biomolecule in

49

biological system. For food systems, fluorescent sensors have been also gradually

50

developed for rapid detection of food additives and harmful substances. For example,

51

Chen’s team constructed a fluorescent sensor for rapidly sensing of foodborne

52

pathogens based on upconversion nanoparticles,

53

carbon dots-based fluorescent sensor for selective detection of tartrazine in food

54

samples.10 Wang’s team have successfully developed optosensing platform for

55

detecting tocopherol in rice20 and Wu established fluorescent sensor for the

56

simultaneous determination of Mycotoxins21 .However, to the best of our knowledge,

57

TBHQ fluorescence sensors applied in food system has never been reported.

16

ascorbic acid,

17

biothiol

19

18

12-14

which make carbon dots

12

folate

and other molecules. These

and Liao’s team

physical,

developed a

58

According to previous reports, the fluorescence of carbon dots can be quenched

59

by Fe(Ⅲ) irons via PET effect17, 22. Meanwhile, it is well known that complexation

60

reaction will probably happen when phenolic hydroxyl exists with Fe(Ⅲ) irons. 4


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Considering that there are two phenolic hydroxyl groups in TBHQ molecule, we

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conclude that it is possible to construct an “on-off-on” fluorescent sensor based on a

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competitive reaction occurring between PET effect and complexation reaction. What's

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more, to the best of our knowledge, there is hardly any available report on the

65

quantification of TBHQ using CDs-based fluorescent sensor.

66

Inspired by the above investigations, this work aimed to construct a novel, rapid

67

and inexpensive switchable fluorescent sensing platform for the rapid detection of

68

TBHQ in edible oil samples based on the competitive interaction between the PET

69

effect and the complexation reaction. Combining the advantages of fluorescent

70

nanomaterial with competitive reaction, this simple, rapid and low-cost analytical

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method offers a very effective fluorescent probe for sensitive and selective detection

72

of TBHQ with a wide linear range, low detection limit and satisfactory recoveries.

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Besides, we further demonstrate the feasibility through employing the developed

74

assay strategy for the detection of TBHQ spiked in edible oil samples and verify

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herein the reliability of the designed sensor by comparing the testing results of

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fluorescent sensor with that of HPLC (high performance liquid chromatograph).

77

MATERIALS AND METHODS

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Reagents. TBHQ, BHA (butylated hydroxyanisole) and BHT (butylated

79

hydroxytoluene) were purchased from Sigma-Aldrich (Sigma-Aldrich, Shanghai,

80

China). Citric acids (CA), ethylenediamine (EDA) and metal salts (AgNO3, CaCl2,

81

MnCl2·4H2O,

CdCl2,

BaCl2,

Cu(NO3)2·5H2O,

CoCl2·6H2O,

5


NiCl2·6H2O,


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Hg(ClO4)·3H2O, CrCl3·3H2O and AlCl3) were obtained from Sinopharm Chemical

83

Reagent Co. Ltd

84

isopropanol used in HPLC were of chromatographic grade and other chemicals were

85

of analytical reagent grade. Besides, the water used in the process of TBHQ detection

86

by HPLC was Wahaha purified water purchased from local market. Doubly distilled

87

water was used throughout other experiments.

(Shanghai, China). Acetic acid, methanol, acetonitrile and

88

Apparatus. Fluorescence spectra were performed on a PE LS-55 spectrometer

89

with a slit width of 5 nm for both excitation and emission to obtain suitable

90

fluorescent

intensity.

91

JEM-3010

transmission electron microscope (TEM). UV / Vis absorption

92

measurements were performed on a shimadzu UV-2550 spectrophotometer (Japan).

93

Fourier transform infrared (FT-IR) spectroscopy was obtained from a Vetex70 FT-IR

94

spectroscope (Bruker Corp., Germany). X-ray photo-electron spectroscopy (XPS)

95

analysis was obtained on a Thermo ESCALAB 250 spectrometer. HPLC

96

measurements were performed

97

liquid chromatograph with an UV/ Vis detector.

Morphology

on

characterizations

were

obtained

with

a

Shimadzu LC-10AVPPLUS high performance

98

Preparation of Fluorescence CDs. CDs were synthesized by microwave

99

assisted pyrolysis method according to previous report with some modifications. 23

100

Briefly, 0.2 g citric acid was dissolved in 5 mL water and 1 mL ethylenediamine was

101

added by the dropwise under vigorous magnetic stirring. After that, the mixture was

102

pyrolyzed in a microwave oven over medium heat for 2 min and high heat for another 6


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2 min to form a brown product. Finally, anhydrous ethanol was added and the sample

104

was sonicated until muddy dispersion was formed. Attributed to the poorer solubility

105

of CDs in ethanol than that of CA and EDA, the purified CDs can be separated after

106

several cycles of centrifugation (10000rpm, 5min) and wash with anhydrous ethanol.

107

Finally, the purified CDs was dried at 60 °C, dissolved with a small amount of water

108

sample and then freezing dried overnight at -50 °C to obtain dried powder.

109

Real samples preparation and HPLC detection. Samples preparation and

110

HPLC detection were based on the standard method GB/T 21512-2008 of China.

111

Briefly, edible oil sample (2 g) was weighed accurately and added to a 40 mL

112

centrifuge tube and 6 mL 95% ethanol was added to the centrifuge tube. Then, the

113

mixture was vibrated for about 1 min and layered under 90 °C water bath. The

114

supernatant was transferred into another centrifuge tube and

115

re-extracted twice. The supernatant was concentrated by vacuum rotatory evaporator

116

and then diluted with acetonitrile and iso-propanol to 10 mL. Finally, the above

117

solution was filtered before for HPLC determination. Chromatographic conditions：

118

flow rate: 0.8 ml/min; Injection Volume: 20 µL; column temperature: room

119

temperature 30 °C; ultraviolet detector wavelength: 280 nm, organic phase:

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methanol-acetonitrile (1:1); aqueous phase: 1.8% acetic acid solution.

the residues was

121

Fluorescence Assay of TBHQ. For fluorescence assay, the standard or real

122

(extracts of edible oils) TBHQ solutions was prepared with anhydrous ethanol. 1 mL

123

of water and 1mL anhydrous ethanol was added with 1 mL of the CD dispersion (0.5 7



124

µg mL-1) at room temperature in 4mL fluorescent cuvette as blank control group. For

125

TBHQ determination, 1 mL of ferric chloride hexahydrate (1mM) was added into 1

126

mL of the CD dispersion ( 0.5 µg mL-1) at room temperature and mixed with 1 mL

127

standard TBHQ solutions with different concentrations or sample extract. After

128

incubation for 10 min, the fluorescence intensity of the solution was measured with an

129

excitation at 360 nm. The fluorescence of CDs could be quenched in the presence of

130

Fe(Ⅲ) due to the photo-induced electron transfer effect occurring between Fe(Ⅲ)

131

ions and CDs. Subsequently, the fluorescence of the CDs–Fe(Ⅲ) ions system was

132

recovered gradually with the addition of TBHQ due to their strong complexation

133

reaction with Fe(Ⅲ) ions and the quantitive detection of TBHQ was achieved

134

according to the degree of fluorescence restoration of CDs. All experiments were

135

conducted at room temperature and repeated at least three times.

136

RESULTS AND DISCUSSION

137

The detection principle of the switchable fluorescent sensor. The principle of

138

this fluorescence sensor is shown in Scheme 1. Emission of a photon, fluorescence,

139

follows HOMO (Highest Occupied Molecular Orbital) to LUMO (Lowest

140

Unoccupied Molecular Orbital) excitation of an electron in a molecule. If this

141

emission is efficient, the molecule may be termed a fluorophore.24 Various other

142

interactions may also affect the emission process, which are significant in regard to

143

analytical applications of fluorescence. When CDs serving as a fluorophore in this

144

work was excited, the electrons on the surface of CDs leap into the excited state from 8


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the ground state. Due to the instability of excited electronic state, these electrons will

146

return to the ground state, accompanied by efficient energy transfer, giving rise to the

147

generation of fluorescence. After the introduction of Fe(Ⅲ)

148

excited state enters the unfilled orbit of Fe(Ⅲ) ions. Such PET provides a mechanism

149

for nonradiative deactivation of the excited state, leading to a decrease in emission

150

intensity or “quenching” of the fluorescence. 25, 26 Subsequently, with the introduction

151

of TBHQ, TBHQ will compete with CDs for Fe(Ⅲ) ions, which will lead to that the

152

fluorescence of the CDs–Fe(Ⅲ )

153

introduction of TBHQ due to their strong complexation reaction with Fe(Ⅲ) ions.

154

According to the above principles, we infer that it is probable to design an

155

“on-off-on” fluorescent sensing platform based on a competitive reaction occurring

156

between PET effect and complexation reaction.

ions, an electron in the

ions system is recovered gradually with the

157

＜Scheme 1＞＞

158

Characterization of Morphology and optical properties of CDs. In general,

159

the optical properties are largely dependent on the size, shape and surface state of

160

nanomaterials.27 TEM was applied to characterize the morphology of the as-prepared

161

CDs by one-step microwave-assisted pyrolysis. As shown in Figure. 1A, CDs

162

distributed evenly and well separated from each other. The corresponding nanoparticle

163

size distribution histogram obtained by counting 100 particles CDs reveals that CDs

164

have a narrow size ranging from 2 to 5 nm, as displayed in Figure 1B and Figure 1C.

165

According to previous reports, these CDs of this size probably have excellent optical 9



166

characteristics and excellent biocompatibility and will be greatly applicable in

167

fluorescence analysis.28-30

168

Furthermore, we investigated the remarkable optical properties of the as-obtained

169

CDs by the UV-vis absorption and the steady-state fluorescent spectra. From Figure

170

1D, it is seen that the maximum fluorescent excitation wavelength and emission

171

wavelength of the CDs were 360 and 455 nm, respectively. Additionally, the UV/Vis

172

spectrum shows that the CDs exhibit a significant UV/Vis absorption band centered at

173

approximately 360 nm (Figure 1D), which is corresponding to the optimum

174

fluorescence excitation peak and should be attributed to the n−π* transition of C=O or

175

C–OH on the surface of the CDs. Another UV/Vis absorption band is centered at

176

approximately 230 nm, which is a typical characteristic of CDs, arising from the π–π*

177

transition of aromatic C=C bonds,27, 31 further indicating the successful synthesis of

178

CDs. FT-IR spectrum was acquired to determine the surface state of CDs. As shown

179

in Figure 1E, the peaks at 3421 cm-1 and 2925 cm-1 are assigned to stretching

180

vibrations of the C−OH and C−H, respectively. The peaks at 1654 and 1560 cm-1 can

181

be attributed to the stretching vibration of C=O and N−H, respectively. Bands at 1294

182

cm-1 are attributed to the stretching vibration of C−O. The above results indicate that

183

the as-prepared CDs are surrounded by –COOH and –OH groups. These hydrophilic

184

surface groups enable the as-prepared CDs to exhibit good water-solubility and can

185

improve the stability and hydrophilicity of the CDs in an aqueous system.13

186

＜Figure 1＞＞ 10


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XPS was used to identify the surface chemical composition and elemental

188

distribution of CDs. As shown in Figure 2, XPS spectra revealed that these

189

nanoparticles are comprised of three main elements carbon, oxygen and nitrogen

190

and a limited mount of Si element which may come from the silicon substrate

191

during XPS test. The high-resolution spectra of C 1s (Figure 2B) displayed three

192

intensive peaks at around 284.5 eV, 285.6 eV, 288.0 eV, which are attributed to

193

C−C, C−N, and C=O, respectively.

194

O 1s spectrum (Figure 2C) should be assigned to C–O and C–OH/C–O–C groups,

195

respectively. In the high-resolution N1s spectrum (Figure 2D), two peaks appeared

196

at 399.4 and 400.3 eV can be ascribed to C–N–C and N–(C)3 groups,

197

respectively.13 The above observations imply that citric acid was oxidized and

198

carbonized during the process of microwave pyrolysis, resulting in the successful

199

synthesis of CDs with lots of hydrophilic groups including hydroxyl, carbonyl and

200

carboxyl group. Importantly, the presence of those hydrophilic groups is not only

201

in favour of achieving higher water solubility but also benefit to facilitate electron

202

transfer between CDs and Fe(Ⅲ) ions.

30

Two peaks at 531.0 eV and 532.2 eV in the

203

＜Figure 2＞＞

204

Optimization of analytical conditions and mechanism analysis. It has been

205

reported that the absorption of the excitation or emission light by absorbers may

206

reduce the fluorescence intensity of the fluorophor.33 Aiming to further explore the

207

optical properties of the as-prepared CDs and optimize analytical condition, we 11



208

investigated the excitation-dependent photoluminescence behavior. As shown in

209

Figure 3A, the highest fluorescent intensity appeared at 360 nm, which was selected

210

as the excitation wavelength for the following experiments. On the other hand, when

211

CDs were excited at different excitation wavelength the photoluminescence peak of

212

the resultant CDs remained almost unchanged, which is of benefit to reducing the

213

effect of auto fluorescence during applications.34 Such excitation-dependent

214

photoluminescence behavior may be related to the different surface states of the

215

CDs.23,31, 34

216

To understand the mechanism of the interaction between the CDs / Fe(Ⅲ)

217

complex and TBHQ, the time-dependent fluorescence responses of the CDs

218

dispersion upon addition of 1mM Fe(Ⅲ) ions and time-dependent fluorescence

219

recovery of CDs in the presence of Fe(Ⅲ) ions upon addition of TBHQ were also

220

measured. As displayed in Figure 3B, When only Fe(Ⅲ) ions were added to the

221

reaction system(1mL CDs and 1mL anhydrous ethanol), the fluorescence intensity

222

instantly decreased at the beginning of incubation, which should be ascribed to the

223

rapid PET effect happened between CDs and Fe(Ⅲ) ions. In details, when a lone

224

electron pair is located in an orbital of the fluorophore itself or an adjacent molecule

225

and the energy of this orbital lies between those of the HOMO and LUMO, efficient

226

electron transfer of one electron of the pair to the hole in the HOMO created by light

227

absorption may occur, followed by transfer of the initially excited electron to the lone

228

pair orbital. Such PET provides a mechanism for nonradiative deactivation of the 12


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24

229

excited state, leading to a decrease in emission intensity.

And then the fluorescence

230

intensity gradually leveled off because the process of electron transfer has been

231

accomplished. In contrast, when 1 mL TBHQ (100 µg mL-1) was added to the CDs /

232

Fe(III) reaction system, the fluorescence responses of the CDs increased dramatically

233

at the initial stage because TBHQ can react with Fe(Ⅲ)

234

from the surface of the CDs, making the fluorescence of CDs restore quickly.

235

More specifically, fluorescence quenching as a result of PET may be recovered if it is

236

possible to involve the lone pair in a bonding interaction. In this work, the recovery of

237

fluorescence should be ascribed the coordination reaction can happen between TBHQ

238

and Fe(III) ions. Thus, on one hand, protonation or binding of a metal ion effectively

239

places the electron pair in an orbital of lower energy and inhibits the electron-transfer

240

process and on the other hand, TBHQ compete with CDs for Fe(III) ions, which make

241

a rapid recovery of the fluorescence. Afterwards, the fluorescence intensity

242

gradually leveled off, indicating that the competitive interaction between CDs / Fe(III)

243

system and TBHQ / Fe(III) system reached equilibrium. This experimental

244

phenomenon well vindicated the feasibility of the principle that we used to design the

245

switchable “on-off-on” sensing platform for TBHQ detection.

ions and take them away

246

The pH value of the solution is another key factor affecting the sensing system,

247

because the initial fluorescence intensity (in the absence of Fe (Ⅲ)) and the quenched

248

fluorescence intensity (in the presence of Fe (Ⅲ)) of the CDs are both

249

pH-dependent.33 Therefore, to achieve sensitive detection, the effect of pH values on 13



250

the fluorescence intensity of CDs was also investigated. As shown in Figure 3C, the

251

fluorescence intensity increases with the increase of pH, which could be ascribed to

252

the protonation of the carboxyl groups on the surface of the CDs, thus weakening the

253

electrostatic repulsion between CDs and rendering them unstable. For another aspect

254

of stability, a basic environment will result in the formation of insoluble ferric

255

hydroxide.25 Hence, we selected a weak acidic condition (pH 5.5, 10 mM) for

256

subsequent detection.

257

To further confirm the feasibility of the strategy we proposed, a control

258

experiment was carried out. As exhibited in Figure 3D, CDs displayed high

259

fluorescence intensity at 455 nm and the fluorescence intensity decreased significantly

260

after the addition of Fe(III) to the CDs. As anticipated, upon the addition of TBHQ to

261

the CDs/Fe(III) system, the fluorescence intensity at 455 nm increased remarkably.

262

During the whole detection procedures, carbon dots acts as PET donor, and Fe(Ⅲ)

263

ions serve both as a PET acceptor and a bridge between PET effect and complexation

264

reaction, thus constructing a switchable “on-off-on” sensor. Additionally, there is no

265

change in fluorescence intensity when the TBHQ solution was added to the CDs

266

dispersion alone. It's worth mentioning that no detectable fluorescence was observed

267

in the TBHQ and Fe(III) solution alone at 455 nm. These results verify that Fe(III)

268

can effectively quench the fluorescence of CDs and then the preferential complexation

269

of TBHQ with Fe(III) ions induces the recovery of fluorescence, which is a good

270

match with the principle of this fluorescence sensor, confirming the feasibility of the 14


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271

strategy we proposed.

272

＜Figure 3＞＞

273

Fluorescence detection of TBHQ. It is well known that linear range, detection

274

limit and sensitivity are three important factors to evaluate the analytical performance

275

of sensors. The calibration curve was constructed under the optimal conditions and as

276

seen in Figure 4A, the fluorescent intensity enhanced with the increasing of the

277

concentrations of TBHQ in the range of 0.5 to 80 µg mL-1. When the concentration of

278

TBHQ beyond 80 µg mL-1, the fluorescent intensity keeps unchanged, which may be

279

due to the fact that the binding between CDs and Fe(III) and the complexing between

280

Fe(III) and TBHQ achieve a balance, leading to maximum recovery of the

281

fluorescence produced by CDs. As shown in Figure 4B, F-F0 was in a linear

282

relationship along with concentrations of TBHQ and the linear regression equation is

283

y (F-F0) = 3.118 x + 3.298 with a correlation coefficient of 0.994, where F0 is the

284

fluorescence intensity of CDs in the presence of 1mM ferric ions and F is the

285

recovered fluorescence intensity after the addition of TBHQ. The limit of detection

286

(LOD) was estimated to be

287

3S0/S criterion (based on three times signal-to-noise ratio), where S is the slope of the

288

calibration curve and S0 represents the standard deviation of a blank (n=6). As

289

exhibited in Table 1, the LOD of the proposed method is much lower than that of

290

previously report obtained by conventional instrumental methods with a much wider

0.01 µg mL-1, as calculated according to the

15



291

linear range. Moreover, the detection limit of the developed fluorescent sensor is quite

292

lower than the maxium level for TBHQ in edible oils permitted by most of countries,

293

implying the developed method has a great potential for further applications.

294

＜Figure 4＞＞

295

＜Table 1＞＞

296

Interference effect. Selectivity is another key parameter to evaluate the

297

analytical performance of fluorescent sensor. Fluorescence response of CDs was

298

inspected in the presence of other similar oil-solubility antioxidants including BHA

299

and BHT. As shown in Figure 5, the introduction of BHA and BHT to CDs/Fe (III)

300

system presents negligibly fluorescent recovery of CDs. Besides, we also examined

301

the fluorescence intensity changes in the presence of representative metal ions under

302

the same conditions, including Ag+, Ba2+, Ca2+, Cr2+, Co2+, Pb2+, Cu2+ and Hg2+. From

303

Figure 5A and Figure 5B, although Cu2+ displays weak fluorescence quenching and

304

Hg2+ induces a substantial decrease in the fluorescence intensity of CDs, the

305

fluorescence intensity of CDs can not be restored after the addition of TBHQ, which

306

should be on account of that there is no chemical reaction between TBHQ and both

307

two metal ions. Thus, these potential oil-soluble antioxidants and metal ion

308

interferences showed negligible effects on the signal for the detection of TBHQ,

309

demonstrating the high specificity of the proposed method for TBHQ determination.

310

＜Figure 5＞＞ 16


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311

Practical applications in edible oils of the fluorescence sensor. It is a very key

312

problem whether the constructed sensing platform could be used in real samples and

313

that

314

can be applied in practical application. To confirm the feasibility of this “on-off-on”

315

fluorescent sensor, the edible samples spiked with standard solutions containing

316

different concentrations of TBHQ at five levels (0, 50, 100, 150 and 200 µg g -1) were

317

detected. As summarized in Table 2, for the three samples, the relative standard

318

deviation (RSD, %) of the repeated measurements ranged from 1.29 to 4.13%,

319

suggesting that this developed method has favorable precision. And the recovery

320

values were also good, ranging from 94.29 % to 105.82 %, providing evidence that

321

the results obtained by the fluorescent sensor were relative accurate and reproducible.

322

These results illustrate that the designed method has a great potential for the

323

quantitive detection of TBHQ in complex food matrix. Besides, to verify the

324

reliability of the results detected by fluorescence sensor, traditional HPLC was used to

325

validate the results obtained by the fluorescent sensing platform. From Figure 6, we

326

can observe that the retention time of TBHQ was about 11.8 min and the intrinsic

327

concentration of edible oil sample detected by HPLC is 112.5 µg g-1, which is well

328

consistent with the result gained by the developed fluorescence sensor, indicating the

329

proposed sensor has a good reliability.

will

determine

whether

this

kind

330

＜Figure 6＞＞

331

＜Table 2＞＞ 17


of fluorescence

sensor


Page 18 of 37

332

In summary, we have successfully designed a facile and effective “on-off-on”

333

fluorescence switchable strategy for the rapid detection of TBHQ. This method

334

combines the advantages of CDs fluorescence with the competitive interaction

335

between the PET effect of carbon dots (CDs) / Fe(Ⅲ) ions and the complexation

336

reaction of TBHQ / Fe(Ⅲ) ions, enabling an ultrasensitive and highly selective

337

determination of TBHQ. The developed approach provides a wide linear range from

338

0.5 µg mL-1 to 80 µg mL-1 and a low detection limit (0.01 µg mL-1), which is much

339

better than that of previously report obtained by conventional instrumental methods

340

and much lower than the maxium level for TBHQ in edible oils permitted by most of

341

countries. Furthermore, favorable accuracy, satisfactory stability and reproducibility

342

are achieved for the detection of TBHQ in edible oils samples, indicating a great

343

potential for monitoring and reducing the risk of TBHQ overuse during food storage.

344

Meantime, the present design combines the advantages of fluorescent nanomaterial

345

with competitive reaction, which may be as reference for the fluorescent sensor

346

design of other food additive.

347

ABBREVIATIONS USED

348

TBHQ, tertiary butylhydroquinone; BHA, butylated hydroxyanisole; BHT, butylated

349

hydroxytoluene; PET, photo-induced electron transfer; XPS, X-ray photoelectron

350

spectroscopy;

351

Ultraviolet–visible spectroscopy; HPLC, high performance liquid chromatography;

352

HPLC–DAD, high performance liquid chromatography with diode array detector;

FT-IR,

Fourier

transform

infrared

18


spectroscopy;

UV-Vis,

Page 19 of 37


353

HPLC–FLD, high performance liquid chromatography with fluorescence detector;

354

AuNPs / GCE, gold nanoparticles modified bare glasy carbon electrode; MWCNT /

355

GCE, Multi walled carbon nanotubes modified bare glasy carbon electrode; HMDE,

356

hanging mercury-drop electrode; MWCNT / SPE, Multi walled carbon nanotubes

357

modified screen printed electrode; RSD, relative standard deviation; SD, standard

358

deviation

359

AUTHOR INFORMATION

360

Corresponding Author

361

*Tel.: +86 29-8709-2275; Fax: +86 29-8709-2275.

362

E-mail address: [email protected] (JL, Wang)

363

Funding Sources

364

This research was financed by Grants from the New Century Excellent Talents in

365

University (NCET-13-0483), Open Fund of State Key Laboratory of Electroanalytical

366

Chemistry (SKLEAC201301), the Shaanxi Provincial Research Fund (2014KJXX-42,

367

2014K02-13-03, 2014K13-10), the Yangling district research fund (2014NY-35) and

368

Fundamental Research Funds for the Northwest A&F University of China

369

(2014YB093, 2452015257).

370

Note

371

The authors declare no competing financial interest.

372 19



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501

26


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502

TABLES

503

Table 1 Comparison of the analytical performance of several commonly selected

504

methods for the determination of TBHQ Linear range

LOD

(µg mL-1)

(ng mL-1)

HPLC–DAD

7.99~22.98

180.0

35

HPLC–FLD

0.40~12.80

20.70

36

AuNPs / GCE

0.20~2.80

79.00

37

MWCNT / GCE

0.66~16.6

5.31

38

HMDE

0.17~1.68

5.69

39

MWCNT / SPE

0.166~1.66

81.00

40

Fluorescence detection

0.5~80

10.00

This work

Methods

Reference

HPLC

electroanalytical method

505

27



Page 28 of 37

Table 2 Comparison of performance: analysis of TBHQ in commercial edible oil samples using calibrations modeled with the HPLC and Fluorescence analysis Added spiked

Detected concentration by

Detected by fluorescence

RSD by fluorescence

Recovery by

concentration (µg

HPLC (mean±SD, µg g-1)

detection (mean±SD, µg

detection (%)

fluorescence detection

g-1)

g-1)

(%)

0

118.35±5.90

119.80±3.22

2.69

101.23

50

170.15±1.97

179.90±7.40

4.13

105.82

100

215.95±4.93

222.28±6.30

2.83

102.93

150

270.50±3.68

278.02±3.59

1.29

102.78

200

309.60±2.17

291.92±7.73

2.40

94.29

28


Page 29 of 37


Scheme and Figure Captions: Scheme 1. The sensing principle of the detection process for TBHQ Figure 1. A: Low-resolution TEM of CDs; B and C: high-resolution TEM of the as-synthesized carbon quantum dots (CQDs). D: UV−vis absorption (black line) , PL excitation (blue line) and emission (red line) spectra of the aqueous dispersion of the nanoparticles ; E: FTIR spectra of CDs. The inset of (B) shows the particle size distribution histogram. Figure 2. A: XPS of CD; B–D: High-resolution XPS spectra of (B) C 1s, (C) O 1s and (D)N Figure 3. A: Emission spectra of fluorescence CDs at different excitation; B: Time-dependent fluorescence responses of the CD dispersion upon addition of 1mM Fe(III) ions (red line) and the time-dependent fluorescence recovery of CDs in the presence of Fe(III)

ions (1mM) upon addition of 100 µg mL-1 TBHQ (black line). C:

Emission spectra of fluorescence CDs at different pH; inset: the relationship between pH and fluorescence intensity. D:Fluorescence emission spectra of CDs under different conditions. Figure 4. A: The fluorescence emission spectra of CDs in the presence of Fe (III) ions (1mM) upon addition of TBHQ with different concentrations. B: Plot of the fluorescence recovery factor (F –F0) versus concentrations of TBHQ (Inset is a linear region). Figure 5. A: Fluorescence response of CDs in the presence of different interfering 29



substances (the concentrations of metal ions and antioxidants are all 100 µg mL-1); B:fluorescence recovery factor (F –F0) versus different interfering substances Figure 6. (A)Representative chromatograms of standard sample containing 100 µg mL-1 TBHQ; (B) HPLC of the real edible oil samples. Experimental details are described in the text.

Scheme 1.

30


Page 30 of 37

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Figure 1.

31



Figure 2.

32


Page 32 of 37

Page 33 of 37


Figure 3.

33



Figure 4.

34


Page 34 of 37

Page 35 of 37


Figure 5.

35



Figure 6.

36


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Page 37 of 37


Table of Contents Graphic ( TOC) In this work, we developed a new analytical method for sensitive and selective detection of TBHQ based on a facile and effective “on-off-on” fluorescence switchable strategy.

37


Highly Sensitive and Selective Determination of Tertiary

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