Accepted Manuscript Spectroscopic investigations on the interaction of thioacetamide with ZnO quantum dots and application for its fluorescence sensing
Dipika Saha, Devendra P.S. Negi PII: DOI: Reference:
S1386-1425(17)30682-0 doi: 10.1016/j.saa.2017.08.053 SAA 15410
To appear in:
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Received date: Revised date: Accepted date:
24 May 2017 4 August 2017 17 August 2017
Please cite this article as: Dipika Saha, Devendra P.S. Negi , Spectroscopic investigations on the interaction of thioacetamide with ZnO quantum dots and application for its fluorescence sensing, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2017), doi: 10.1016/j.saa.2017.08.053
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ACCEPTED MANUSCRIPT Spectroscopic investigations on the interaction of thioacetamide with ZnO quantum dots and application for its fluorescence sensing
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Dipika Saha, Devendra P. S. Negi*
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Centre for Advanced Studies in Chemistry, North-Eastern Hill University, Shillong 793022,
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India
*
Corresponding author
E-mail address: [email protected]
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Abstract The purpose of the present work was to develop a method for the sensing of thioacetamide by using spectroscopic techniques. Thioacetamide is a carcinogen and it is important to detect its
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presence in food-stuffs. Semiconductor quantum dots are frequently employed as sensing probes since their absorption and fluorescence properties are highly sensitive to the
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interaction with substrates present in the solution. In the present work, the interaction
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between thioacetamide and ZnO quantum dots has been investigated by using UV-visible,
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fluorescence and infrared spectroscopy. Besides, dynamic light scattering (DLS) has also been utilized for the interaction studies. UV-visible absorption studies indicated the bonding
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of the lone pair of sulphur atom of thioacetamide with the surface of the semiconductor. The fluorescence band of the ZnO quantum dots was found to be quenched in the presence of
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micromolar concentrations of thioacetamide. The quenching was found to follow the Stern-
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Volmer relationship. The Stern-Volmer constant was evaluated to be 1.20 x 105 M-1. Infrared spectroscopic measurements indicated the participation of the –NH2 group and the sulphur
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atom of thioacetamide in bonding with the surface of the ZnO quantum dots. DLS measurements indicated that the surface charge of the semiconductor was shielded by the
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thioacetamide molecules.
Keywords: zinc oxide quantum dots, thioacetamide, fluorescence, quenching, semiconductor
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1. Introduction Studies on the interaction of semiconductor nanoparticles (NPs) with organic/biological molecules is useful from the viewpoint of sensing [1-6] and biological labelling [7-8].
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Analytical techniques such as infra- red, fluorescence and UV-visible spectroscopy are powerful tools for the characterization of the semiconductor-organic molecule interaction.
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Infra-red spectroscopy provides useful information on the functional groups of the organic
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molecule involved in the bonding with the semiconductor NPs. For example, Chatterjee et al.
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reported the interaction of biofunctionalized CdS NPs with adenine. They suggested the coordination of the N-7 nitrogen of adenine to the cadmium atoms attached to the surface of
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the CdS NPs on the basis of the infra-red spectroscopic studies [9]. Fluorescence spectroscopy is a useful tool for studying the interaction of fluorescent semiconductor NPs
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with the substrate molecules. The fluorescence of the semiconductor NPs may be quenched
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or enhanced by its interaction with an analyte [10]. For example, the selective sensing of 1,2,4 – trihydroxybenzene was reported using histidine-stabilized ZnS nanospheres as a
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fluorescent probe [6]. UV-visible spectroscopy provides useful information on the groundstate interaction between the additive and the semiconductor.
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ZnO is a wide band gap semiconductor and is considered to be more environment friendly in comparison to CdS because of the toxicity associated with cadmium. Recently, some groups have reported studies on the interaction of this semiconductor with biomolecules [1113 ]. Thioacetamide is a carcinogen and prolonged exposure to such a substance causes bile duct proliferation and liver cirrhosis with histology similar to that produced by chronic viral hepatitis in humans [14]. The experimental hepatotoxicity of thioacetamide in rodents was first studied in 1948 in response to its detection in orange juice following its use as a
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ACCEPTED MANUSCRIPT fungicide in orange groves [15]. Therefore, it is important to develop a method for the detection of thioacetamide in low concentration range. To the best of our knowledge, there is no previous report in the literature on fluorescence based method for thioacetamide sensing. In the present work, we have investigated the interaction of thioacetamide with colloidal ZnO quantum dots by using UV-visible, fluorescence and infra-red spectroscopic techniques.
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DLS measurements were also carried out to get further insights into the interaction between
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thioacetamide and the semiconductor.
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2. Material and methods
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2.1. Materials
All the chemicals used for the research work were of analytical reagent grade. Zinc acetate
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dihydrate and thioacetamide were purchased from Himedia (Mumbai, India). Potassium
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hydroxide pellets were purchased from Merck (India) Limited (Mumbai). Methanol was obtained from Sisco Research Laboratory (Mumbai India).
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2.2. Instrumentation
UV-visible absorption spectra were recorded on a PerkinElmer Lambda 25 (Singapore)
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absorption spectrophotometer. Hitachi FL 4500 (Japan) spectrofluorimeter was used to obtain the steady state fluorescence spectra. Infrared spectra were recorded on a PerkinElmer BX FTIR system (Singapore). Transmission electron microscopy (TEM) measurements were carried out using a JEM 2100 (USA) instrument operating at 100 kV. A drop of the ZnO colloidal solution was dropped on a copper grid and allowed to dry at room temperature. The hydrodynamic diameter of the colloidal particles was determined by DLS measurements by using Malvern Zetasizer Nano ZS90 instrument (UK). The instrument operated at 633 nm which was fixed at 900 scattering angle and the temperature was maintained by the built-in 4
ACCEPTED MANUSCRIPT Peltier temperature control unit of the instrument at 21 0C. X-ray Diffraction measurements were carried out using an Explorer powder diffractometer from GNR Analytical Instruments Group (Italy).
2.3. Synthesis of colloidal ZnO solution
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ZnO quantum dots were prepared by using a reported sol-gel method [16] with slight
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modifications. Briefly, zinc acetate dihydrate (0.9790 g) and potassium hydroxide (0.4859 g) were dissolved in 42 ml and 23 ml of methanol (solvent) respectively. The zinc acetate
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solution was stirred and refluxed to 60 0C for half an hour. The solution of potassium
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hydroxide was added drop wise to the zinc acetate suspension. The solution was stirred for five minutes to ensure proper mixing of the two components and then the solution was
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refluxed for 15 min at 60 0C. During the reaction, the solution turned from turbid to transparent. The transparent solution was then cooled to room temperature and stored in
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refrigerator for further use. The concentration of ZnO thus prepared was calculated to be 8.8
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x 10-2 M on the basis of the concentration of zinc precursor used in the synthesis.
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2.4. Preparation of samples for fluorescence measurements Freshly prepared thioacetamide stock solutions were prepared and different concentrations
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(2 x 10-6 to 1 x 10-3 M) were added to the sample tubes containing 10 ml of colloidal ZnO solution. The sample tubes were shaken for 10 min in a water bath cum incubator shaker with a speed of 80 strokes per minute. After that, the samples were kept undisturbed for an hour. Then, the fluorescence spectra of the samples were recorded. The excitation wavelength was chosen such that the substrates did not absorb any light.
3. Results and discussion
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ACCEPTED MANUSCRIPT 3.1. Characterization of the colloidal ZnO solution The UV-visible absorption spectrum of the ZnO particles in methanol has been displayed in Fig. 1. The onset of absorption of the colloidal particles was ~ 360 nm which was
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Fig. 1. UV-visible absorption spectrum of the ZnO particles in methanol.
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considerably blue-shifted from that of the bulk ZnO (370 nm) [16]. It indicates the phenomenon of quantum size effect [17] in the formation of the semiconductor particles.
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Such semiconductor particles are called quantum dots. The fluorescence spectrum of the ZnO quantum dots has been displayed in Fig. 2. A broad peak at 512 nm was observed. This
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green fluorescence band has been attributed to the photogenerated trapped electrons tunneling to pre-existing trapped holes [18].
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6000
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Fig. 2. Fluorescence spectrum of the ZnO quantum dots in methanol. Excitation wavelength = 350
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nm.
Transmission electron microscopy was used in order to determine the shape and size of the
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ZnO quantum dots. The TEM image of the semiconductor particles has been displayed in
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Fig. 3.
Fig. 3. TEM image of the ZnO quantum dots.
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ACCEPTED MANUSCRIPT The average diameter of the ZnO quantum dots was calculated to be 3.7 nm from the image shown in Fig. 3. The higher magnification TEM image shows the lattice planes in a single
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ZnO particle (Fig. 4).
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Fig. 4 Higher magnification TEM image of the ZnO quantum dots.
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The crystalline nature of the ZnO particles was ascertained by the selected area electron
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diffraction (SAED) pattern shown in Fig. 5.
Fig. 5. SAED pattern of the ZnO quantum dots.
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ACCEPTED MANUSCRIPT The concentric rings observed in Fig. 5 may be attributed to the diffraction from various planes of crystalline ZnO [19]. The ZnO quantum dots were further characterized by powder X-ray diffraction (XRD) measurements. The XRD pattern of the quantum dots has been displayed in Fig. 6. The peaks in the pattern indicated high crystallinity of the material
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(202)
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(112)
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(102)
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Fig. 6 XRD pattern of the ZnO quantum dots.
and were assigned to the (100), (002), (101), (102), (110), (103), (112) and (202) planes of
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the wurtzite-type ZnO structure [16].
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3.2. Spectroscopic investigation of the interaction between thioacetamide and the ZnO quantum dots
3.2.1. UV-visible spectroscopic measurements The ground state interaction between thioacetamide and the ZnO quantum dots was investigated by using UV-visible absorption spectroscopy. The structure of thioacetamide has been displayed in Fig. 7. The lone pair of electrons present on the nitrogen and sulphur atoms of thioacetamide are potential sites for bonding with another chemical species.
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Fig. 7. Chemical structure of thioacetamide.
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The original colloidal ZnO solution was diluted by adding excess methanol in order to bring the value of absorbance within 1.0. The UV-visible absorption spectra of the ZnO quantum
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dots, 20 µM thioacetamide and their reaction mixture have been displayed in Fig. 8.
ZnO 20 M TA ZnO + 20 M TA
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Fig. 8. UV-visible absorption spectra of ZnO quantum dots, 20 µM thioacetamide (TA) and their reaction mixture.
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ACCEPTED MANUSCRIPT There was no shift in the position of the absorption band (λmax ~ 322 nm) of the ZnO quantum dots upon addition of thioacetamide. The position of the 263 nm absorption band of thioacetamide remained unchanged in the presence of the semiconductor. However, the 223 nm absorption band of thioacetamide was found to disappear in the presence of the ZnO quantum dots. Earlier studies on the electronic transitions in thioacetamide have assigned the
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223 nm absorption band to the excitation of the lone pair electrons on the sulphur atom to the
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anti-bonding pi orbital ( 1ns → 1п* ) [20]. The fact that the 223 nm band of thioacetamide
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disappeared in the presence of the ZnO quantum dots indicates that lone pair of electrons on the sulphur atom was bonded to the ZnO quantum dots. As a result, the electronic transition
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associated with this lone pair ( 1ns → 1п* ) was not observed.
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3.2.2. Fluorescence spectroscopic measurements
Steady-state fluorescence spectroscopy was used in order to further probe the interaction
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between thioacetamide and the ZnO quantum dots. The fluorescence properties of the
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semiconductor was investigated in the presence of wide range of concentrations of the substrate. The fluorescence spectra of the ZnO quantum dots in the absence and presence of
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different concentrations of thioacetamide have been displayed in Fig. 9.
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700 0 M 2 M 4 M 6 M 8 M 10 M
500 400 300 200
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Intensity (a.u.)
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Wavelength (nm)
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Fig. 9. Fluorescence spectra of the ZnO quantum dots in the absence and presence of various
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concentrations of thioacetamide as indicated in the inset. Excitation wavelength = 340 nm.
The emission band of the ZnO quantum dots was increasingly quenched by increasing
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concentration of thioacetamide without any change in the position of its λmax. The quenching
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of the fluorescence of a semiconductor is generally attributed to the transfer of the photogenerated electron or hole to the substrate molecules present in the solution.
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Thioacetamide is likely to be a good hole acceptor due the presence of a lone pair of electrons on the nitrogen and sulphur atoms (Fig. 7). Molecular oxygen is known to be scavenger of
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electrons [21]. Therefore, the photogenerated electrons might be scavenged by the oxygen molecules present in the solution. The quenching data was fitted to the Stern-Volmer relationship [22], given by I0 / I = 1 + KSV [Q] where I0 and I are the fluorescence intensity of the fluorophore in the absence and presence of the quencher, Q. The Stern-Volmer constant for the quenching process is denoted by KSV.
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ACCEPTED MANUSCRIPT The Stern-Volmer plot for the quenching of the ZnO fluorescence by thioacetamide has been displayed in Fig. 10.
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y = 0.120x + 1 R2 = 0.999
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I0 / I
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[Thioacetamide] (M)
Fig. 10. Stern-Volmer plot for the quenching of the ZnO fluorescence by thioacetamide.
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From this plot, the value of KSV was calculated to be 1.20 x 105 M-1. The linear dynamic
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range (LDR) of the method is thus 0-10 µM. The fluorescence quenching method described above provides a platform for the sensing of thioacetamide in the micromolar concentration
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range. The concentration of thioacetamide in an unknown sample could be determined by using Fig. 10 from the extent of quenching (I0/I) of ZnO fluorescence caused by the substrate.
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The limit of detection (LOD) and limit of quantification (LOQ) of the method were calculated using the formulae 3σ/S and 10σ/S, where σ is the standard deviation of the responses of the blank solutions and S is the slope of the calibration curve [23, 24]. The calibration curve has been displayed as Fig. S4 of the Supplementary data. On the basis of the calculations, the LOD and LOQ for thioacetamide on the basis of the fluorescence quenching method has been calculated to be 2.14 nM and 7.14 nM respectively. The comparison of the
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ACCEPTED MANUSCRIPT LOD and LDR of the present fluorescence quenching method with the other analytical methods reported in the literature has been summarized in Table 1. Table 1 Comparison of the various analytical methods for the detection of thioacetamide. LOD
LDR
Reference
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Voltammetry
0.84 µM
5-60 µM
[25]
2.
Amperometry
3 µM
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[26]
3.
Fluorimetry
2.14 nM
0-10 µM
Present work
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S.No. Analytical Method
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From Table 1 it is evident that the present method of thioacetamide sensing is much more sensitive compared to the earlier reported methods.
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Upon further increasing the concentration of thioacetamide (20-100 µM), the extent of quenching was also found to increase (Fig. S1, Supplementary data). The Stern-Volmer plot
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for the quenching process has been displayed in Fig. S2 of the supplementary data. The value
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of KSV was determined to be 5.4 x 104 M-1 from Fig. S2. We were interested to determine whether the fluorescence of the ZnO quantum dots could be completely quenched by thioacetamide.
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Therefore, the fluorescence quenching experiments were carried out by keeping the concentration of thioacetamide in the 200 -1000 µM range. Approximately, 800 µM of thioacetamide completely
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quenched the fluorescence intensity of the ZnO quantum dots (Fig. S3, Supplementary data).
3.2.3. Infra-red spectroscopic measurements Infra-red spectroscopy was used in order to further probe the interaction between thioacetamide and the ZnO quantum dots. The infra-red spectra of ZnO, thioacetamide and ZnO-thioacetamide reaction mixture have been displayed in Figs. 11, 12 and 13 respectively.
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Fig. 11. Infra-red absorption spectrum the ZnO quantum dots.
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Fig. 12. Infra-red absorption spectrum of thioacetamide.
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Fig. 13. Infra-red absorption spectrum of thioacetamide in the presence of the ZnO quantum dots.
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The Zn-O stretching vibration of ZnO has been reported to be in the 450-600 cm-1 region [27]. Therefore, the absorption band at 519 cm-1 in Fig.11 may be attributed to the vibration
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of the Zn-O bond. The other bands observed in the infra-red spectrum could be assigned to
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the different vibrational modes of methanol (used during the synthesis) adsorbed on the surface of the quantum dots. The sharp absorption band observed at 1650 cm-1 in Fig. 12 has
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been assigned to the in-plane bending mode of the –NH2 group of thioacetamide by Suzuki [28]. The 1650 cm-1 vibrational band of the substrate was not observed in the presence of the
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ZnO quantum dots (Fig. 13). However, a broad band was observed at 1564 cm-1 which could be due to the shift of the 1650 cm-1 band to lower wavenumber. This observation suggests that the -NH2 group of the thioacetamide may be bonded to the surface of the ZnO quantum dots. The sharp absorption band observed at 718 cm-1 in Fig. 12 has been assigned to the C=S stretching mode of thioacetamide by Bala and co-workers [29]. This band was found to disappear in the presence of the ZnO quantum dots (Fig.13). Additionally, new bands were seen in the range of 624-674 cm-1, which may be attributed to the shift of the 718 cm-1 band to lower wavenumber. It indicated the bonding of the sulphur atom of thioacetamide with the 16
ACCEPTED MANUSCRIPT surface of the ZnO quantum dots. The bonding might be through the lone pair of electrons as was indicated in the UV-visible absorption studies described under Section 3.2.1. Therefore, on the basis of the infra-red spectroscopic measurements, it may be suggested that the –NH2 group and the sulphur atom of thioacetamide participated in the bonding with the surface of
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the ZnO quantum dots.
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3.2.4. Dynamic light scattering (DLS) measurements
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DLS measurements were carried out in order to further probe the interaction of thioacetamide with the ZnO quantum dots. The plot of the intensity versus the hydrodynamic diameter of
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the ZnO quantum dots has been displayed in Fig. 14. The maxima in the figure corresponds
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Fig. 14. Plot of the intensity versus the hydrodynamic diameter of the ZnO quantum dots.
to a value of 11.4 nm, the hydrodynamic diameter of the majority of the particles. However, in the presence of 20 µM thioacetamide, the hydrodynamic diameter of the ZnO quantum dots increased to 15.2 nm (Fig. 15). The increase in the size of the ZnO quantum
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Fig. 15. Plot of the intensity versus the hydrodynamic diameter of the ZnO quantum dots in the
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presence of 20 µM thioacetamide.
dots in the presence of thioacetamide suggested a bonding between the two. Moreover, the
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zeta potential of the ZnO quantum dots was -0.63 mV which increased to -0.32 mV in the
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presence of 20 µM thioacetamide. It suggested that the thioacetamide molecules partially shield the surface charge on the ZnO quantum dots [30]. This shielding of the surface charge
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could be attributed to the presence of the thioacetamide molecules on the surface of the ZnO quantum dots. The DLS measurements were also carried out with other higher concentrations
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(60 and 100 µM) of thioacetamide. However, the hydrodynamic diameter of the ZnO did not increase beyond 15.2 nm upon increasing the concentration of thioacetamide. It appears that the surface of the ZnO quantum dots was already saturated with thioacetamide at a concentration of 20 µM. Hence further increase in the concentration of thioacetamide did not increase the hydrodynamic diameter of the quantum dots.
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4. Conclusions The UV-visible absorption studies suggested the bonding of the lone pair of electrons on the sulphur atom of thioacetamide with the surface of the ZnO quantum dots. The fluorescence band of the semiconductor was efficiently quenched by the addition of
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micromolar concentrations of thioacetamide. The quenching data was found to fit the Stern-
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Volmer relationship. The fluorescence quenching method provides a platform for the sensing of thioacetamide in the micromolar concentration range. The LOD of the method
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was calculated to be 2.14 nM. The infra-red spectroscopic measurements indicated the
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participation of both the amino group and the sulphur atom of thioacetamide in the bonding with the surface of the ZnO quantum dots. DLS measurements indicated that the
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thioacetamide molecules shielded the surface charge of the semiconductor.
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Acknowledgments
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The research work was funded by the University Grants Commission, New Delhi. We thank the Sophisticated Analytical Instrumentation Facility of our University for the TEM
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measurements. We acknowledge the Head, Department of Nanotechnology of our university
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for the XRD measurements.
Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version.
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ACCEPTED MANUSCRIPT [27] A.M. Pourrahimi, D. Liu, V. Ström, M.S. Hedenqvist, R.T. Olsson, U.W. Gedde, Heat treatment of Zno nanoparticles: new methods to achieve high-purity nanoparticles for highvoltage applications, J. Mater. Chem. A 3 (2015) 17190-17200. [28] I. Suzuki, Infrared spectra and normal vibrations of thioamides. II. Thioacetamide, B. Chem. Soc. Jpn. 35 (1962) 1449-1456.
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[29] S.S. Bala, P.K. Panja, P.N. Ghosh, Vibrational spectra of acetamide and thioacetamide
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at low temperature: Fermi resonance and hydrogen bond studies, J. Mol. Struct. 157 (1987) 339-351.
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[30] D. Xue, H. Wang, Y. Zhang, Specific and sensitive colorimetric detection of Al 3+ using
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5- mercaptomethyltetrazole capped gold nanoparticles in aqueous solution, Talanta 119
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(2014) 306-311.
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Graphical abstract
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ACCEPTED MANUSCRIPT Highlights
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Fluorescence of ZnO was quenched by micromolar concentration of thioacetamide. Quenching was found to follow the Stern-Volmer relationship. Surface charge of ZnO was shielded by the thioacetamide molecules. Amino group and sulphur atom of thioacetamide interact with the ZnO surface.
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Dipika Saha, Devendra P.S. Negi PII: DOI: Reference:
S1386-1425(17)30682-0 doi: 10.1016/j.saa.2017.08.053 SAA 15410
To appear in:
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Received date: Revised date: Accepted date:
24 May 2017 4 August 2017 17 August 2017
Please cite this article as: Dipika Saha, Devendra P.S. Negi , Spectroscopic investigations on the interaction of thioacetamide with ZnO quantum dots and application for its fluorescence sensing, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2017), doi: 10.1016/j.saa.2017.08.053
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ACCEPTED MANUSCRIPT Spectroscopic investigations on the interaction of thioacetamide with ZnO quantum dots and application for its fluorescence sensing
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Dipika Saha, Devendra P. S. Negi*
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Centre for Advanced Studies in Chemistry, North-Eastern Hill University, Shillong 793022,
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India
*
Corresponding author
E-mail address: [email protected]
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Abstract The purpose of the present work was to develop a method for the sensing of thioacetamide by using spectroscopic techniques. Thioacetamide is a carcinogen and it is important to detect its
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presence in food-stuffs. Semiconductor quantum dots are frequently employed as sensing probes since their absorption and fluorescence properties are highly sensitive to the
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interaction with substrates present in the solution. In the present work, the interaction
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between thioacetamide and ZnO quantum dots has been investigated by using UV-visible,
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fluorescence and infrared spectroscopy. Besides, dynamic light scattering (DLS) has also been utilized for the interaction studies. UV-visible absorption studies indicated the bonding
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of the lone pair of sulphur atom of thioacetamide with the surface of the semiconductor. The fluorescence band of the ZnO quantum dots was found to be quenched in the presence of
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micromolar concentrations of thioacetamide. The quenching was found to follow the Stern-
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Volmer relationship. The Stern-Volmer constant was evaluated to be 1.20 x 105 M-1. Infrared spectroscopic measurements indicated the participation of the –NH2 group and the sulphur
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atom of thioacetamide in bonding with the surface of the ZnO quantum dots. DLS measurements indicated that the surface charge of the semiconductor was shielded by the
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thioacetamide molecules.
Keywords: zinc oxide quantum dots, thioacetamide, fluorescence, quenching, semiconductor
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1. Introduction Studies on the interaction of semiconductor nanoparticles (NPs) with organic/biological molecules is useful from the viewpoint of sensing [1-6] and biological labelling [7-8].
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Analytical techniques such as infra- red, fluorescence and UV-visible spectroscopy are powerful tools for the characterization of the semiconductor-organic molecule interaction.
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Infra-red spectroscopy provides useful information on the functional groups of the organic
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molecule involved in the bonding with the semiconductor NPs. For example, Chatterjee et al.
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reported the interaction of biofunctionalized CdS NPs with adenine. They suggested the coordination of the N-7 nitrogen of adenine to the cadmium atoms attached to the surface of
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the CdS NPs on the basis of the infra-red spectroscopic studies [9]. Fluorescence spectroscopy is a useful tool for studying the interaction of fluorescent semiconductor NPs
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with the substrate molecules. The fluorescence of the semiconductor NPs may be quenched
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or enhanced by its interaction with an analyte [10]. For example, the selective sensing of 1,2,4 – trihydroxybenzene was reported using histidine-stabilized ZnS nanospheres as a
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fluorescent probe [6]. UV-visible spectroscopy provides useful information on the groundstate interaction between the additive and the semiconductor.
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ZnO is a wide band gap semiconductor and is considered to be more environment friendly in comparison to CdS because of the toxicity associated with cadmium. Recently, some groups have reported studies on the interaction of this semiconductor with biomolecules [1113 ]. Thioacetamide is a carcinogen and prolonged exposure to such a substance causes bile duct proliferation and liver cirrhosis with histology similar to that produced by chronic viral hepatitis in humans [14]. The experimental hepatotoxicity of thioacetamide in rodents was first studied in 1948 in response to its detection in orange juice following its use as a
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ACCEPTED MANUSCRIPT fungicide in orange groves [15]. Therefore, it is important to develop a method for the detection of thioacetamide in low concentration range. To the best of our knowledge, there is no previous report in the literature on fluorescence based method for thioacetamide sensing. In the present work, we have investigated the interaction of thioacetamide with colloidal ZnO quantum dots by using UV-visible, fluorescence and infra-red spectroscopic techniques.
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DLS measurements were also carried out to get further insights into the interaction between
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thioacetamide and the semiconductor.
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2. Material and methods
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2.1. Materials
All the chemicals used for the research work were of analytical reagent grade. Zinc acetate
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dihydrate and thioacetamide were purchased from Himedia (Mumbai, India). Potassium
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hydroxide pellets were purchased from Merck (India) Limited (Mumbai). Methanol was obtained from Sisco Research Laboratory (Mumbai India).
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2.2. Instrumentation
UV-visible absorption spectra were recorded on a PerkinElmer Lambda 25 (Singapore)
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absorption spectrophotometer. Hitachi FL 4500 (Japan) spectrofluorimeter was used to obtain the steady state fluorescence spectra. Infrared spectra were recorded on a PerkinElmer BX FTIR system (Singapore). Transmission electron microscopy (TEM) measurements were carried out using a JEM 2100 (USA) instrument operating at 100 kV. A drop of the ZnO colloidal solution was dropped on a copper grid and allowed to dry at room temperature. The hydrodynamic diameter of the colloidal particles was determined by DLS measurements by using Malvern Zetasizer Nano ZS90 instrument (UK). The instrument operated at 633 nm which was fixed at 900 scattering angle and the temperature was maintained by the built-in 4
ACCEPTED MANUSCRIPT Peltier temperature control unit of the instrument at 21 0C. X-ray Diffraction measurements were carried out using an Explorer powder diffractometer from GNR Analytical Instruments Group (Italy).
2.3. Synthesis of colloidal ZnO solution
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ZnO quantum dots were prepared by using a reported sol-gel method [16] with slight
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modifications. Briefly, zinc acetate dihydrate (0.9790 g) and potassium hydroxide (0.4859 g) were dissolved in 42 ml and 23 ml of methanol (solvent) respectively. The zinc acetate
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solution was stirred and refluxed to 60 0C for half an hour. The solution of potassium
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hydroxide was added drop wise to the zinc acetate suspension. The solution was stirred for five minutes to ensure proper mixing of the two components and then the solution was
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refluxed for 15 min at 60 0C. During the reaction, the solution turned from turbid to transparent. The transparent solution was then cooled to room temperature and stored in
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refrigerator for further use. The concentration of ZnO thus prepared was calculated to be 8.8
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x 10-2 M on the basis of the concentration of zinc precursor used in the synthesis.
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2.4. Preparation of samples for fluorescence measurements Freshly prepared thioacetamide stock solutions were prepared and different concentrations
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(2 x 10-6 to 1 x 10-3 M) were added to the sample tubes containing 10 ml of colloidal ZnO solution. The sample tubes were shaken for 10 min in a water bath cum incubator shaker with a speed of 80 strokes per minute. After that, the samples were kept undisturbed for an hour. Then, the fluorescence spectra of the samples were recorded. The excitation wavelength was chosen such that the substrates did not absorb any light.
3. Results and discussion
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ACCEPTED MANUSCRIPT 3.1. Characterization of the colloidal ZnO solution The UV-visible absorption spectrum of the ZnO particles in methanol has been displayed in Fig. 1. The onset of absorption of the colloidal particles was ~ 360 nm which was
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Fig. 1. UV-visible absorption spectrum of the ZnO particles in methanol.
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considerably blue-shifted from that of the bulk ZnO (370 nm) [16]. It indicates the phenomenon of quantum size effect [17] in the formation of the semiconductor particles.
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Such semiconductor particles are called quantum dots. The fluorescence spectrum of the ZnO quantum dots has been displayed in Fig. 2. A broad peak at 512 nm was observed. This
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green fluorescence band has been attributed to the photogenerated trapped electrons tunneling to pre-existing trapped holes [18].
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Fig. 2. Fluorescence spectrum of the ZnO quantum dots in methanol. Excitation wavelength = 350
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Transmission electron microscopy was used in order to determine the shape and size of the
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ZnO quantum dots. The TEM image of the semiconductor particles has been displayed in
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Fig. 3. TEM image of the ZnO quantum dots.
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ACCEPTED MANUSCRIPT The average diameter of the ZnO quantum dots was calculated to be 3.7 nm from the image shown in Fig. 3. The higher magnification TEM image shows the lattice planes in a single
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ZnO particle (Fig. 4).
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Fig. 4 Higher magnification TEM image of the ZnO quantum dots.
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The crystalline nature of the ZnO particles was ascertained by the selected area electron
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diffraction (SAED) pattern shown in Fig. 5.
Fig. 5. SAED pattern of the ZnO quantum dots.
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ACCEPTED MANUSCRIPT The concentric rings observed in Fig. 5 may be attributed to the diffraction from various planes of crystalline ZnO [19]. The ZnO quantum dots were further characterized by powder X-ray diffraction (XRD) measurements. The XRD pattern of the quantum dots has been displayed in Fig. 6. The peaks in the pattern indicated high crystallinity of the material
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and were assigned to the (100), (002), (101), (102), (110), (103), (112) and (202) planes of
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the wurtzite-type ZnO structure [16].
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3.2. Spectroscopic investigation of the interaction between thioacetamide and the ZnO quantum dots
3.2.1. UV-visible spectroscopic measurements The ground state interaction between thioacetamide and the ZnO quantum dots was investigated by using UV-visible absorption spectroscopy. The structure of thioacetamide has been displayed in Fig. 7. The lone pair of electrons present on the nitrogen and sulphur atoms of thioacetamide are potential sites for bonding with another chemical species.
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Fig. 7. Chemical structure of thioacetamide.
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The original colloidal ZnO solution was diluted by adding excess methanol in order to bring the value of absorbance within 1.0. The UV-visible absorption spectra of the ZnO quantum
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dots, 20 µM thioacetamide and their reaction mixture have been displayed in Fig. 8.
ZnO 20 M TA ZnO + 20 M TA
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Fig. 8. UV-visible absorption spectra of ZnO quantum dots, 20 µM thioacetamide (TA) and their reaction mixture.
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ACCEPTED MANUSCRIPT There was no shift in the position of the absorption band (λmax ~ 322 nm) of the ZnO quantum dots upon addition of thioacetamide. The position of the 263 nm absorption band of thioacetamide remained unchanged in the presence of the semiconductor. However, the 223 nm absorption band of thioacetamide was found to disappear in the presence of the ZnO quantum dots. Earlier studies on the electronic transitions in thioacetamide have assigned the
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223 nm absorption band to the excitation of the lone pair electrons on the sulphur atom to the
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anti-bonding pi orbital ( 1ns → 1п* ) [20]. The fact that the 223 nm band of thioacetamide
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disappeared in the presence of the ZnO quantum dots indicates that lone pair of electrons on the sulphur atom was bonded to the ZnO quantum dots. As a result, the electronic transition
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associated with this lone pair ( 1ns → 1п* ) was not observed.
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3.2.2. Fluorescence spectroscopic measurements
Steady-state fluorescence spectroscopy was used in order to further probe the interaction
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between thioacetamide and the ZnO quantum dots. The fluorescence properties of the
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semiconductor was investigated in the presence of wide range of concentrations of the substrate. The fluorescence spectra of the ZnO quantum dots in the absence and presence of
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700 0 M 2 M 4 M 6 M 8 M 10 M
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Fig. 9. Fluorescence spectra of the ZnO quantum dots in the absence and presence of various
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concentrations of thioacetamide as indicated in the inset. Excitation wavelength = 340 nm.
The emission band of the ZnO quantum dots was increasingly quenched by increasing
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concentration of thioacetamide without any change in the position of its λmax. The quenching
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of the fluorescence of a semiconductor is generally attributed to the transfer of the photogenerated electron or hole to the substrate molecules present in the solution.
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Thioacetamide is likely to be a good hole acceptor due the presence of a lone pair of electrons on the nitrogen and sulphur atoms (Fig. 7). Molecular oxygen is known to be scavenger of
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electrons [21]. Therefore, the photogenerated electrons might be scavenged by the oxygen molecules present in the solution. The quenching data was fitted to the Stern-Volmer relationship [22], given by I0 / I = 1 + KSV [Q] where I0 and I are the fluorescence intensity of the fluorophore in the absence and presence of the quencher, Q. The Stern-Volmer constant for the quenching process is denoted by KSV.
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ACCEPTED MANUSCRIPT The Stern-Volmer plot for the quenching of the ZnO fluorescence by thioacetamide has been displayed in Fig. 10.
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Fig. 10. Stern-Volmer plot for the quenching of the ZnO fluorescence by thioacetamide.
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From this plot, the value of KSV was calculated to be 1.20 x 105 M-1. The linear dynamic
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range (LDR) of the method is thus 0-10 µM. The fluorescence quenching method described above provides a platform for the sensing of thioacetamide in the micromolar concentration
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range. The concentration of thioacetamide in an unknown sample could be determined by using Fig. 10 from the extent of quenching (I0/I) of ZnO fluorescence caused by the substrate.
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The limit of detection (LOD) and limit of quantification (LOQ) of the method were calculated using the formulae 3σ/S and 10σ/S, where σ is the standard deviation of the responses of the blank solutions and S is the slope of the calibration curve [23, 24]. The calibration curve has been displayed as Fig. S4 of the Supplementary data. On the basis of the calculations, the LOD and LOQ for thioacetamide on the basis of the fluorescence quenching method has been calculated to be 2.14 nM and 7.14 nM respectively. The comparison of the
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ACCEPTED MANUSCRIPT LOD and LDR of the present fluorescence quenching method with the other analytical methods reported in the literature has been summarized in Table 1. Table 1 Comparison of the various analytical methods for the detection of thioacetamide. LOD
LDR
Reference
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Voltammetry
0.84 µM
5-60 µM
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Amperometry
3 µM
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Fluorimetry
2.14 nM
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From Table 1 it is evident that the present method of thioacetamide sensing is much more sensitive compared to the earlier reported methods.
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Upon further increasing the concentration of thioacetamide (20-100 µM), the extent of quenching was also found to increase (Fig. S1, Supplementary data). The Stern-Volmer plot
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for the quenching process has been displayed in Fig. S2 of the supplementary data. The value
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of KSV was determined to be 5.4 x 104 M-1 from Fig. S2. We were interested to determine whether the fluorescence of the ZnO quantum dots could be completely quenched by thioacetamide.
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Therefore, the fluorescence quenching experiments were carried out by keeping the concentration of thioacetamide in the 200 -1000 µM range. Approximately, 800 µM of thioacetamide completely
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quenched the fluorescence intensity of the ZnO quantum dots (Fig. S3, Supplementary data).
3.2.3. Infra-red spectroscopic measurements Infra-red spectroscopy was used in order to further probe the interaction between thioacetamide and the ZnO quantum dots. The infra-red spectra of ZnO, thioacetamide and ZnO-thioacetamide reaction mixture have been displayed in Figs. 11, 12 and 13 respectively.
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Fig. 11. Infra-red absorption spectrum the ZnO quantum dots.
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Fig. 12. Infra-red absorption spectrum of thioacetamide.
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Fig. 13. Infra-red absorption spectrum of thioacetamide in the presence of the ZnO quantum dots.
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The Zn-O stretching vibration of ZnO has been reported to be in the 450-600 cm-1 region [27]. Therefore, the absorption band at 519 cm-1 in Fig.11 may be attributed to the vibration
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of the Zn-O bond. The other bands observed in the infra-red spectrum could be assigned to
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the different vibrational modes of methanol (used during the synthesis) adsorbed on the surface of the quantum dots. The sharp absorption band observed at 1650 cm-1 in Fig. 12 has
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been assigned to the in-plane bending mode of the –NH2 group of thioacetamide by Suzuki [28]. The 1650 cm-1 vibrational band of the substrate was not observed in the presence of the
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ZnO quantum dots (Fig. 13). However, a broad band was observed at 1564 cm-1 which could be due to the shift of the 1650 cm-1 band to lower wavenumber. This observation suggests that the -NH2 group of the thioacetamide may be bonded to the surface of the ZnO quantum dots. The sharp absorption band observed at 718 cm-1 in Fig. 12 has been assigned to the C=S stretching mode of thioacetamide by Bala and co-workers [29]. This band was found to disappear in the presence of the ZnO quantum dots (Fig.13). Additionally, new bands were seen in the range of 624-674 cm-1, which may be attributed to the shift of the 718 cm-1 band to lower wavenumber. It indicated the bonding of the sulphur atom of thioacetamide with the 16
ACCEPTED MANUSCRIPT surface of the ZnO quantum dots. The bonding might be through the lone pair of electrons as was indicated in the UV-visible absorption studies described under Section 3.2.1. Therefore, on the basis of the infra-red spectroscopic measurements, it may be suggested that the –NH2 group and the sulphur atom of thioacetamide participated in the bonding with the surface of
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the ZnO quantum dots.
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3.2.4. Dynamic light scattering (DLS) measurements
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DLS measurements were carried out in order to further probe the interaction of thioacetamide with the ZnO quantum dots. The plot of the intensity versus the hydrodynamic diameter of
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the ZnO quantum dots has been displayed in Fig. 14. The maxima in the figure corresponds
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to a value of 11.4 nm, the hydrodynamic diameter of the majority of the particles. However, in the presence of 20 µM thioacetamide, the hydrodynamic diameter of the ZnO quantum dots increased to 15.2 nm (Fig. 15). The increase in the size of the ZnO quantum
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Fig. 15. Plot of the intensity versus the hydrodynamic diameter of the ZnO quantum dots in the
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dots in the presence of thioacetamide suggested a bonding between the two. Moreover, the
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zeta potential of the ZnO quantum dots was -0.63 mV which increased to -0.32 mV in the
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presence of 20 µM thioacetamide. It suggested that the thioacetamide molecules partially shield the surface charge on the ZnO quantum dots [30]. This shielding of the surface charge
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could be attributed to the presence of the thioacetamide molecules on the surface of the ZnO quantum dots. The DLS measurements were also carried out with other higher concentrations
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(60 and 100 µM) of thioacetamide. However, the hydrodynamic diameter of the ZnO did not increase beyond 15.2 nm upon increasing the concentration of thioacetamide. It appears that the surface of the ZnO quantum dots was already saturated with thioacetamide at a concentration of 20 µM. Hence further increase in the concentration of thioacetamide did not increase the hydrodynamic diameter of the quantum dots.
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4. Conclusions The UV-visible absorption studies suggested the bonding of the lone pair of electrons on the sulphur atom of thioacetamide with the surface of the ZnO quantum dots. The fluorescence band of the semiconductor was efficiently quenched by the addition of
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micromolar concentrations of thioacetamide. The quenching data was found to fit the Stern-
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Volmer relationship. The fluorescence quenching method provides a platform for the sensing of thioacetamide in the micromolar concentration range. The LOD of the method
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was calculated to be 2.14 nM. The infra-red spectroscopic measurements indicated the
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participation of both the amino group and the sulphur atom of thioacetamide in the bonding with the surface of the ZnO quantum dots. DLS measurements indicated that the
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thioacetamide molecules shielded the surface charge of the semiconductor.
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Acknowledgments
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The research work was funded by the University Grants Commission, New Delhi. We thank the Sophisticated Analytical Instrumentation Facility of our University for the TEM
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measurements. We acknowledge the Head, Department of Nanotechnology of our university
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for the XRD measurements.
Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version.
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[21] P.F. Schwarz, N.J. Turro, S.H. Bossmann, A.M. Braun, A-M.A. Abdel Wahab, H. Durr, J. Phys. Chem. B 101 (1997) 7127.
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[22] J.R. Lackowicz, Principles of fluorescence spectroscopy, third ed., Springer, Singapore, 2006.
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[23] R. İlktaç, N. Aksuner, E. Henden, Selective and sensitive fluorimetric determination of carbendazim in apple and orange after preconcentration with magnetite-molecularly
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imprinted polymer, Spectrochim. Acta A 174 (2017) 86-93.
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[24] B. Sen, S.K. Sheet, R. Thounaojam, R. Jamatia, A.K. Pal, K. Aguan, S. Khatua, A coumarin based Schiff base probe for selective fluorescence detection of Al3+ and its
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ACCEPTED MANUSCRIPT [27] A.M. Pourrahimi, D. Liu, V. Ström, M.S. Hedenqvist, R.T. Olsson, U.W. Gedde, Heat treatment of Zno nanoparticles: new methods to achieve high-purity nanoparticles for highvoltage applications, J. Mater. Chem. A 3 (2015) 17190-17200. [28] I. Suzuki, Infrared spectra and normal vibrations of thioamides. II. Thioacetamide, B. Chem. Soc. Jpn. 35 (1962) 1449-1456.
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at low temperature: Fermi resonance and hydrogen bond studies, J. Mol. Struct. 157 (1987) 339-351.
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[30] D. Xue, H. Wang, Y. Zhang, Specific and sensitive colorimetric detection of Al 3+ using
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5- mercaptomethyltetrazole capped gold nanoparticles in aqueous solution, Talanta 119
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ACCEPTED MANUSCRIPT Highlights
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Fluorescence of ZnO was quenched by micromolar concentration of thioacetamide. Quenching was found to follow the Stern-Volmer relationship. Surface charge of ZnO was shielded by the thioacetamide molecules. Amino group and sulphur atom of thioacetamide interact with the ZnO surface.
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