Synthesis and characterization of novel molecularly imprinted polymer – coated Mn-doped ZnS quantum dots for specific fluorescent recognition of cocaine

Author’s Accepted Manuscript Synthesis and characterization of novel molecularly imprinted polymer – coated Mn-doped ZnS quantum dots for specific fluorescent recognition of cocaine María Pilar Chantada-Vázquez, Juan SánchezGonzález, Elena Peña-Vázquez, María Jesús Tabernero, Ana María Bermejo, Pilar Bermejo– Barrera, Antonio Moreda–Piñeiro

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S0956-5663(15)30348-1 http://dx.doi.org/10.1016/j.bios.2015.08.022 BIOS7918

To appear in: Biosensors and Bioelectronic Received date: 18 June 2015 Revised date: 11 August 2015 Accepted date: 12 August 2015 Cite this article as: María Pilar Chantada-Vázquez, Juan Sánchez-González, Elena Peña-Vázquez, María Jesús Tabernero, Ana María Bermejo, Pilar Bermejo–Barrera and Antonio Moreda–Piñeiro, Synthesis and characterization of novel molecularly imprinted polymer – coated Mn-doped ZnS quantum dots for specific fluorescent recognition of cocaine, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2015.08.022 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1

Synthesis and characterization of novel molecularly imprinted polymer – coated

2

Mn-doped ZnS quantum dots for specific fluorescent recognition of cocaine

3

María Pilar Chantada-Vázquez1, Juan Sánchez-González1, Elena Peña-Vázquez1, María Jesús

4

Tabernero2, Ana María Bermejo2, Pilar Bermejo–Barrera1, Antonio Moreda–Piñeiro1*

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(1) Department of Analytical Chemistry, Nutrition and Bromatology. Faculty of Chemistry.

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University of Santiago de Compostela. Avenida das Ciencias, s/n. 15782 – Santiago de

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Compostela. Spain. (2) Department of Pathologic Anatomy and Forensic Sciences. Faculty of

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Medicine. University of Santiago de Compostela. Rúa de San Francisco, s/n. 15782 –

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Santiago de Compostela. Spain.

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Abstract

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Mn-doped ZnS quantum dots (QDs) coated with a molecularly imprinted polymer (MIP)

12

material selective toward cocaine and its metabolites have been prepared and applied to

13

cocaine (COC) and metabolites assessment by spectrofluorimetry. Ultrasound irradiation (37

14

kHz) was novelty used for performing the Mn-doped ZnS QDs synthesis as well as for

15

preparing the QD based MIP-coated composite by precipitation polymerization (imprinting

16

process). This fact allowed the synthesis to be accomplished in four hours. In addition, the

17

use of ultrasound irradiation during MIP-QDs synthesis increased the homogeneity of the

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QDs size, and reduced nanoparticles agglomeration. MIP was synthesized using COC as a

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template molecule, ethylene dimethacrylate (EDMA) as a functional monomer,

20

divinylbenzene (DVB) as a cross-linker, and 2,2´-azobisisobutyronitrile (AIBN) as an

21

initiator. The fluorescence of MIP-coated QDs was quenched by the template (COC) and also

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by metabolites from COC such as benzoylecgonine (BZE), and ecgonine methyl ester

23

(EME). Quenching was not observed when performing experiments with non-imprinted

*

Corresponding author: Telephone number: 00 34 881814375; Fax number: 00 34 981547141; E–mail address: [email protected]

1

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polymer (NIP)-coated QDs; and also, fluorescence quenching of MIP-coated QDs was not

25

observed by other drugs of abuse and metabolites (heroin and cannabis abuse). This fact

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indicates that the prepared material recognize only COC (template) and metabolites.

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Keywords: Mn-doped ZnS quantum dot, molecularly imprinted polymer, cocaine,

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spectrofluorimetry.

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1. Introduction

30

The remarkable unique properties of semiconductive nanocrystal quantum dots (QDs), such

31

as narrow emission spectra (and broad excitation spectra), strong signal intensity, and size

32

tunability (Algar et al., 2011), explain the several application of these nanostructures when

33

developing sensor probes and also biomedical markers. Although early developments, even

34

for biological labeling, were based on CdSe QDs, toxicity of cadmium for biological systems

35

and for the environment (Algar et al., 2011) led to the development of QDs such as ZnSe and

36

ZnS QDs (Pradhan et al., 2005; Suyvere et al., 2001), in which zinc replaces cadmium,

37

avoiding toxicity and environmental problems. Transition metals or rare-earth metal ions

38

have been used for preparing mainly doped ZnS nanocrystals, and Mn- and Cu-doped ZnS

39

are two of the most well studied doped QDs (Ren et al., 2015). These doped nanocrystals

40

were found to offer advantages over undoped CdSe QDs such as lower self-quenching, and

41

greater strength to thermal, chemical and photochemical disturbances (Xiao and Xiao, 2008),

42

and have been commonly proposed as suitable fluorescent and room temperature

43

phosphorescent probes (Bol and Meijerink, 2000).

44

In order to increase the response (photoluminescence-activation and quenching effect) of

45

QDs, chemical or physical interactions between certain chemical species and the surface of

46

the nanoparticles have been used, and several luminescent probes have been proposed for

47

detecting mainly inorganic ions. However, lack of selectivity of QDs based probes was

48

commonly reported, and research on the development of novel and selective QDs based

2

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sensors is a current developing area. A way to improve selectivity of QDs for a certain

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analyte has successfully reached by coating the QD core with a film layer of a molecularly

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imprinted polymer (MIP). The high selectivity of MIPs is attributed to the recognition

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cavities generated after analyte (template molecule) reaction with an adequate monomer,

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polymerization, and template removal stages. These cavities are complementary to the

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template molecule in shape, size and chemical functionality, and selectivity is therefore

55

ensured. Several attempts have been performed for synthesizing MIP-QD composites for

56

which the high sensitivity of luminescent QDs is combined with the high selectivity of MIP.

57

Although the first proposals consisted of surface functionalization with 4-vinylpyridine

58

before MIP synthesis (Lin et al., 2004a; Lin et al., 2004b), most of the surface modification

59

was

60

aminopropyltriethoxysilane (APTES), tetra-ethoxysilane (TEOS) and ammonia, and

61

mercaptopropyltriethoxysilane (MPTS) were mainly used for preparing capped QDs (Wang

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et al., 2009; Tan et al., 2014; Liu et al., 2010; Xu et al., 2012; Chen et al., 2012; Kang et al.

63

2013; Wei et al., 2014; Ren and Chen, 2015). As a result, Mn-doped ZnS QDs were coated

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with a –NH2 surface, which offers binding sites for reacting with the template when

65

performing MIP synthesis. Mn-doped ZnS QDs surface modification can also be performed

66

with oleic acid (Ren et al., 2015), polyethyleneimine (PEI) and TEOS (Dan and Wang, 2013),

67

and L-cysteine (Lei et al., 2012). Finally, developments involving MIP synthesis over un-

68

treated Mn-doped ZnS QDs have also been reported (Zhao et al., 2012).

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Based on these developments, fluorescent probes were mainly developed for sensing proteins

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(Tan et al., 2014; Xu et al., 2012; Kang et al. 2013), organophosphate and pyrethroid

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insecticides (Ren et al., 2015; Ren and Chen, 2015), pesticides (Zhao et al., 2012), and

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tetrabromobisphenol A (Chen et al., 2012). Chemiluminescence sensing was also proposed

73

for assessing 4-nitrophenol (Liu et al., 2010), and developments for determining

via

capped

Mn-doped

ZnS

QDs

synthesis.

Reagents

such

as

3-

3

74

chlorophenols (Wang et al., 2009; Wei et al., 2014), domoic acid (Dan and Wang, 2013),

75

proteins (Kang et al. 2013), and mercury ions (Lei et al., 201) based on room temperature

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phosphorescent probes were also reported. However, the potential of quantum dots coated

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MIPs for sensing drugs of abuse has not yet been shown.

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Although recent data from the United Nations Office on Drugs and Crime (UNODC) state a

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decrease in cocaine use between 2010 and 2011 (United Nations Office on Drugs and Crime,

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2013), cocaine is one of the most widely used illicit substances worldwide. Rapid and low-

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cost methodologies for assessing cocaine abuse are therefore needed as screening and

82

confirmative methods. MIP-QDs probes can allow a rapid and un-expensive screening of

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cocaine abuse, and the aim of the current work has been the synthesis and characterization of

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novel MIP-Mn-dopped ZnS QDs for the selective fluorescent recognition of cocaine.

85

Improvements have been addressed in increasing the water solubility of the prepared material

86

by using ethylene glycol (PEG) for quantum dots surface modification before MIP synthesis.

87

In addition, quantum dots surface modification with PEG as well as MIP synthesis, as

88

proposed by Zhao et al. (Zhao et al., 2012), was assisted by ultrasound irradiation, which

89

allowed a fast preparation procedure. Preliminary studies were then addressed to obtain the

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optimum settings for allowing an efficient analyte [COC, and also metabolites (BZE, and

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EME)] interaction with the fluorescent nanoparticles. In addition, selectivity of the prepared

92

material was fully evaluated.

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2. Materials and methods

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2.1. Instrumentation

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Fluorescence determinations were performed with a Hitachi F-2500 fluorescence

96

spectrometer (Schaumburg, IL, USA) equipped with a xenon lamp and 10 mm quartz cells.

97

Template removal confirmation was performed with a 3200 Q TRAP LC/MS/MS system

98

(ABSciex, Concord, Canada), equipped with a Kinetex 5µ C18 100 Å reverse phase column

4

99

(100 mm length × 2.10 mm i.d., 5.0 µm particle diameter) from Phenomenex (Torrance, CA,

100

USA) connected to a Phenomenex C8 guard column (4 mm length × 3.0 mm i.d), a Flexar

101

FX-15 UHPLC binary chromatographic pump (Perkin Elmer, Waltham, MA, USA), and a

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Flexar UHPLC autosampler (Perkin Elmer). A Raypa Model UCI-150 ultrasonic cleaner bath

103

from R. Espinar S.L. (Barcelona, Spain) programmable for temperature and time, frequency

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of 35 kHz for the ultrasound energy, was used for synthesizing QD-MIP composites. An

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Agimatic-N magnetic stirrer with controllable temperature and speed from Selecta

106

(Barcelona, Spain) was also used for QDs synthesis. QD-MIP characterization was performed

107

by analytical transmission electron microscopy (Libra 200 FE OMEGA, Zeiss, Oberkochem,

108

Germany), energy dispersive X-ray fluorescence spectrometry (Philips PW1710, PANalytical

109

B.V., Almelo, Netherlands) provided with a PW1820/00 goniometer (PANalytical B.V.) and

110

IR spectrometry (Spectrum Two FT-IR, Perkin Elmer). Other laboratory devices were:

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Basic20 pH–meter (Crison, Barcelona, Spain), Reax 2000 mechanical stirrer (Heidolph,

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Kelheim, Germany), vacuum pump (Millipore Co), and VLM EC1 metal block thermostat

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and N2 sample concentrator from VLM (Leopoldshöhe-Greste, Germany).

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2.2. Reagents

115

Ultrapure water 18 MΩcm of resistivity from a Milli-Q purification device (Millipore,

116

Bedford, MA, USA). Drug stock standard solutions were prepared from COC, BZE, and

117

EME (1000 mg L-1) purchased from Cerilliant (Round Rock, TX, USA). Other drug stock

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standard solutions (1000 mg L-1) were codeine, morphine, 6-monoacetylmorphine (6-MAM),

119

Δ9-tetrahydrocannabinol (Δ9-THC), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (Δ9-THC-

120

COOH), 11-hydroxy-Δ9-tetrahydrocannabinol (Δ9-THC-OH), and cannabinol (CBN), also

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from Cellirant. Cannabidiol (CBN), 2000 mg L-1, was prepared by dissolving 10 mg of CBD

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(National Measurement Institute Australian Government, Sidney, Australia) in 5 mL of

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methanol. Mn-doped ZnS QDs were synthesized by using heptahydrate zinc sulfate (Panreac,

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Barcelona, Spain), sodium sulphide (Fluka, Buchs, Switzerland), and manganese dichloride

125

(Merck, Darmstadt, Germany). Polyethylene glycol, PEG 6000, dimethyl sulfoxide (DMSO),

126

2-propanol, toluene, ammonium hydroxide, and silica gel 2, 5-6 mm were from Panreac. MIP

127

was synthesized by using divinylbenzene-80 (DVB) from Sigma-Aldrich (Steinhelm,

128

Germany), and ethylene dimethacrylate (EDMA) and 2,2´-azobisisobutyronitrile (AIBN)

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from Fluka. Acetonitrile and methanol (supragradient HPLC grade), ammonium acetate,

130

neutral alumina, and sodium hydroxide were from Merck. Potassium dihydrogen phosphate

131

was from BDH (Poole, UK). Other used consumables were: Durapore 0.20 µm membrane

132

filters (Millipore), 0.20 µm cellulose acetate syringe filters (LLG, Meckenheim, Germany),

133

and ACCUREL PP membrane (Membrana, Wuppertal, Germany).

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2.3. Synthesis of PEG-Mn-doped ZnS QDs

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Mn-doped ZnS QDs were synthesized following two different procedures: (a) magnetic

136

stirring procedure according to Wang et al. (Wang et al., 2009), although MPTS was replaced

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by polyethylene glycol (PEG) for modifying QDs surface (capped Mn-Doped ZnS); and (b)

138

ultrasound irradiation procedure. Ethylene glycol has previously been proposed for magnetite

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nanoparticles surface modification (Zhang et al., 2009; Hu et al., 2011; Wang et al., 2011),

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and as when using oleic acid for Mn-doped ZnS QDs (Ren et al., 2015), solubility is expected

141

to be increased, since hydroxyl-terminated QDs have good solubility and low non-specific

142

binding (Kuang et al., 2011).

143

(a) Magnetic stirring mode. A three-neck flask was used for performing QDs synthesis. One

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of the necks was attached to a pressure-equalized addition funnel containing 10 mL of a

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freshly prepared 1.25 M sodium sulphide solution, other neck was used for purging with N2,

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and the remaining neck was used for adding 12.5 mmol (3.595 g) of ZnSO4·7 H2O and 1.0

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mmol (0.1259 g) of MnCl2, and 40 mL of ultrapure water. After reagent addition, the open

148

neck was stopped, and the mixture was allowed to react under N2 (stirring speed at 100 rpm,

6

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room temperature) for 10 min. Sodium sulphide solution was dropwise added, and the

150

mixture was allowed to react for 30 min. Finally, 10 mL of an aqueous solution containing

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3.3 g of PEG was added, and the mixture was stirred (N2, stirring speed at 100 rpm, room

152

temperature) for 20 hours for allowing PEG surface modification of Mn-doped-ZnS QDs.

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(b) Ultrasound irradiation mode. Synthesis of Mn-doped-ZnS QDs was performed as shown

154

above. The difference in this method regards to QDs surface modification by PEG. After

155

PEG addition (10 mL of an aqueous solution containing 3.3 g of PEG), the mixture inside the

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three-neck flask was subjected to ultrasound irradiation at 37 kHz for 4 hours. Although the

157

ultrasound-water bath allows temperature control, the temperature of the water-bath was

158

slightly increased from room temperature up to 30°C due to the long irradiation time used.

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The proposed method for PEG-QDs preparation avoid high temperatures as well as long

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heating times as previously described (Zhao et al., 2012).

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After PEG-Mn-doped-ZnS QDs synthesis (both magnetic and ultrasound modes), the

162

mixtures were centrifuged at 3000 rpm for 20 min, and the prepared material was rinsed three

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times by adding 5 mL of methanol and isolating the solid nanoparticles by centrifugation

164

(3000 rpm, 20 min). The synthesized QDs were finally dried at room temperature inside a

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desiccator (silica gel as a desiccant) for 48 hours. Oven-drying (40°C) was also tried but a

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high degree of particle agglomeration was observed, which diminished the further QDs

167

dispersion/dissolution. The dried synthesized material was kept at 4 °C in the dark.

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2.4. Synthesis of MIP-coated PEG-QDs

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MIP synthesis onto the PEG-Mn-doped-ZnS QDs was performed using COC (0.20 g) as a

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template and EDMA (126 µL) as a monomer. These two reagents were dissolved in 4 mL of

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DMSO, sparged with argon, and kept at room temperature in the dark for 12 h for allowing a

172

self-assembly of the template and the monomer. PEG-Mn-doped-ZnS particles (0.50 g) were

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dispersed in 25 mL of ultrapure water, and were then mixed with the pre-polymerization

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mixture (COC-EDMA in DMSO) before adding the cross-linker (1.25 mL of DVB) and the

175

initiator (0.10 g of AIBN), and starting the polymerization by two different synthesis modes.

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(a) Magnetic stirring polymerization: sealed bakers containing the reaction mixture subjected

177

to magnetic stirring (100 rpm) at 50°C for 20 hours;

178

(b) Ultrasound irradiation polymerization: sealed bakers containing the reaction mixture were

179

sonicated (ultrasound frequency of 37 kHz) at room temperature for 4 hours. The proposed

180

procedure is performed at room temperature, and in contrast to previously described methods

181

(Zhao et al., 2012), organic solvent (DMSO) removal during the synthesis is not needed.

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DVB was previously treated to remove the polymerization inhibitor by passing a few

183

milliliters of the reagent through a mini-column containing 0.50 g of neutral alumina.

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Similarly, AIBN was purified by crystallization at -20°C after dissolving the reagent in

185

methanol at 50–60°C.

186

Once polymerization was finished, the synthesized material was washed several times with

187

methanol (centrifugation at 3000 rpm, 20 min), and finally dried at room temperature inside a

188

desiccator for 24 hours. The dried synthesized material was kept at 4°C in the dark. Non

189

imprinted polymers (NIPs)-coated PEG-QDs were also prepared as above but without using

190

the template. The NIP-coated PEG-QDs were subjected to the washing/drying treatment

191

described above.

192

As a summary, a schematic diagram showing MIP-coated PEG-QDs preparation can be seen

193

in Figure 1.

194

2.5. Template removal procedure

195

COC was removed from the prepared MIP-coated PEG-QDs by subjecting approximately

196

200 mg of the dried MIP-coated PEG-QD to ultrasound assisted extraction using a hexane/2-

197

propanol/ammonium hydroxide (70:20:10) extracting mixture (ten 30-min cycles at 37 kHz

198

with 10 mL of fresh extracting solution). To avoid MIP-coated PEG-QD loss, the synthesized

8

199

material was enclosed inside a rectangular in shape (3.0 × 2.0 cm) polypropylene (PP)

200

membrane prepared after successive folds and edges heat-sealing. Negligible COC

201

concentrations were found in the tenth washing solution (HPLC-MS/MS analysis after eluate

202

evaporation to dryness under stream of N2 at 40°C, and re-dissolution with 1 mL of 2 mM

203

ammonium acetate in methanol). The MIP-coated PEG-QD particles were rinsed two times

204

with methanol and three times with ultrapure water, and dried at room temperature inside a

205

desiccator for 24 hours. The dried synthesized material was kept at 4°C in the dark.

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Under optimum conditions, fluorescence measurements were performed using NIP/MIP-

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coated PEG-QDs (100 mg) redispersed in 250 mL of 0.1M/0.1M potassium

208

dihydrogenphosphate-sodium hydroxide buffer, pH 5.5 (NIP/MIP-coated PEG-QD

209

concentration of 200 mg L-1). These solutions were stored at 4°C in the dark, and they were

210

stable (constant fluorescence intensity) over a period of 5-6 months (time in which the

211

material was used until finished).

212

2.6. Fluorescence measurements

213

Fluorescence detections (photomultiplier tube voltage set at 700 V) were performed using an

214

excitation wavelength of 296 nm (slit width of 0.2 nm), and recording an emission range of

215

400–800 nm (emission slit of 0.2 nm, maximum fluorescence emission at 590 nm).

216

Measurements were performed by mixing 1.5 mL of MIP- or NIP-coated PEG-QDs solutions

217

(200 mg L-1), volumes within the 0-0.5 mL range of COC previously prepared in 0.1M/0.1M

218

potassium dihydrogenphosphate-sodium hydroxide buffer, pH 5.5, and volumes within the

219

0.5-0 mL range of the 0.1M/0.1M potassium dihydrogenphosphate-sodium hydroxide buffer

220

(fixed volume of 2 mL). The mixtures were kept at 4°C for at least 15 min before

221

fluorescence scanning. Three replicates were performed for each COC concentration tried.

222 223

2.7. Liquid chromatography-tandem mass spectrometry measurements

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COC assessment in eluates after template removal was performed by high performance liquid

225

chromatography – tandem mass spectrometry (HPLC-MS/MS) using gradient mode (2 mM

226

ammonium acetate in methanol and 2 mM ammonium acetate in ultrapure water as mobiles

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phases, flow rate of 0.4 mL min-1) under optimum acquisition settings.

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3. Results and discussion

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3.1. Characterization of the composite particles

230

3.1.1. Transmission electron microscopy characterization

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Figure S1 (supplementary section) shows the transmission electron microscopy (TEM)

232

images of PEG-QDs synthesized by magnetic stirring (a) and ultrasound irradiation (b). As

233

shown, the particle size of QDs, PEG-QDs obtained by magnetic stirring and ultrasound

234

irradiation, and MIP-coated PEG-QDs synthesized under ultrasound irradiation (Figure

235

S1(d), supplementary section) were quite similar (within the nanometer range).

236

3.1.2. Fourier transform infra red spectrometry characterization

237

Figure S2 (supplementary section) shows the Fourier transform infra red spectrometry (FT-

238

IR) spectra for QDs (a), and also PEG-QDs obtained by magnetic stirring (b) and ultrasound

239

irradiation (c) methods. Characteristic peaks at 610, 985, 1080, 1150, and 1650 cm-1 were

240

observed for QDs (Figure S2(a)). According to Rema Devi et al. (Rema Devi et al, 2007), the

241

peak at 610 cm-1 can be assigned to the ZnS band (i.e., corresponding to sulphides). In

242

addition, bands at 985, 1080, 1150, and 1650 cm-1 are due to the characteristic frequency of

243

inorganic ions, and they indicate the presence of resonance interaction between vibrational

244

modes of sulphide ions in the nanostructure (Kurian et al., 2004).

245

FT-IR spectra of PEG-QDs (Figure S2(b,c)) show bands at 1350 and 1460 cm-1 (C-H

246

bending) and 2900 cm-1 (C-H stretch), which suggest that PEG was successfully modified

247

onto the surface of the QDs. In addition, the characteristic band of ZnS (610 cm-1) was

10

248

weaker after PEG modification, as well as bands regarding the characteristic frequency of

249

inorganic ions (985, 1080, and 1150 cm-1).

250

FT-IR for PEG-QDs, NIP-coated PEG-QDs, and MIP-coated PEG-QDs synthesized by

251

ultrasound irradiation are shown in the supplementary section as Figure S3(a-c)

252

(supplementary section). In addition, FT-IR spectra for MIP-coated PEG-QDs synthesized

253

under ultrasound irradiation after template (COC) removal is given (Figure S3(d)). The

254

presence of the bands at 1350 and 1460 cm-1 (C-H bending) and 2900 cm-1 (C-H stretch) are

255

present in NIP/MIP-coated PEG-QDs. In addition, a narrow band at 1727 cm−1 is also

256

observed, which is attributed to the C=O (-CO-NH) stretch. The characteristic band at 610

257

cm-1 is not observed in NIP/MIP-coated PEG-QDs (Figure S3(b-d)). In addition, as obtained

258

for PEG-QDs, bands regarding the characteristic frequency of inorganic ions (985, 1080, and

259

1150 cm-1) are weaker. These findings prove that MIP/NIP layer is efficiently anchored onto

260

the surface of the PEG-QDs.

261

3.1.3. X ray diffraction spectrometry characterization

262

From the X ray diffraction spectrometry (XRD) patterns of QDs, PEG-QDs (magnetic

263

stirring and ultrasound irradiation modes), and MIP-coated PEG-QDs (Figure S4,

264

supplementary section), the crystalline size of the prepared material were calculated by

265

applying the Debye-Scherrer method. Calculated parameters in Table S1 (supplementary

266

section) are quite similar to those reported by to Rema Devi et al. (Rema Devi et al., 2007).

267

The average size of Mn-doped ZnS QDs was 1.50 nm; whereas, the average size of PEG-

268

QDs was 1.50 and 1.67 nm when using magnetic stirring and ultrasound irradiation,

269

respectively. Finally, the mean size of MIP-coated PEG-QDs (ultrasound irradiation mode) is

270

1.66 nm. The slightly high mean sizes found for MIP-coated PEG-QDs and PEG-QDs

271

synthesized by ultrasound assistance were not statistically significant at a 95% confidence

11

272

range. This fact was verified after applying the Multiple Range Test for comparing mean

273

particle sizes (results listed in Table S2, supplementary section).

274

3.2. Fluorescence study

275

The fluorescence excitation spectra of the MIP-coated PEG-QDs (before and after template

276

removal), NIP-PEG-QDs, and PEG-QD, were recorded by fixing the emission wavelength at

277

550 nm, and varying the excitation wavelength from 400 to 800 nm. Two maximum were

278

obtained at 258 and 296 nm. As shown in Fig. S5(a) (supplementary section) a strong

279

fluorescence signal (514.5 nm) and an overtone (weak signal at 770 nm) were generated

280

when using an excitation wavelength of 258 nm. The high signal at 514.5 nm, however,

281

saturates the detector when using high MIP-coated PEG-QDs concentrations. On the other

282

hand, excitation at wavelength at 296 nm (Fig. S5(b), supplementary section) generates a

283

moderate emission signal at 590 nm (adequate fluorescence intensity when varying the MIP-

284

coated PEG-QDs concentration within the 50–400 mg L-1 range without saturation), and

285

overtones were not observed). In addition, the fluorescence signal was sharp, indicating that

286

the sizes of MIP-coated PEG-QDs were very homogeneous. As shown in Fig. S5(a,b),

287

excitation and emission wavelengths are the same when measuring PEG-QDs, NIP-coated

288

PEG-QDs, and MIP-coated PEG-QDs before and after template removal.

289

3.3. Optimization of the response of MIP-coated PEG-QDs for cocaine

290

Preliminary studies showed that the fluorescence of MIP-coated PEG-QDs is quenched when

291

COC (template for MIP synthesis) is present. Since COC and metabolites (BZE and EME)

292

are slightly alkaline in aqueous medium, the MIP-coated PEG-QDs were suspended in

293

aqueous 0.1M/0.1M K2HPO4/NaOH buffer solutions at fixed acid pHs for allowing an

294

efficient interaction with the composite nanoparticles. In addition, higher fluorescence

295

intensities were observed when using cold MIP-coated PEG-QDs solutions, and experiments

296

were performed by placing the vials containing the MIP-coated PEG-QDs and COC mixtures

12

297

in an ice-bath. Three operating conditions (pH, MIP-coated PEG-QDs concentration, and

298

time for COC and MIP-coated PEG-QDs interaction) were therefore fully optimized.

299

3.3.1 Effect of the MIP-coated PEG-QDs concentration

300

Several masses of MIP-coated PEG-QDs (12.5, 25, 50, and 100 mg) were dissolved in 250

301

mL of K2HPO4/NaOH buffer, pH 5.5, giving MIP-coated PEG-QDs concentrations of 50,

302

100, 200, and 400 mg L-1, respectively. By fixing the volume of the MIP-coated PEG-QDs

303

solution at 1.5 mL, several volumes of a solution containing 10 mg L-1 COC (0, 0.04, 0.08,

304

0.12, 0.16, and 0.20 mL, prepared in K2HPO4/NaOH buffer, pH 5.5), and volumes of

305

K2HPO4/NaOH buffer pH 5.5 (0.50, 0.46, 0.42, 0.38, 0.34, and 0.30 mL) were added. The

306

fluorescence emission was recorded after 10 min. Each COC concentration level (within the

307

0.0 – 1.0 mg L-1) was measured in triplicate, and results (mean fluorescence peak height) are

308

plotted in Figure 2. Bad linear regression (regression coefficient of 0.682) was obtained when

309

using the smallest MIP-coated PEG-QDs concentration; whereas, a linear relationship

310

between the fluorescence quenching and the concentration of COC was observed when using

311

the higher MIP-coated PEG-QDs concentrations. In addition, the slope of the lineal curve

312

was higher when using more concentrated MIP-coated PEG-QDs solutions. Therefore, the

313

highest MIP-coated PEG-QDs concentrations tested (200 and 400 mg L-1) can be

314

successfully used, and a concentration of 200 mg L-1 was selected for further experiments.

315

3.3.2 Effect of pH

316

Because of the alkaline nature of COC in aqueous solution, a better COC-MIP interaction is

317

expected when working at acid pHs. Therefore, MIP-coated PEG-QDs solutions (200 mg L-1)

318

were prepared in K2HPO4/NaOH buffer solutions at acidic pHs (5.0, 5.5, 6.0, and 6.5).

319

Several volumes of COC solutions (10 mg L-1) within the 0 – 0.20 mL (prepared in

320

K2HPO4/NaOH buffer at the tested pH) were mixed with 1.5 mL of 200 mg L-1 MIP-coated

321

PEG-QDs solutions at the tested pH, and volumes of K2HPO4/NaOH buffer at the selected

13

322

pH (from 0.50 to 0.30 mL). After a delay time of 10 min for allowing COC and MIP-coated

323

PEG-QD assembly, the fluorescence was recorded, and results (also three independent

324

measurements for each COC concentration and pH tested) are plotted in Figure S6

325

(supplementary section). Good linearity was observed between fluorescence quenching and

326

COC concentration. However, the highest slope (the highest fluorescence quenching) was

327

observed when fixing a pH of 5.5. A pH of 5.5 was therefore used for further experiments.

328

Fluorescence intensity of the blank (K2HPO4/NaOH buffer, pH 5.5) was measured when

329

performing each further experiment. Values ranged from 25 to 45 fluorescence units.

330

3.3.3 Effect of the interaction time between COC and MIP-coated PEG-QDs

331

Preliminary fluorescence quenching experiments showed bad precision when measurements

332

were performed just before mixing COC and MIP-coated PEG-QDs solutions. This is

333

because a certain time is needed for allowing an efficient interaction between COC and the

334

composite material. Therefore, several experiments using 1.5 mL of 200 mg L-1 of MIP-

335

coated PEG-QDs and COC solutions at 0.50 mg L-1 (all solutions prepared in K2HPO4/NaOH

336

buffer, pH 5.5) were preformed by recording the fluorescence quenching just before mixing

337

the solution (interaction time of 0 min), and after every two minutes (the highest interaction

338

time tested was 20 min). Results (mean fluorescence height, three replicates) in Figure 3

339

show instability (bad precision) within the first 4 min. The fluorescence signal decreases

340

linearly within the 4 - 14 min range, and it remain then constant up to 20 min. Precision

341

(small standard deviation bars in Figure 3) is highly improved when increasing the interaction

342

time between COC and the composite nanoparticles. Therefore, an interaction time (delay

343

time) of 15 min was finally selected for further experiments.

344

3.4. MIP- and NIP-coated PEG-QDs response with cocaine and its metabolites: imprinting

345

effect

14

346

Experiments were performed for establishing the responses of MIP- and NIP-coated PEG-

347

QDs with several template (COC) and metabolites (BZE and EME) concentrations under

348

optimized operating conditions (structures of analytes are given in Table S3, supplementary

349

section). As shown in Figure 4(a-c), the fluorescence intensity of the MIP-coated PEG-QDs

350

was quenched lineally with the increasing concentration of template COC up to 1 mg L-1;

351

whereas, a linear fluorescence decrease was observed up to 1.5 and 5.0 mg L-1 of EME and

352

BZE, respectively. Fluorescence quenching depends on the recognition capacity through the

353

imprinted cavities of the particles with the template. In this case, MIP-coated PEG-QDs show

354

also affinity for two cocaine metabolites which are structurally similar to COC. Figure 4(d-f)

355

shows the Stern–Volmer equation analysis [F0/F ratio versus the quencher (COC, BZE and

356

EME) concentration] for the MIP-coated PEG-QDs. The Stern–Volmer constants (KSV) are

357

the slope of the linear curves in Figure 4(d-f), which were 0.073, 0.035, and 0.031 for COC,

358

BZE and EME, respectively. The KSV constant for COC (template) is twice than the KSV

359

constant for BZE and EME. This gives KSV,MIP(COC)/KSV,MIP(BZE) and KSV,MIP(COC)/KSV,MIP(EME)

360

ratios of 2.1, and 2.4, respectively (Table S4), which implies therefore a good recognition

361

capacity of the prepared composite for BZE and EME.

362

To prove the existence of specific interactions between COC (and also BZE and EME) and

363

MIP-coated PEG-QDs, similar experiments were performed by recording the response of

364

NIP-coated PEG-QDs with several COC, BZE and EME concentrations. Results plotted in

365

Figure 5(a-c) show that fluorescence quenching is not observed when using NIP-coated PEG-

366

QDs and COC, BZE and EME concentrations within the 0 – 3.0 mg L-1 range. This proves

367

that the interaction of COC and metabolites with the MIP layer occurs through the

368

recognition cavities. Figure 5(d-f) also shows the Stern–Volmer equation analysis for NIP-

369

coated PEG-QDs. The imprinting factor (IF), expressed as the KSV,MIP/KSV,NIP ratio (KSV,MIP

370

and KSV,NIP were the slopes in Figure 4(d-f) and Figure 5(d-f)) was used to evaluate the

15

371

imprinting effect of the MIP-coated PEG-QD composite. Table S4 (supplementary section)

372

lists the calculated KSV,MIP/KSV,NIP ratios. The high values for this ratio, mainly for COC (23),

373

but also for BZE (7.9) and EME (9.1), prove the specific interaction of COC and its

374

metabolites through the recognition cavities in the MIP layer. Finally, fluorescence changes

375

in MIP-coated PEG-QDs when varying the concentration of COC, BZE, and EME are shown

376

in Figure 6(a). Fluorescence from NIP-coated PEG-QDs was found to be constant at all COC,

377

BZE, and EME concentrations.

378

3.5. MIP- and NIP-coated PEG-QDs response with other drugs of abuse: selectivity study

379

Stern–Volmer constants were calculated by recording the fluorescence intensity under

380

optimum conditions but using MOR, COD, and 6-MAM (heroin abuse), and Δ9-THC, Δ9-

381

THC-OH, CBD, and CBN (cannabis abuse) within the 0.0 – 3.0 mg L-1 range as fluorescence

382

quenchers. Table S3 lists the structures of the drugs/metabolites to test selectivity of the

383

prepared material. Table S4 (supplementary section) lists the Stern–Volmer constants for

384

experiments by using the MIP-coated PEG-QDs (KSV(MIP)) and the NIP-coated PEG-QDs

385

(KSV(NIP)) for all quenchers. In addition, Table S4 also lists the selectivity factors expressed as

386

the ratio between the Stern–Volmer constant obtained for MIP-coated PEG-QDs using COC

387

(template) as a quencher (KSV(MIP)COC) and the Stern–Volmer constants obtained for MIP-

388

coated PEG-QDs using the other drugs/metabolites (KSV(MIP)Q). Low ratios (2.1 and 2.4) were

389

obtained for BZE and EME: whereas, higher ratios were obtained for the other drugs,

390

especially for MOR (121) and COD (81), which verifies that the prepared material is highly

391

selective to cocaine and its metabolites. Finally, Figure 6(b) shows that MIP/NIP-coated

392

PEG-QDs fluorescence is not changed in the presence of several drugs/metabolites at

393

different concentrations.

394

3.6. Application to urine samples.

16

395

In order to evaluate the feasibility of the method, a drug-free urine sample (analytical

396

recovery study) and three urine samples from cocaine abusers (concentration of cocaine and

397

metabolites previously measured by COBAS INTEGRA 400 analyzer) were analyzed. For all

398

cases, urine samples were 1:20 diluted by mixing 100 µL of urine with 1.5 mL of 200 mg L-1

399

MIP-coated PEG-QDs, and 400 µL of the aqueous 0.1M/0.1M K2HPO4/NaOH buffer

400

solution (pH 5.5). The standard addition method (use of a drug-free urine sample) was used

401

for all analysis. The standard addition method uses an urine sample volume of 100 µL, 1.5

402

mL of 200 mg L-1 MIP-coated PEG-QDs solution, variable volumes of a 10 mg L-1 cocaine

403

standard solution (from 0 to 400 µL, which give cocaine concentrations within the 0 – 2 mg

404

L-1 range), and variable volumes of aqueous 0.1M/0.1M K2HPO4/NaOH buffer solution, pH

405

5.5 (from 400 to 0 µL). Intra-day precision (n=7) was 8, 7, and 3% for cocaine concentrations

406

of 0.5, 1.0 and 2.0 mg L-1, respectively; whereas, intra-day analytical recovery (n=7) was

407

97±8, 99±7, and 104±3 % for cocaine concentrations of 0.5, 1.0 and 2.0 mg L-1, respectively.

408

The analysis of three urine samples from cocaine abusers gave concentrations of cocaine

409

(COC+BZE+EME)

410

(COC+BZE+EME) concentrations given by the reference method (COBAS INTEGRA 400

411

analyzer) were 0.97, 1.6, and 0.81 mg L-1, respectively. The results showed that the

412

fluorescent probe based on MIP-coated PEG-QDs has the potential applicability for cocaine

413

assessment in urine samples.

414

Conclusions

415

A novel MIP-coated Mn-doped-ZnS probe was developed through a precipitation

416

polymerization method involving ultrasound irradiation for enhancing MIP coating and for

417

shortening the synthesis. The prepared material offers molecular imprinting capabilities for

418

the detection of COC and metabolites (BZE, and EME), and a selective and sensitive

419

determination can be achieved on the basis of an electron-transfer-induced fluorescence

of

1.1±0.077,

1.7±0.10,

and

0.79±0.061

mg

L-1.

Cocaine

17

420

quenching mechanism. The MIP-coated Mn-doped-ZnS was fully characterized and

421

selectivity was proved by studying the fluorescence responses against drugs other than

422

cocaine. The simple, rapid, and reliable MIP-coated Mn-doped-ZnS sensing strategy opens

423

up attractive perspectives for screening and confirmation analysis of cocaine abuse.

424

Acknowledgements

425

The authors wish to thank the Dirección Xeral de I+D – Xunta de Galicia (Project number

426

10CSA209042PR) for financial support.

427

18

428

Figures’ captions

429 430

Figure 1. Schematic diagram for the preparation of MIP-coated PEG-QDs: DVB is

431

divinylbencene; and AIBN is 2,2´-azobisisobutyronitrile.

432 433

Figure 2. Effect of the concentration of MIP-coated PEG-QDs on the fluorescence quenching

434

by several COC concentrations.

435 436

Figure 3. Effect of interaction time (delay time for fluorescence measurement) on the

437

fluorescence quenching by COC

438 439

Figure 4. Effect of the concentration of COC (a), BZE (b), and EME (c) on the fluorescence

440

quenching of MIP-coated PEG-QDs, and Stern–Volmer equations for COC (d), BZE (e), and

441

EME (f).

442 443

Figure 5. Effect of the concentration of COC (a), BZE (b), and EME (c) on the fluorescence

444

quenching of NIP-coated PEG-QDs, and Stern–Volmer equations for COC (d), BZE (e), and

445

EME (f).

446 447

Figure 6. Fluorescence changes from MIP-coated PEG-QDs and NIP-coated PEG-QDs at

448

increasing COC, BZE, and EME concentrations (a), and at increasing MOR, COD, 6-MAM,

449

Δ9-THC, Δ9-THCOH, CBD and CBN (b).

450

19

451

References

452

Algar, W. R., Susumu, K., Delehanty, J. B., Medintz, I. L., 2011. Anal. Chem. 83, 8826–

453

8837.

454

Bol, A. A., Meijerink, A., 2000. J. Lumin. 87–89, 315–318.

455

Chen, Y.-P., Wang, D.-N., Yin, Y.-M., Wang, L.-Y., Wang, X.-F., Xie, M.-X., 2012. J.

456

Agric. Food Chem. 60, 10472−10479.

457

Dan, L., Wang, H.-F., 2013. Anal. Chem. 85, 4844−4848.

458

Hu, Y., Li, Y., Liu, R., Tan, W., Li, G., 2011. Talanta 84, 462–470.

459

Kuang, H., Zhao, Y., Ma, W., Xu, L., Wang, L., Xu, C., 2011. Trends Anal. Chem. 30, 1620-

460

1636.

461

Kurian, S., Sebastian, S., Mathew, J., George, K. C., 2004. Indian J. Pure Appl. Phys. 42,

462

926-933.

463

Lin, C. L., Joseph, A. K., Chang, C. K., Lee, Y. D., 2004a. Biosens. Bioelectron. 20, 127–

464

131.

465

Lin, C. L., Joseph, A. K., Chang, C. K., Lee, Y. D., 2004b. J. Chromatogr. A 1027, 259–262.

466

Liu, J., Chen, H., Lin, Z., Lin, J.-M., 2010. Anal. Chem. 82, 7380–7386.

467

Pradhan, N., Goorskey, D., Thessing, J., Peng, X., 2005. J. Am. Chem. Soc. 127, 17586-

468

17587.

469

Rema Devi, B. S., Raveendran, R., Vaidyan, A. V., 2007. Pramana J. Phys. 68, 679-687.

470

Ren, X., Liu, H., Chen, L., 2015. Microchim Acta 182, 193–200.

471

Ren, X., Chen, L., 2015. Biosens. Bioelectron. 64, 182–188.

472

Suyvere, J. F., Wuister, S. F., Kelly, J. J., Meijerink, A., 2001. Nano Lett. 1, 429-433.

473

Tan, L., Huang, C., Peng, R., Tang, Y., Li, W., 2014. Biosens. Bioelectron. 61, 506–511.

474

Tan, L., Kang, C., Xu, S., Tang, Y., 2013. Biosens. Bioelectron. 48, 216–223.

20

475

Tan, L., Li, Y., Tang, Y., Kang, C., Yu, Z., Xu, S. 2012. J. Nanosci. Nanotech. 12, 7788-

476

7795.

477

United Nations Office on Drugs and Crime (2013) World Drug Report 2013. Available at:

478

http://www.unodc.org/wdr. Accessed May 28th 2015.

479

Wang, H.-F., He, Y., Ji, T.-R., Yan, X.-P., 2009. Anal. Chem. 81, 1615–1621

480

Wang, X., Mao, H., Huang, W., Guan, W., Zou, X., Pan, J., Yan, Y., 2011. Chem. Eng. J.

481

178, 85–92.

482

Wei, X., Zhou, Z., Dai, J., Hao, T., Li, H., Xu, Y., Gao, L., Pan, J., Li, C., Yan, Y., 2014. J.

483

Lumin. 155, 298–304.

484

Xiao, Q., Xiao, C., 2008. Appl. Surf. Sci. 254, 6432-6435.

485

Xu, M. B., Ye, T., Lu, S. Y., Hu, Q. Q., Zhou, J., Lu, J. Q., 2012. Chinese Chem. Lett. 23,

486

1403–1406.

487

Zhang, Y., Liu, R., Hu, Y., Li, G., 2009. Anal. Chem. 81, 967–976.

488

Zhao, Y., Ma, Y., Li, H., Wang, L., 2012. Anal. Chem. 84, 386–395.

489

21

5000

(a)

4500 4000 3500

-1

F.I.

0 µg L

-1

3000

0.2 µg L

2500

0.4 µg L

-1 -1

1.0 µg L

2000 1500 1000 COC MIP COC NIP BZE MIP BZE NIP EME MIP EME NIP

4000

(b)

3500 3000

F.I.

-1

0 µg L

2500

-1

0.2 µg L 2000

-1

0.4 µg L

-1

1500

1.0 µg L

1000

490

OH CH2

Mn-ZnS QD

N

CH2

ultrasounds n Ethylene glycol (PEG)

O

DVB / AIBN O

ultrasounds

O

O

491

22

8000 (d) F.I. = - 980.9 [COC] + 7039 R2 = 0.995 7000 6000

(c) F.I. = - 1204 [COC] + 5404 R2 = 0.996

F.I.

5000 4000 3000

2000

(b) F.I. = - 286.4 [COC] + 1449 R2 = 0.951

1000

(a) F.I. = - 108.0 [COC] + 636.0 R2 = 0.681

0 0.0

0.2

0.4

0.6

0.8

1.0

[COC] / mg L-1

492

5250

5000

F.I.

4750

4500

4250

4000 0

2

4

6

8

10

12

14

16

18

20

Delay time / min

493

23

4650

(a)

(d)

4600

1.10

F0/F = 0.073 [COC] + 1.0 R2 = 0.987

4550 1.08

4500 1.05

F.I.

F0/F

4450

1.03

4400 4350

1.00

4300

0.98

4250

0.95

4200

0.0

0.2

0.4

0.6

0.8

1.0

4.0

5.0

[COC] / mg L-1

4150 0.0

1.0

2.0

3.0

4.0

5.0

[COC] mg L-1

(b)

5400 5200

1.25

(e) F0/F = 0.035 [BZE] + 1.0 R2=0.984

1.20

5000

F0/F

F.I.

1.15

4800

1.10 1.05

4600

1.00

4400

0.95 0.0

1.0

2.0

4200

3.0

[BZE] / mg L-1

0.0

1.0

2.0

3.0

4.0

5.0

[BZE] mg L -1

6050

(c)

6000

5950

1.10

5900 5850

F0/F = 0.031 [EME] + 1.0 R2 = 0.959

1.05

5800 F0/F

F.I.

(f)

1.08

5750

1.03

5700

1.00

5650

0.98

5600

0.95 0.0

0.25

5550

494

0.50

0.75

1.00

1.25

[EME] / mg L-1

0

0.5

1.0

1.5

2.0

[EME] mg L-1

2.5

1.5

3.0

2200 (a) 2150

1.10

2100

(d) F0/F = 0.0032 [COC] + 1.0 R2 = 0.0236

1.06

F0/F

F.I.

2050 2000

1.02 0.98

1950 0.94

1900

0.90

1850

0.0

0.5

1.0

1.5

2.0

2.5

3.0

2.5

3.0

[COC] / mg L-1

1800

0.0 1700 (b)

0.5

1.0

1.5 [COC] mg L -1

2.0

2.5

3.0

(e)

1650

1.01 F /F = --0.0045 [BZE] + 0.97 R2 = 0.212 0

1600

1.00

F0/F

F.I.

0.99

1550

0.98 0.97

1500

0.96

1450 1400 0.0

0.95 0.0

0.5

1.0

1.5

[BZE] mg 2900

2.0

2.5

1.0

1.5

2.0

[BZE] / mg L-1

L-1

(c) 1.05

2850 2800

(f) F0/F = 0.0034 [EME] + 1.0 R2 = 0.0914

1.03

F0/F

2750

F.I.

0.5

3.0

2700 2650

1.00

0.98

2600 0.95 0.0

2550

0.0

495

0.5

1.0

1.5

2.0

2.5

3.0

[EME] / mg L-1

2500 0.5

1.0

1.5

2.0

2.5

3.0

[EME] mg L-1

496 497

Highlights:

498

> Molecularly imprinted polymer – coated Mn-ZnS QDs for cocaine recognition

499

> Ultrasound irradiation assistance for improving homogeneity of the composite material

24

500

> Ultrasound irradiation assistance for speeding up the time synthesis of the composite

501

material

502

> Fast cocaine and metabolites assessment by spectrofluorimetry

503

> High sensitivity and selectivity of the prepared composite material

504

25