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Cadmium chalcogenide derived fluorescent quanta-sensor for melamine detection
Accepted Manuscript Title: Cadmium chalcogenide derived fluorescent quanta-sensor for melamine detection Authors: Suman Singh, Vishaldeep Kaur, Nishant Kumar, Mayank Garg, Satish Kumar Pandey, Vijay Kumar Meena PII: DOI: Reference:
S0925-4005(18)31169-9 https://doi.org/10.1016/j.snb.2018.06.063 SNB 24900
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
5-12-2017 4-6-2018 13-6-2018
Please cite this article as: Singh S, Kaur V, Kumar N, Garg M, Pandey SK, Meena VK, Cadmium chalcogenide derived fluorescent quanta-sensor for melamine detection, Sensors and Actuators: B. Chemical (2018), https://doi.org/10.1016/j.snb.2018.06.063 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 proof before it is published in its final 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.
Cadmium chalcogenide derived fluorescent quanta-sensor for melamine detection Suman Singh1,2*, Vishaldeep Kaur1#, Nishant Kumar
1,2#
, Mayank Garg1,2, Satish Kumar
Pandey1, Vijay Kumar Meena1 Central Scientific Instruments Organisation (CSIR-CSIO), Chandigarh, India
2
Academy of Scientific and Innovative Research (AcSIR-CSIO), New Delhi, India
*
Corresponding author: [email protected], [email protected]
#
Equal contribution of authors
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Graphical abstract
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Highlights:
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Synthesis and characterization of functionalized cadmium selenide (CdSe) quantum dots (QDs), referred as quanta-sensor Application of quanta-sensor for melamine estimation Optimization of parameters for quanta-sensor
Abstract:
The present work reports the detection of melamine (artificial protein) in milk using mercaptopropionic acid capped cadmium selenide (m-CdSe) quantum dots (QDs) as fluorescent labels, denoted as quanta-sensor. The m-CdSe QDs were prepared using aqueous chemical synthesis
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method and were characterized to get insight about their structural and functional properties. The
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formation of m-CdSe QDs was confirmed by using Transmission electron microscopy (TEM), Xray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) and X-ray
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photoelectron spectroscopy (XPS). The optical properties (absorption and emission) of m-CdSe
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QDs were studied as a function of cadmium salt concentration, selenide salt concentration and pH. The effect of incubation time with melamine on fluorescence of QDs was also studied in order to
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check the stability of quanta-sensor. The linearity range of this quanta-sensor was found to be 0.01
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nM to 60 μM, with LOD of 0.013 nM. The quanta-sensor exhibited good reproducibility,
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selectivity and acted as a potential probe for detection of melamine in milk samples.
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Keywords: Chalcogenides, melamine, quanta-sensor, cadmium, optical probe
1.
Introduction
Melamine (C3H6N6) 1,3,5-triazine-2,4,6-triamine, is an organic base chemical, used for the production of melamine formaldehyde resins, primarily for manufacturing of plastics, laminates, cooking utensils and adhesives [1]. However, owing to its high nitrogen content (66 % nitrogen 2
by mass) and analytical characteristics similar to that of protein, it is deliberately added to the milk, infant formula and other dairy-based products to artificially inflate the protein levels [2]. The protein content of food increases to about 4% on addition of 1% melamine. The prolong intake of melamine is associated with health hazards like nephrolithiasis, chronic kidney inflammation, and bladder carcinoma [3]. In body, melamine can get hydrolyzed to cyanuric acid which in turn can
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result in the formation of kidney stones depending upon the urine pH. This might cause organ failure and even death in humans and animals. In 2007, melamine was reported in wheat gluten and rice protein concentrate exported from the China, which was for manufacturing the pet food in the United States. The intake of food led to the renal failure and subsequent deaths of many pets [4]. Much worse, death of six infants and many cases of kidney stones in Chinese infants and children was reported in September 2008, due to the ingestion of the milk adulterated with
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melamine [4]. In China and US, the maximum residue limit (MRL) for infant formula has been set
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at 1.0 mg/kg and at 2.5 mg/kg for milk and other milk products, while in Europe, the Food Safety Authority has set the limit to 2.5 mg/kg [5].
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At present, a lot of confirmation and screening methods are available for the determination
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of melamine and its analogues which include liquid chromatography-mass spectrometry (LC-MS) [6], gas chromatography-mass spectrometry (GC-MS) [7], capillary zone electrophoresis (CE) [8],
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enzyme-linked immunoassay (ELISA) [9], waveguide fluorescence immunosensor [10-12] and
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surface enhanced Raman spectroscopy (SERS) [13]. Despite the high sensitivity of these conventional methods for melamine analysis, these strategies are generally not favored due to the necessity of complicated, expensive and labor-intensive instrumentation. Hence, a sensitive, facile,
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rapid and effective method is required for the investigation of melamine. In comparison to the aforementioned strategies, the fluorescent methods have emerged as
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promising tool for the analysis of melamine in recent years due to the reliability, simple instrumentation and ease of operation. A variety of molecular interactions can result in
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fluorescence quenching which can be implemented as a signal for the detection of analytes. These include excited-state reactions, molecular rearrangements, energy transfer, ground-state complex formation, and collisional quenching [14]. Among various fluorescent probes, the quantum dots (QDs), are extensively studied for the past few years as fluorescent labels owing to their excellent optical and electronic properties. The QDs exhibit broad absorption spectrum, size tunability due to quantum confinement and excellent photo-stability. In this work, a sensitive method is proposed 3
for the determination of melamine in milk using mercaptopropionic acid capped cadmium selenide (m-CdSe) quantum dots as fluorescent labels, referred as quanta-sensor in the manuscript. The fluorescence intensity of QDs got quenched on addition of melamine and quenching was proportional to the concentration of melamine. The quenching is the result of the interaction bewtween negatively charged QDs and positively charged melamine. Interference study performed
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with different constituents present in milk showed negligible interference. The real sample analysis with satisfactory results proved the system to be appropriate for the analysis of melamine in milk. 2. Materials and Methods 2.1 Chemicals
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All the chemicals used in the experiments were of analytical grade. Cadmium Chloride (CdCl2),
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manganese sulphate GR (MnSO4.H2O), potassium bromide (KBr), potassium fluoride (KF), selenium powder (Se) and sodium chloride (NaCl) were purchased from Loba Chemie Pvt. Ltd.,
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Mumbai. Calcium nitrate tetrahydrate (Ca(NO3)2.4H2O) was purchased from MolyChem,
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Mumbai, and ferrous chloride was procured from NICE Chemicals Pvt. Ltd., sodium hydroxide (NaOH) from Merck Specialties Pvt. Ltd. and sodium borohydride (NaBH4) from Spectrochem
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Pvt. Ltd., Mumbai were used without further purification. Mercaptopropionic acid (MPA) procured from ACROS, Belgium, melamine and trichloroacetic acid (TCA) acquired from
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2.2 Instrumentation
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Himedia Lab Pvt. Ltd., Mumbai, were used in the experiments.
Fluorescence studies were performed on a Varian Cary Eclipse Fluorescence Spectrophotometer
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that employs xenon lamp technology using excitation and emission slit of 5 nm. UV-Vis absorption spectra were acquired on a HITACHI U-3900H Spectrophotometer. The morphology and size of
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QDs was studied with the help of transmission electron microscope (TEM), JEOL make, ModelJEM 2100. The FTIR spectrum of m-CdSe QDs was recorded on Varian FTIR system (600 series, USA). The crystal structure of quantum dots was studied by recording their diffraction pattern on X-ray diffractometer (XRD) from Rigaku Ultima IV Type II. X-ray photon spectroscropy (XPS) was performed on PHI 5000 Versa probe II system, equipped with Kα monochromatic source and zeta potential studies were performed on Dynamic Light Scattering (DLS) system from Malvern. 4
2.3 Synthesis of m-CdSe quantum dots Fluorescent m-CdSe QDs were prepared using aqueous synthesis method. In this method, first sodium biselenide (NaHSe) aqueous solution was prepared by mixing 0.02g sodium borohydride (NaBH4) and 0.01 g selenide powder in 10 mL of nitrogen purged distilled water under ice bath
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condition, followed by stirring for about 30 minutes. A small outlet was connected to the flask in order to release the pressure from the resulting hydrogen. The dark red color of selenium powder disappeared and clear NaHSe is formed after 30 min. The resulting clear aqueous solution was used as selenium precursor solution. Secondly, the cadmium precursor solution was prepared by mixing 0.25 g of cadmium chloride and 350 µL mercaptopropionic acid (MPA) in 100 mL distilled water. The freshly prepared NaHSe solution was then transferred to the flask containing cadmium
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precursor solution. Under vigorous stirring, when the prepared oxygen-free NaHSe solution was injected, the m-CdSe QDs started forming as seeds. This solution was heated for 60 minutes at 80
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°C temperature. The appearance of pale yellow color confirmed the formation of m-CdSe QDs.
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The process of m-CdSe QDs formation was optimized by varying the concentrations of cadmium
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precursor and selenide as well as varying their pH. The results are discussed in subsequent section.
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2.4 Melamine detection using quanta-sensor
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For melamine detection study, first a stock solution of melamine (100 μM) was prepared in deionized water. Various concentrations of melamine were obtained by serial dilution of the stock solution. The fluorescence of QDs was recorded and then different concentrations of melamine
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were added sequentially. The change in fluorescence intensity of QDs was monitored as signal for
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melamine detection.
2.5 Pretreatment of the milk sample
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Milk samples were prepared according to a published report with a minor modification [15]. Typically, 1 mL of 2 M trichloroacetic acid (TCA) was added to 5 mL of raw milk. Afterwards, the solution was sonicated for 10 minutes to precipitate the protein. The precipitated protein was removed from the solution using centrifugation at 10,000 rpm for 10 minutes. The pH of supernatant was adjusted to 7.0 with NaOH, further filtered with 0.22 µm filter and diluted the solution 25-fold before using for experiment. For real sample analysis experiment, known 5
concentration of melamine was spiked into the milk sample and was assayed (pretreated) according to the above pre-treatment procedure. A known amount of this sample was incubated with m-CdSe QDs solution and fluorescence was recorded.
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3. Results and discussion Before going into the details of characterization data, the mechanism of QDs formation needs to be considered. Though the mechanism of formation of QDs is not well identified but it is presumed that the mechanism of m-CdSe formation consists of three steps; i) dissolution and equilibrium, ii) nucleation in the form formation of seed and iii) growth of final particles by homogeneous solution precipitation [16, 17]. The Cd2+ and Se2- ions are generated from the hydrolysis reaction of precursors. During chemical reaction, there is the possibility of formation of cadmium hydroxide
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[Cd(OH)2] which can act as nucleating sites for CdSe formation as mentioned in the literature [18,
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19]. After synthesis of QDs, the physical and optical characterizations were performed to
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understand their size, morphology, crystallinity and optical behavior.
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3.1 Physical characterization of m-CdSe QDs
The synthesized m-CdSe QDs were characterized for their size using TEM imaging. The TEM
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image (figure 1 (a)) shows synthesis of well dispersed QDs of size 3-5 nm. X-ray Diffraction
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(XRD) spectroscopy was used to study the size and crystalline structure of the QDs. From XRD study [figure 1 (b)], the quantum dots were found to exhibit cubic crystal structure having miller indices {111}, {220} (Joint Committee on Powder Diffraction Standards Card No. 19-0191). The
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maximum intensities were observed at 2θ values of 25.52° and 43.26°. The lattice constant as calculated from the miller indices is found to be 6.03 Å. The peaks exhibited significant line
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broadening, which shows that particles formed are small in size [20]. The size calculated for peak at 2θ values of 25.52° using Scherer formula is 0.50 nm. The size obtained from XRD is generally
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smaller than the size obtained by other methods due to the fact that XRD gives crystallite size, not actual particle size. Other techniques like scanning electron microscopy (SEM) and TEM measure particle size and one particle may contain several nano-crystallites. Further, X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR) were also used to understand the composition and elemental chemical states, the results of which are discussed in supplementary file and data is shown in Figure S1(a-c). 6
3.2 Optical characterization of m-CdSe QDs: Figure 2 (a) shows the absorption and emission spectra of mercaptopropionic acid capped cadmium selenide (m-CdSe) QDs. These spectra correspond to QDs, synthesized with final optimized parameters. The absorbance spectrum shows presence of broad band with maximum
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absorption at 400 nm, which is expected to arise from π-molecular orbitals for UV-visible transitions and its first excitonic peak. This absorption position is sensitive to the size of quantum dots and can cover wavelengths covering colors red to violet. Smaller the particle, the shorter will be the wavelength. The spectrum profile of m-CdSe QDs is smooth towards low wavelengths, indicating continuous absorption of photons by creating electrons and holes higher in their respective bands [21]. The band gap energy of m-CdSe QDs has been calculated from the tauc plot
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(data not shown) and is found to be 2.7 eV, comparatively blue shift from the bulk CdSe band energy (1.78 eV), which may be attributed to the small size of QDs. The size of QDs has been
–(2.6575×10-6)
+ (1.6242×10-3)
– (0.4277)λ + (41.57) Eq 1
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D = (1.6122×10-9)
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calculated using Peng’s equation which is as follows:
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where, D (nm) is the size of CdSe QDs, λ is the wavelength of the first excitonic peak. The size obtained using this equation is found to be 1.43 nm, which confirms the formation of small sized
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QDs.
The emission spectrum of m-CdSe QDs on the other hand exhibits a peak centered at 532
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nm, when excited at the wavelength of 360 nm [figure 2 (b)]. When QDs are irradiated, electrons get excited to conduction band and form excitons with pairing holes. And as a result of electron-
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hole combination, band edge emission appears. However, for small sized quantum dots, there is the possibility of holes being trapped in mid-gap states existing on surface [22]. Emission from
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these states is generally red-shifted relative to the absorption peak which can be observed in present case as well. The shift in emission w.r.t absorbance is referred as stokes effect, which is contributed by shape, structure and local chemical environments, experienced by QDs. The emission peak is
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also large as compared to absorption edge, this also confirms that the m-CdSe QDs have trap state emissions rather than band edge emissions. Further for optimization studies, the optical properties (absorption and emission) of m-CdSe QDs were studied and discussed as a function of cadmium salt concentration, selenide salt concentration and pH (Figure S2[I-II]; Supplementary data). 3.3 Application of quanta-sensor for melamine detection 7
Among various food adulterants, melamine is recognized as one of the major adulterants in India and many other countries. It is used widely as adulterant in milk powder, wheat gluten, chicken feed, and processed foods. Its prolonged consumption is a health threat to both humans as well as animals. Fluorescence spectroscopy is one of the easy and user friendly approach for being considered in sensing/detection of various adulterants. In this study, photoluminescence exhibited
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by m-CdSe QDs have been used as sensing approach for melamine detection. The change in fluorescence intensity of m-CdSe QDs in presence of melamine is taken as a signal for melamine sensing. The synthesized m-CdSe quantum dots exhibit maximum fluorescence intensity centered at 532 nm, (figure 2 b). When melamine was added to the quantum dots solution, the fluorescence intensity dramatically quenched. This quenching can be due to energy transfer or due to surface adsorption, which can contribute to change in surface state of quantum dots [23]. This quenching
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is attributed to the formation of strong complex between melamine and quantum dots as shown in
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pictorial in figure 3 (a). The capping of the as-synthesized CdSe QDs with mercaptopropionic acid (MPA) induces a negative charge on the QDs. The analyte ‘melamine’ on the other hand, is
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positively charged, hence complex forms easily between negatively charged QDs and positively
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charged melamine. Further to support this, zeta potential was also measured for m-CdSe QDs, melamine and m-CdSe QDs –melamine complex and the result is shown in figure 3 (b) i-iii. The
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m-CdSe QDs exhibited negative charged of -27.2 mV and melamine showed zeta potential of 14.9
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mV. On complex formation between quantum dots and melamine, the overall negative charge of m-CdSe QDs got reduced to -10.10 mV, which indicates the formation of complex between oppositely charged species. Moreover, the melamine is able to form self-assembling, high
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molecular weight complexes via organized intramolecular networks of hydrogen bonds and π-π aromatic ring stacking. These electrostatic charges as well as hydrogen bonding help in strong
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complex formation between QDs and melamine, as a result of which the process of energy transfer will take place. This energy transfer is experienced in the form of intensity quenching. Besides, at
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excessive concentration of melamine, there are chances of cross linking between NH…N hydrogen bonds between melamine molecules, which will hurdle the emission coming from quantum dots, hence a very high quenching will be observed (experimentally demonstrated in coming section). The effect of melamine concentration on the response of quanta-sensor has also been studied and result is shown in figure 4 (a - c). The fluorescence intensity of QDs continued to quench as the concentration of melamine was increased from 0.01 nM to 60.0 μM, with limit of 8
detection to be 0.013 nm, calculated using relationship LOD = 3xSD/slope (SD = standard deviation). The approved safe limits of melamine by U.S. Food and Drug Administration (FDA) is 8 μM for infant formula in China and 20 μM in both the USA and EU and 1.2 μM in Codex Alimentarius Commission (CAC) [24, 25]. The excited state reactions, energy transfer, collisional quenching and complex formation are some of the factors which are responsible for the quenching
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process. The response of these synthesized optical probes has been compared with other probes also and has been shown in table S1, supplementary data. The response of m-CdSe as fluorescent quanta-sensor covers wide dynamic range. The reaction followed linear relation y = 249.5 + (16.3)x, with regression coefficient of 0.986. Figure 3 (c) shows plot between relative fluorescence intensity (F0/F) and quencher concentration which is melamine in present case. The plot has
such cases, the system follows the relation:
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Fo/F = (1 + Kqτo[Q]) (1 + Ka[Q])
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upward curvature which indicates that the system has both dynamic as well as static quenching. In
Eq 2.
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where Fo fluorescence intensity in absence of quencher, F is fluorescence intensity in presence of
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quencher, where Kq is the bimolecular quenching rate constant, τo is the excited state lifetime in the absence of quencher and Ka is the association constant of the complex. Dynamic quenching,
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also known as collision quenching, occurs when the excited fluorophore experiences contact with an atom or molecule that can facilitate non-radiative transitions to the ground state. Static
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quenching, on the other hand, occurs when fluorophore forms a stable complex with another molecule. The effect of incubation time of melamine with m-CdSe on fluorescence intensity has
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also been studied [figure 4 (d)]. For this, the QDs were incubated with a known concentration of melamine for different intervals (0 to 30 minutes) of time and monitored the fluorescence intensity
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of m-CdSe QDs. The graph shows that the reaction between melamine and QDs is instant and there is no change in fluorescence intensity with increase in incubation time. The fluorescence intensity of a system is highly sensitive to the pH of a solution. Therefore,
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effect of pH has been studied on complex formation between QDs and melamine [figure 5 (a)]. The melamine solution was prepared at different pH (3.0 to 11.0) using phosphate buffer and was incubated with known amount of m-CdSe QDs. At pH 3.0, the QDs-melamine complex showed minimum fluorescence intensity and as the pH was incremented from 3.0 to 7.0, the intensity started increasing, with maximum intensity at pH 7.0. The fluorescence intensity m-CdSe-
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melamine complex thereafter started decreasing, reaching saturation at pH 9.0 and 11.0. Thus, the optimal pH of 7.0 was chosen for further experiments. The optical probes (QDs) have been studied for their reproducibility towards melamine estimation [Figure 5 (b)]. For the study, five identical samples of QDs-melamine complex were prepared and recorded the fluorescence. As can be seen, all the five samples exhibited identical
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response. This shows that these optical probes are highly efficient for being used for melamine estimation.
The interferences of the common constituents of milk such as amino acids, proteins, sugar were investigated to demonstrate the selectivity of the prepared m-CdSe QDs towards the detection of melamine. For the study, the known concentration of cysteine, glycine, sucrose, tyrosine, BSA (bovine serum albumin), sodium arsenate and lead nitrate were added in the mixture consisting of
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QDs and melamine. As shown in figure 5 (c), the presence of these interferents caused slight
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variation in the fluorescence intensity of QDs-melamine complex, which is within the acceptable range of variation. No significant affect has been observed. This indicates high selectivity of the
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quanta-sensor towards the detection of melamine.
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Reliability of quanta-sensor to specifically detect melamine in real samples has also been studied for which spiking method was opted [Figure 5 (d)]. Before performing this study,
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melamine of different known concentrations (0.1 μM, 10.0 μM, & 20 μM) were spiked in the milk
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sample and it was pre-treated as explained in experimental section. These samples were incubated with constant volume of m-CdSe QDs. The change in their fluorescence intensity was monitored.
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With real samples also, the probes are effective in confirming the presence of melamine. Conclusion:
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A rapid, simple and economical assay has been demonstrated for melamine detection using fluorescent optical probes which are cadmium selenide QDs. The advantage of these optical probes
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is their easy synthesis and application as fluorescent sensor at room temperature. The probes doesn’t require any specific functionalization as the capping agent itself is capable of providing required chemistry for complex formation with melamine. The melamine detection is based on fluorescence quenching of m-CdSe QDs on complex formation between negatively charged QDs and positively charged melamine as well as through hydrogen bonding. The reaction between mCdSe QDs and melamine occurs immediately as confirmed from effect of reaction time studies, 10
which asserts fast reaction. Also, as compared to traditional methods, the complete procedure of melamine estimation including sample pre-treatment can be performed within 25-30 minutes. Therefore, it appears to be a promising select for onsite monitoring of melamine. For further future studies, we are targeting on development of miniaturized fluorescence reader which can be coupled
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with our quanta-sensor.
Acknowledgement:
The authors acknowledge Director, CSIR-CSIO for his constant encouragement and support. The work was financially supported by Department of Science & Technology (DST), New Delhi through its grant in aid scheme (Project No: GAP 0352). N.K acknowledges DST for his junior
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research fellowship (JRF) from the project GAP-0352. S.K.P acknowledges National Post-doc
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fellowship (NPDF) from SERB-DST, New Delhi.
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Biographies: Suman Singh is working as senior scientist in CSIR-Central Scientific Instruments Organisation (CSIR-CSIO), Chandigarh, India and did her PhD from Panjab University, Chandigarh, India. She
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is also a faculty of chemistry and member of Welfare association at Academy of Scientific and Innovative Research (AcSIR) at CSIR-CSIO, Chandigarh. Her area of research work is advanced materials, nanotechnology and(bio)sensors. She is working on synthesis and application of nanomaterials and their composites for application in bio/chemo/immunosensing of analytes for applications like health, environment, orthopaedic and security. Her work is published in reputed SCI journals and as book chapters.
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Vishaldeep Kaur has done her Masters (M.Tech) in Nanoscience and Nanotechnology from
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Panjab University, Chandigarh, India. She pursued her Masters training at Central Scientific
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and sensing applications of nanomaterials.
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Research Organization (CSIR-CSIO), Chandigarh, India. Her research interest include synthesis
Nishant Kumar has done his integrated dual degree (B.Tech-M.Tech) in Nanotechnology from
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Amity, Noida. He did his Masters Training at CSIR-Central Scientific Instruments Organisation (CSIR-CSIO), Chandigarh, India. Presently he is pursuing his PhD from Academy of Scientific
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and Innovative Research (AcSIR) and is recipient of senior research fellowship from CSIR, New Delhi. His interest is material synthesis and their electro-chemical applications. He has published
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his work in reputed SCI journals.
Mr. Mayank Garg has done his B.Tech in Biotechnology from UIET, Chandigarh. Presently he
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is pursuing his PhD from Academy of Scientific and Innovative Research (AcSIR) and is recipient of GATE- Junior Research Fellowship (GATE-JRF) from CSIR, New Delhi. His area of interest
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is synthesis of layered materials and their applications for pathogen detection and disease biomarkers. Dr. Satish Kumar Pandey did his Ph.D jointly from Panjab University and IMTECH, Chandigarh, India in 2015. He is recipient of BIRAC Young Innovator award, 2015 and National Post-Doc fellowship (NPDF) from SERB DST, New Delhi. Presently he is working as Pool
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Scientist in CSIR-CSIO, Chandigarh and his expertise is developing platforms for food borne pathogens by exploring surface components as specific markers. Vijay Kumar Meena is working as senior scientist in CSIR-Central Scientific Instruments Organisation (CSIR-CSIO), Chandigarh, India. He has done his Bachelors from PEC University
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of Technology, Chandigarh and M.Tech in Manufacturing Management from Birla Institute of Technology & Science (BITS), Pilani, Rajasthan, India. His research interest is design of
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orhtopedic implants and materials for biomedical and environmental applications.
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Figure Captions:
Figure 1: (a) TEM of m-CdSe QDs (b) XRD of m-CdSe QDs
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Figure 2: (a) Overlapped spectra of absorbance and emission of m-CdSe QDs (b) Emission spectra of m-CdSe QDs and its complex with melamine (concentration 20.0 μM, pH 7.0.
Figure 3: (a) Pictorial representation of complex formation between m-CdSe QDs and
melamine (b) Zeta potential of (i) m-CdSe QDs (ii) Melamine (iii) m-CdSe QDsmelamine concentration
Figure 4: (a) Fluorescence spectra recorded with quantasensor in the presence of different
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concentrations of melamine [0.01 nM to 60 μM] (b) Linearity graph (c) Fo/F vs
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quencher (melamine) concentration (d) Effect of incubation time with melamine on
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fluorescence signal of quant-sensor
Figure 5: (a) Fluorescence response of quanta-sensor as a function of pH for melamine
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detection (b) Reproducibility study of quanta-sensor (c) Selectivity study of
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quantasensor (d) Melamine estimation in spiked samples using quanta-sensor
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S0925-4005(18)31169-9 https://doi.org/10.1016/j.snb.2018.06.063 SNB 24900
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
5-12-2017 4-6-2018 13-6-2018
Please cite this article as: Singh S, Kaur V, Kumar N, Garg M, Pandey SK, Meena VK, Cadmium chalcogenide derived fluorescent quanta-sensor for melamine detection, Sensors and Actuators: B. Chemical (2018), https://doi.org/10.1016/j.snb.2018.06.063 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 proof before it is published in its final 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.
Cadmium chalcogenide derived fluorescent quanta-sensor for melamine detection Suman Singh1,2*, Vishaldeep Kaur1#, Nishant Kumar
1,2#
, Mayank Garg1,2, Satish Kumar
Pandey1, Vijay Kumar Meena1 Central Scientific Instruments Organisation (CSIR-CSIO), Chandigarh, India
2
Academy of Scientific and Innovative Research (AcSIR-CSIO), New Delhi, India
*
Corresponding author: [email protected], [email protected]
#
Equal contribution of authors
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Graphical abstract
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Highlights:
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Synthesis and characterization of functionalized cadmium selenide (CdSe) quantum dots (QDs), referred as quanta-sensor Application of quanta-sensor for melamine estimation Optimization of parameters for quanta-sensor
Abstract:
The present work reports the detection of melamine (artificial protein) in milk using mercaptopropionic acid capped cadmium selenide (m-CdSe) quantum dots (QDs) as fluorescent labels, denoted as quanta-sensor. The m-CdSe QDs were prepared using aqueous chemical synthesis
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method and were characterized to get insight about their structural and functional properties. The
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formation of m-CdSe QDs was confirmed by using Transmission electron microscopy (TEM), Xray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) and X-ray
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photoelectron spectroscopy (XPS). The optical properties (absorption and emission) of m-CdSe
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QDs were studied as a function of cadmium salt concentration, selenide salt concentration and pH. The effect of incubation time with melamine on fluorescence of QDs was also studied in order to
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check the stability of quanta-sensor. The linearity range of this quanta-sensor was found to be 0.01
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nM to 60 μM, with LOD of 0.013 nM. The quanta-sensor exhibited good reproducibility,
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selectivity and acted as a potential probe for detection of melamine in milk samples.
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Keywords: Chalcogenides, melamine, quanta-sensor, cadmium, optical probe
1.
Introduction
Melamine (C3H6N6) 1,3,5-triazine-2,4,6-triamine, is an organic base chemical, used for the production of melamine formaldehyde resins, primarily for manufacturing of plastics, laminates, cooking utensils and adhesives [1]. However, owing to its high nitrogen content (66 % nitrogen 2
by mass) and analytical characteristics similar to that of protein, it is deliberately added to the milk, infant formula and other dairy-based products to artificially inflate the protein levels [2]. The protein content of food increases to about 4% on addition of 1% melamine. The prolong intake of melamine is associated with health hazards like nephrolithiasis, chronic kidney inflammation, and bladder carcinoma [3]. In body, melamine can get hydrolyzed to cyanuric acid which in turn can
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result in the formation of kidney stones depending upon the urine pH. This might cause organ failure and even death in humans and animals. In 2007, melamine was reported in wheat gluten and rice protein concentrate exported from the China, which was for manufacturing the pet food in the United States. The intake of food led to the renal failure and subsequent deaths of many pets [4]. Much worse, death of six infants and many cases of kidney stones in Chinese infants and children was reported in September 2008, due to the ingestion of the milk adulterated with
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melamine [4]. In China and US, the maximum residue limit (MRL) for infant formula has been set
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at 1.0 mg/kg and at 2.5 mg/kg for milk and other milk products, while in Europe, the Food Safety Authority has set the limit to 2.5 mg/kg [5].
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At present, a lot of confirmation and screening methods are available for the determination
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of melamine and its analogues which include liquid chromatography-mass spectrometry (LC-MS) [6], gas chromatography-mass spectrometry (GC-MS) [7], capillary zone electrophoresis (CE) [8],
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enzyme-linked immunoassay (ELISA) [9], waveguide fluorescence immunosensor [10-12] and
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surface enhanced Raman spectroscopy (SERS) [13]. Despite the high sensitivity of these conventional methods for melamine analysis, these strategies are generally not favored due to the necessity of complicated, expensive and labor-intensive instrumentation. Hence, a sensitive, facile,
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rapid and effective method is required for the investigation of melamine. In comparison to the aforementioned strategies, the fluorescent methods have emerged as
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promising tool for the analysis of melamine in recent years due to the reliability, simple instrumentation and ease of operation. A variety of molecular interactions can result in
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fluorescence quenching which can be implemented as a signal for the detection of analytes. These include excited-state reactions, molecular rearrangements, energy transfer, ground-state complex formation, and collisional quenching [14]. Among various fluorescent probes, the quantum dots (QDs), are extensively studied for the past few years as fluorescent labels owing to their excellent optical and electronic properties. The QDs exhibit broad absorption spectrum, size tunability due to quantum confinement and excellent photo-stability. In this work, a sensitive method is proposed 3
for the determination of melamine in milk using mercaptopropionic acid capped cadmium selenide (m-CdSe) quantum dots as fluorescent labels, referred as quanta-sensor in the manuscript. The fluorescence intensity of QDs got quenched on addition of melamine and quenching was proportional to the concentration of melamine. The quenching is the result of the interaction bewtween negatively charged QDs and positively charged melamine. Interference study performed
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with different constituents present in milk showed negligible interference. The real sample analysis with satisfactory results proved the system to be appropriate for the analysis of melamine in milk. 2. Materials and Methods 2.1 Chemicals
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All the chemicals used in the experiments were of analytical grade. Cadmium Chloride (CdCl2),
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manganese sulphate GR (MnSO4.H2O), potassium bromide (KBr), potassium fluoride (KF), selenium powder (Se) and sodium chloride (NaCl) were purchased from Loba Chemie Pvt. Ltd.,
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Mumbai. Calcium nitrate tetrahydrate (Ca(NO3)2.4H2O) was purchased from MolyChem,
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Mumbai, and ferrous chloride was procured from NICE Chemicals Pvt. Ltd., sodium hydroxide (NaOH) from Merck Specialties Pvt. Ltd. and sodium borohydride (NaBH4) from Spectrochem
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Pvt. Ltd., Mumbai were used without further purification. Mercaptopropionic acid (MPA) procured from ACROS, Belgium, melamine and trichloroacetic acid (TCA) acquired from
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2.2 Instrumentation
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Himedia Lab Pvt. Ltd., Mumbai, were used in the experiments.
Fluorescence studies were performed on a Varian Cary Eclipse Fluorescence Spectrophotometer
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that employs xenon lamp technology using excitation and emission slit of 5 nm. UV-Vis absorption spectra were acquired on a HITACHI U-3900H Spectrophotometer. The morphology and size of
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QDs was studied with the help of transmission electron microscope (TEM), JEOL make, ModelJEM 2100. The FTIR spectrum of m-CdSe QDs was recorded on Varian FTIR system (600 series, USA). The crystal structure of quantum dots was studied by recording their diffraction pattern on X-ray diffractometer (XRD) from Rigaku Ultima IV Type II. X-ray photon spectroscropy (XPS) was performed on PHI 5000 Versa probe II system, equipped with Kα monochromatic source and zeta potential studies were performed on Dynamic Light Scattering (DLS) system from Malvern. 4
2.3 Synthesis of m-CdSe quantum dots Fluorescent m-CdSe QDs were prepared using aqueous synthesis method. In this method, first sodium biselenide (NaHSe) aqueous solution was prepared by mixing 0.02g sodium borohydride (NaBH4) and 0.01 g selenide powder in 10 mL of nitrogen purged distilled water under ice bath
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condition, followed by stirring for about 30 minutes. A small outlet was connected to the flask in order to release the pressure from the resulting hydrogen. The dark red color of selenium powder disappeared and clear NaHSe is formed after 30 min. The resulting clear aqueous solution was used as selenium precursor solution. Secondly, the cadmium precursor solution was prepared by mixing 0.25 g of cadmium chloride and 350 µL mercaptopropionic acid (MPA) in 100 mL distilled water. The freshly prepared NaHSe solution was then transferred to the flask containing cadmium
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precursor solution. Under vigorous stirring, when the prepared oxygen-free NaHSe solution was injected, the m-CdSe QDs started forming as seeds. This solution was heated for 60 minutes at 80
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°C temperature. The appearance of pale yellow color confirmed the formation of m-CdSe QDs.
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The process of m-CdSe QDs formation was optimized by varying the concentrations of cadmium
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precursor and selenide as well as varying their pH. The results are discussed in subsequent section.
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2.4 Melamine detection using quanta-sensor
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For melamine detection study, first a stock solution of melamine (100 μM) was prepared in deionized water. Various concentrations of melamine were obtained by serial dilution of the stock solution. The fluorescence of QDs was recorded and then different concentrations of melamine
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were added sequentially. The change in fluorescence intensity of QDs was monitored as signal for
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melamine detection.
2.5 Pretreatment of the milk sample
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Milk samples were prepared according to a published report with a minor modification [15]. Typically, 1 mL of 2 M trichloroacetic acid (TCA) was added to 5 mL of raw milk. Afterwards, the solution was sonicated for 10 minutes to precipitate the protein. The precipitated protein was removed from the solution using centrifugation at 10,000 rpm for 10 minutes. The pH of supernatant was adjusted to 7.0 with NaOH, further filtered with 0.22 µm filter and diluted the solution 25-fold before using for experiment. For real sample analysis experiment, known 5
concentration of melamine was spiked into the milk sample and was assayed (pretreated) according to the above pre-treatment procedure. A known amount of this sample was incubated with m-CdSe QDs solution and fluorescence was recorded.
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3. Results and discussion Before going into the details of characterization data, the mechanism of QDs formation needs to be considered. Though the mechanism of formation of QDs is not well identified but it is presumed that the mechanism of m-CdSe formation consists of three steps; i) dissolution and equilibrium, ii) nucleation in the form formation of seed and iii) growth of final particles by homogeneous solution precipitation [16, 17]. The Cd2+ and Se2- ions are generated from the hydrolysis reaction of precursors. During chemical reaction, there is the possibility of formation of cadmium hydroxide
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[Cd(OH)2] which can act as nucleating sites for CdSe formation as mentioned in the literature [18,
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19]. After synthesis of QDs, the physical and optical characterizations were performed to
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understand their size, morphology, crystallinity and optical behavior.
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3.1 Physical characterization of m-CdSe QDs
The synthesized m-CdSe QDs were characterized for their size using TEM imaging. The TEM
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image (figure 1 (a)) shows synthesis of well dispersed QDs of size 3-5 nm. X-ray Diffraction
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(XRD) spectroscopy was used to study the size and crystalline structure of the QDs. From XRD study [figure 1 (b)], the quantum dots were found to exhibit cubic crystal structure having miller indices {111}, {220} (Joint Committee on Powder Diffraction Standards Card No. 19-0191). The
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maximum intensities were observed at 2θ values of 25.52° and 43.26°. The lattice constant as calculated from the miller indices is found to be 6.03 Å. The peaks exhibited significant line
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broadening, which shows that particles formed are small in size [20]. The size calculated for peak at 2θ values of 25.52° using Scherer formula is 0.50 nm. The size obtained from XRD is generally
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smaller than the size obtained by other methods due to the fact that XRD gives crystallite size, not actual particle size. Other techniques like scanning electron microscopy (SEM) and TEM measure particle size and one particle may contain several nano-crystallites. Further, X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR) were also used to understand the composition and elemental chemical states, the results of which are discussed in supplementary file and data is shown in Figure S1(a-c). 6
3.2 Optical characterization of m-CdSe QDs: Figure 2 (a) shows the absorption and emission spectra of mercaptopropionic acid capped cadmium selenide (m-CdSe) QDs. These spectra correspond to QDs, synthesized with final optimized parameters. The absorbance spectrum shows presence of broad band with maximum
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absorption at 400 nm, which is expected to arise from π-molecular orbitals for UV-visible transitions and its first excitonic peak. This absorption position is sensitive to the size of quantum dots and can cover wavelengths covering colors red to violet. Smaller the particle, the shorter will be the wavelength. The spectrum profile of m-CdSe QDs is smooth towards low wavelengths, indicating continuous absorption of photons by creating electrons and holes higher in their respective bands [21]. The band gap energy of m-CdSe QDs has been calculated from the tauc plot
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(data not shown) and is found to be 2.7 eV, comparatively blue shift from the bulk CdSe band energy (1.78 eV), which may be attributed to the small size of QDs. The size of QDs has been
–(2.6575×10-6)
+ (1.6242×10-3)
– (0.4277)λ + (41.57) Eq 1
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D = (1.6122×10-9)
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calculated using Peng’s equation which is as follows:
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where, D (nm) is the size of CdSe QDs, λ is the wavelength of the first excitonic peak. The size obtained using this equation is found to be 1.43 nm, which confirms the formation of small sized
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QDs.
The emission spectrum of m-CdSe QDs on the other hand exhibits a peak centered at 532
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nm, when excited at the wavelength of 360 nm [figure 2 (b)]. When QDs are irradiated, electrons get excited to conduction band and form excitons with pairing holes. And as a result of electron-
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hole combination, band edge emission appears. However, for small sized quantum dots, there is the possibility of holes being trapped in mid-gap states existing on surface [22]. Emission from
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these states is generally red-shifted relative to the absorption peak which can be observed in present case as well. The shift in emission w.r.t absorbance is referred as stokes effect, which is contributed by shape, structure and local chemical environments, experienced by QDs. The emission peak is
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also large as compared to absorption edge, this also confirms that the m-CdSe QDs have trap state emissions rather than band edge emissions. Further for optimization studies, the optical properties (absorption and emission) of m-CdSe QDs were studied and discussed as a function of cadmium salt concentration, selenide salt concentration and pH (Figure S2[I-II]; Supplementary data). 3.3 Application of quanta-sensor for melamine detection 7
Among various food adulterants, melamine is recognized as one of the major adulterants in India and many other countries. It is used widely as adulterant in milk powder, wheat gluten, chicken feed, and processed foods. Its prolonged consumption is a health threat to both humans as well as animals. Fluorescence spectroscopy is one of the easy and user friendly approach for being considered in sensing/detection of various adulterants. In this study, photoluminescence exhibited
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by m-CdSe QDs have been used as sensing approach for melamine detection. The change in fluorescence intensity of m-CdSe QDs in presence of melamine is taken as a signal for melamine sensing. The synthesized m-CdSe quantum dots exhibit maximum fluorescence intensity centered at 532 nm, (figure 2 b). When melamine was added to the quantum dots solution, the fluorescence intensity dramatically quenched. This quenching can be due to energy transfer or due to surface adsorption, which can contribute to change in surface state of quantum dots [23]. This quenching
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is attributed to the formation of strong complex between melamine and quantum dots as shown in
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pictorial in figure 3 (a). The capping of the as-synthesized CdSe QDs with mercaptopropionic acid (MPA) induces a negative charge on the QDs. The analyte ‘melamine’ on the other hand, is
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positively charged, hence complex forms easily between negatively charged QDs and positively
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charged melamine. Further to support this, zeta potential was also measured for m-CdSe QDs, melamine and m-CdSe QDs –melamine complex and the result is shown in figure 3 (b) i-iii. The
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m-CdSe QDs exhibited negative charged of -27.2 mV and melamine showed zeta potential of 14.9
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mV. On complex formation between quantum dots and melamine, the overall negative charge of m-CdSe QDs got reduced to -10.10 mV, which indicates the formation of complex between oppositely charged species. Moreover, the melamine is able to form self-assembling, high
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molecular weight complexes via organized intramolecular networks of hydrogen bonds and π-π aromatic ring stacking. These electrostatic charges as well as hydrogen bonding help in strong
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complex formation between QDs and melamine, as a result of which the process of energy transfer will take place. This energy transfer is experienced in the form of intensity quenching. Besides, at
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excessive concentration of melamine, there are chances of cross linking between NH…N hydrogen bonds between melamine molecules, which will hurdle the emission coming from quantum dots, hence a very high quenching will be observed (experimentally demonstrated in coming section). The effect of melamine concentration on the response of quanta-sensor has also been studied and result is shown in figure 4 (a - c). The fluorescence intensity of QDs continued to quench as the concentration of melamine was increased from 0.01 nM to 60.0 μM, with limit of 8
detection to be 0.013 nm, calculated using relationship LOD = 3xSD/slope (SD = standard deviation). The approved safe limits of melamine by U.S. Food and Drug Administration (FDA) is 8 μM for infant formula in China and 20 μM in both the USA and EU and 1.2 μM in Codex Alimentarius Commission (CAC) [24, 25]. The excited state reactions, energy transfer, collisional quenching and complex formation are some of the factors which are responsible for the quenching
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process. The response of these synthesized optical probes has been compared with other probes also and has been shown in table S1, supplementary data. The response of m-CdSe as fluorescent quanta-sensor covers wide dynamic range. The reaction followed linear relation y = 249.5 + (16.3)x, with regression coefficient of 0.986. Figure 3 (c) shows plot between relative fluorescence intensity (F0/F) and quencher concentration which is melamine in present case. The plot has
such cases, the system follows the relation:
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Fo/F = (1 + Kqτo[Q]) (1 + Ka[Q])
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upward curvature which indicates that the system has both dynamic as well as static quenching. In
Eq 2.
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where Fo fluorescence intensity in absence of quencher, F is fluorescence intensity in presence of
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quencher, where Kq is the bimolecular quenching rate constant, τo is the excited state lifetime in the absence of quencher and Ka is the association constant of the complex. Dynamic quenching,
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also known as collision quenching, occurs when the excited fluorophore experiences contact with an atom or molecule that can facilitate non-radiative transitions to the ground state. Static
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quenching, on the other hand, occurs when fluorophore forms a stable complex with another molecule. The effect of incubation time of melamine with m-CdSe on fluorescence intensity has
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also been studied [figure 4 (d)]. For this, the QDs were incubated with a known concentration of melamine for different intervals (0 to 30 minutes) of time and monitored the fluorescence intensity
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of m-CdSe QDs. The graph shows that the reaction between melamine and QDs is instant and there is no change in fluorescence intensity with increase in incubation time. The fluorescence intensity of a system is highly sensitive to the pH of a solution. Therefore,
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effect of pH has been studied on complex formation between QDs and melamine [figure 5 (a)]. The melamine solution was prepared at different pH (3.0 to 11.0) using phosphate buffer and was incubated with known amount of m-CdSe QDs. At pH 3.0, the QDs-melamine complex showed minimum fluorescence intensity and as the pH was incremented from 3.0 to 7.0, the intensity started increasing, with maximum intensity at pH 7.0. The fluorescence intensity m-CdSe-
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melamine complex thereafter started decreasing, reaching saturation at pH 9.0 and 11.0. Thus, the optimal pH of 7.0 was chosen for further experiments. The optical probes (QDs) have been studied for their reproducibility towards melamine estimation [Figure 5 (b)]. For the study, five identical samples of QDs-melamine complex were prepared and recorded the fluorescence. As can be seen, all the five samples exhibited identical
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response. This shows that these optical probes are highly efficient for being used for melamine estimation.
The interferences of the common constituents of milk such as amino acids, proteins, sugar were investigated to demonstrate the selectivity of the prepared m-CdSe QDs towards the detection of melamine. For the study, the known concentration of cysteine, glycine, sucrose, tyrosine, BSA (bovine serum albumin), sodium arsenate and lead nitrate were added in the mixture consisting of
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QDs and melamine. As shown in figure 5 (c), the presence of these interferents caused slight
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variation in the fluorescence intensity of QDs-melamine complex, which is within the acceptable range of variation. No significant affect has been observed. This indicates high selectivity of the
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quanta-sensor towards the detection of melamine.
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Reliability of quanta-sensor to specifically detect melamine in real samples has also been studied for which spiking method was opted [Figure 5 (d)]. Before performing this study,
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melamine of different known concentrations (0.1 μM, 10.0 μM, & 20 μM) were spiked in the milk
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sample and it was pre-treated as explained in experimental section. These samples were incubated with constant volume of m-CdSe QDs. The change in their fluorescence intensity was monitored.
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With real samples also, the probes are effective in confirming the presence of melamine. Conclusion:
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A rapid, simple and economical assay has been demonstrated for melamine detection using fluorescent optical probes which are cadmium selenide QDs. The advantage of these optical probes
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is their easy synthesis and application as fluorescent sensor at room temperature. The probes doesn’t require any specific functionalization as the capping agent itself is capable of providing required chemistry for complex formation with melamine. The melamine detection is based on fluorescence quenching of m-CdSe QDs on complex formation between negatively charged QDs and positively charged melamine as well as through hydrogen bonding. The reaction between mCdSe QDs and melamine occurs immediately as confirmed from effect of reaction time studies, 10
which asserts fast reaction. Also, as compared to traditional methods, the complete procedure of melamine estimation including sample pre-treatment can be performed within 25-30 minutes. Therefore, it appears to be a promising select for onsite monitoring of melamine. For further future studies, we are targeting on development of miniaturized fluorescence reader which can be coupled
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with our quanta-sensor.
Acknowledgement:
The authors acknowledge Director, CSIR-CSIO for his constant encouragement and support. The work was financially supported by Department of Science & Technology (DST), New Delhi through its grant in aid scheme (Project No: GAP 0352). N.K acknowledges DST for his junior
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research fellowship (JRF) from the project GAP-0352. S.K.P acknowledges National Post-doc
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fellowship (NPDF) from SERB-DST, New Delhi.
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Biographies: Suman Singh is working as senior scientist in CSIR-Central Scientific Instruments Organisation (CSIR-CSIO), Chandigarh, India and did her PhD from Panjab University, Chandigarh, India. She
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is also a faculty of chemistry and member of Welfare association at Academy of Scientific and Innovative Research (AcSIR) at CSIR-CSIO, Chandigarh. Her area of research work is advanced materials, nanotechnology and(bio)sensors. She is working on synthesis and application of nanomaterials and their composites for application in bio/chemo/immunosensing of analytes for applications like health, environment, orthopaedic and security. Her work is published in reputed SCI journals and as book chapters.
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Vishaldeep Kaur has done her Masters (M.Tech) in Nanoscience and Nanotechnology from
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Panjab University, Chandigarh, India. She pursued her Masters training at Central Scientific
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and sensing applications of nanomaterials.
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Research Organization (CSIR-CSIO), Chandigarh, India. Her research interest include synthesis
Nishant Kumar has done his integrated dual degree (B.Tech-M.Tech) in Nanotechnology from
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Amity, Noida. He did his Masters Training at CSIR-Central Scientific Instruments Organisation (CSIR-CSIO), Chandigarh, India. Presently he is pursuing his PhD from Academy of Scientific
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and Innovative Research (AcSIR) and is recipient of senior research fellowship from CSIR, New Delhi. His interest is material synthesis and their electro-chemical applications. He has published
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his work in reputed SCI journals.
Mr. Mayank Garg has done his B.Tech in Biotechnology from UIET, Chandigarh. Presently he
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is pursuing his PhD from Academy of Scientific and Innovative Research (AcSIR) and is recipient of GATE- Junior Research Fellowship (GATE-JRF) from CSIR, New Delhi. His area of interest
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is synthesis of layered materials and their applications for pathogen detection and disease biomarkers. Dr. Satish Kumar Pandey did his Ph.D jointly from Panjab University and IMTECH, Chandigarh, India in 2015. He is recipient of BIRAC Young Innovator award, 2015 and National Post-Doc fellowship (NPDF) from SERB DST, New Delhi. Presently he is working as Pool
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Scientist in CSIR-CSIO, Chandigarh and his expertise is developing platforms for food borne pathogens by exploring surface components as specific markers. Vijay Kumar Meena is working as senior scientist in CSIR-Central Scientific Instruments Organisation (CSIR-CSIO), Chandigarh, India. He has done his Bachelors from PEC University
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of Technology, Chandigarh and M.Tech in Manufacturing Management from Birla Institute of Technology & Science (BITS), Pilani, Rajasthan, India. His research interest is design of
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orhtopedic implants and materials for biomedical and environmental applications.
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Figure Captions:
Figure 1: (a) TEM of m-CdSe QDs (b) XRD of m-CdSe QDs
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Figure 2: (a) Overlapped spectra of absorbance and emission of m-CdSe QDs (b) Emission spectra of m-CdSe QDs and its complex with melamine (concentration 20.0 μM, pH 7.0.
Figure 3: (a) Pictorial representation of complex formation between m-CdSe QDs and
melamine (b) Zeta potential of (i) m-CdSe QDs (ii) Melamine (iii) m-CdSe QDsmelamine concentration
Figure 4: (a) Fluorescence spectra recorded with quantasensor in the presence of different
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concentrations of melamine [0.01 nM to 60 μM] (b) Linearity graph (c) Fo/F vs
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quencher (melamine) concentration (d) Effect of incubation time with melamine on
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fluorescence signal of quant-sensor
Figure 5: (a) Fluorescence response of quanta-sensor as a function of pH for melamine
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detection (b) Reproducibility study of quanta-sensor (c) Selectivity study of
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quantasensor (d) Melamine estimation in spiked samples using quanta-sensor
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