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Facile synthesis of nitrogen and sulfur co-doped carbon dots and application for Fe(III) ions detection and cell imaging
Accepted Manuscript Title: Facile Synthesis of Nitrogen and Sulfur Co-doped Carbon Dots and Application for Fe(III) Ions Detection and Cell Imaging Author: Yanfen Chen Yuanya Wu Bo Weng Bin Wang Changming Li PII: DOI: Reference:
S0925-4005(15)30388-9 http://dx.doi.org/doi:10.1016/j.snb.2015.09.081 SNB 19066
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
6-7-2015 4-9-2015 15-9-2015
Please cite this article as: Y. Chen, Y. Wu, B. Weng, B. Wang, C. Li, Facile Synthesis of Nitrogen and Sulfur Co-doped Carbon Dots and Application for Fe(III) Ions Detection and Cell Imaging, Sensors and Actuators B: Chemical (2015), http://dx.doi.org/10.1016/j.snb.2015.09.081 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.
Facile Synthesis of Nitrogen and Sulfur Co-doped Carbon Dots and Application for Fe(III) Ions Detection and Cell Imaging Yanfen Chena, Yuanya Wua, Bo Wenga, Bin Wangab and Changming Lia a.
Institute for Clean Energy & Advanced Materials, Faculty of Materials and Energy, Southwest
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University, Chongqing 400715, China; Chongqing Key Laboratory for Advanced Materials and
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Technologies of Clean Energies, Chongqing 400715, China; Chongqing Engineering Research Center for Rapid diagnosis of Fatal Diseases, Chongqing 400715, China. Tel: 08623-68254947. E-mail:
Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education),
an
b.
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[email protected].
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ed
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Wuhan University, Wuhan 430072, China.
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ABSTRACT
Novel fluorescent nitrogen and sulfur co-doped carbon dots (N,S,C-dots) were synthesized by hydrothermal treatment of garlic. It was confirmed that the prepared fluorescent nanodots were doped with nitrogen and sulfur in the format of pyridinic-N, pyrrolic-N and thiophene-S. The
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N,S,C-dots displayed strong fluorescence with quantum yield of 13 %, and the fluorescence can be efficiently quenched by Fe3+. Based on the results, Fe3+ sensor was successfully developed and
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utilized to detect Fe3+ in environmental waters with excellent sensitivity and repeatability.
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Eventually, the carbon dots were applied for cell imaging, demonstrating their potential towards
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diverse applications.
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Graphical abstract
KEYWORDS: carbon dots ∙ Fe3+ ∙ cell imaging ∙ optical sensor
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Introduction
Carbon dots (C-dots) have attracted tremendous attentions owing to their captivating properties, such as excellent photostability, favorable biocompatibility, low toxicity, and good water solubility.[1] Until now, various methods for C-dots preparation have been reported. Generally, C-dots can be made by etching from large carbon materials (such as graphite, carbon nanotubes, activate carbon, and carbon fibers[2]) or hydrothermal or microwave treatment of organic compounds such as glucose, citric acid, glycerol, and
Page 2 of 32
glycol.[3] C-dots made from both methods have abundant hydroxy, carboxyl or epoxy groups generated by the oxidization of raw materials (bulk carbon materials) or partially carbonized organic carbohydrates which resulted in oligosaccharide and aliphatic chains condensed on the surface of C-dots. These functional groups stabilized the surface energy traps and made
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C-dots emissive.[4] Using similar method, nitrogen-doped C-dots (N,C-dots) can be prepared by chemical of
nitrogenous
propane-1,3-diol
such (TRIS),
as
amino
acid,
chitosan,
ethylenediaminetetraacetic
acid
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2-amino-2-hydroxymethyl-
compounds
cr
treatment
(EDTA), and diamine-terminated poly-(ethylene glycol).[5] Most N,C-dots exhibited better
an
fluorescence properties than C-dots due to the nitrogenous groups on the surface of C-dots.
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Recently, more effective nitrogen and sulfur co-doped C-dots (N,S,C-dots) was reported to be fabricated by hydrothermal treatment of citric acid and l-cysteine, which displays
ed
excellent fluorescent properties with quantum yields (QY) of 73%.[6] It was suggested that this high fluorescence QY is mainly attributed to the nitrogen-doping changing surface state
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and facilitating a high yield of radiative recombination. In comparison to C-dots and N,C-dots, N,S,C-dots possess more abundant nitrogen and sulfur-containing groups. These
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wide varieties of surface functional groups may bring the fluorescent materials novel performance and more extensive application prospect. Various natural raw materials such as hair, grass, cow manure, solanum tuberosum, apple juice, and punica granatum fruits, etc. are also used as carbon source for the fabrication of C-dots, N,C-dots or N,S,C-dots.[7] Natural materials widely distribute in nature and easily available to replace usual chemicals, which is conducive to the development and application of C-dots. On the other hand, most natural materials possess complex component, so C-dots made from these materials usual possess various surface groups and special performance. Page 3 of 32
Nowadays, C-dots have been widely studied and found to have great potentials in a wide range of applications including bio-imaging, drug delivery, photodynamic therapy, catalysis, and sensing.[1,8] For chemical sensing application, C-dots have been designed for the detection of protein, DNA, dopamine, glucose, ascorbic acid, NO 2 gas[9] and some heavy
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metal ions (such as Hg 2+, Ag+, Pb2+, and Cu2+).[1] For example, Yan and co-workers synthesized two types of amino-functionalized C-dots using citric acid with 1,2-ethyldiamine
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(C-dots-1) and N-(b-aminoethyl)-g-aminopropyl (C-dots-2). The obtained C-dots possessed
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high QY of 65.5 and 55.4%, respectively, and showed sensitive fluorescence quenching to Hg2+ due to the reaction between carboxyl groups on C-dots surface and Hg 2+ to form non
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fluorescent complexes.[10] Tian et al. built Cu 2+ sensor using C-dots as fluorescent probe with amino-TPEA [N-(2-aminoethyl) -N,N,N' -tris (pyridin -2-ylmethyl) ethane -1,2-diamine]
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modified on C-dots acted as a receptor for Cu 2+ detection. The fabricated sensor was
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successfully applied in intracellular sensing and in vivo imaging of Cu 2+.[11] In addition, C-dots-based Fe3+ sensors have also been reported. Qu and co-workers
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synthesized C-dots by the hydrothermal treatment of dopamine.[12] The hydroquinone groups on the prepared C-dots could be oxidized to the quinone species by Fe 3+ and resulted
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in the fluorescence of C-dots quenching. Based on this mechanism, a sensitive Fe 3+ sensor was fabricated with the detection limit of 0.32 µM; The C-dots synthesized by oxidizing β-cyclodextrin exhibited peroxidase like activity towards H 2O2. Meanwhile, the prepared C-dots could be used for the detection of Fe 3+ with the detect limitation of 0.8 µM.[13] There were also some other C-dots sensors for Fe3+ detection reported to be prepared using graphite, ionic liquids, konjac flour and the mixture of isoleucine and citric acid as carbon precursors.[14] These prepared C-dots illustrated good selectivity to Fe 3+ attributed to the binding affinity of hydroxyl group on C-dots’ surface to Fe 3+. But the possibility of using Page 4 of 32
these specific C-dots in environmental Fe 3+ detection is still in doubt and needs further investigations. Garlic was often regarded as world's healthiest food as it contains allicin, which can help to improve the body’s iron metabolism and protect human beings from both cardiovascular
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diseases and cancer. Also, abundant carbon, nitrogen, sulfur and oxygen elements from crude protein, amino acid, allicin, niacin, lipid, carbohydrates, citral and vitamin in garlic made
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C-dots fabricated from garlic easy to be functionalized. In addition, garlic is quite common in
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everyday life, low cost, thus quite suitable for large-scale fabrication of C-dots.
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Meanwhile, as the increasing amount of Fe3+ in environmental water became great threats for human health, the needs to develop sensitive, selective and inexpensive sensor for Fe 3+
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detection aroused. In the present work, an easy, low cost method was developed to fabricate N,S,C-dots via one-step hydrothermal treatment of garlic to work as environmental Fe 3+
ed
sensor. The prepared fluorescent materials demonstrated strong fluorescence with QY of
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13 % and excellent selectivity and sensitivity to Fe 3+ in acid solution. The detection limit for Fe3+ could achieve as low as 0.22 nM, which made this material a suitable candidate for environmental Fe3+ detection. The results from the measurement of Fe 3+ in lake water and tap
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water verified our assumption and manifested their potential applications in practi cal applications. Meanwhile, the carbon dots were employed for cell imaging, demonstrating their potential in biomedical fields.
Experimental section Materials and Instrumentation.
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All the chemicals in this work were used as purchased from Sigma-Aldrich. The garlic was purchased from local supermarket. Deionized water (18.2 MU) was used throughout for all the experiments. UV−vis spectra were characterized by Shimadzu UV-2550 spectrophotometer with one
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pair of 10mm quartz cell. Fourier Transform infrared (FTIR) spectrums were performed on an FTIR spectrophotometer (Thermo-Fisher, Nicolet 6700) in the range of 400−4000 cm −1.
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Transmission electron microscopy (TEM, JEOL, JEM-2100) measurements were carried out
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using an accelerating voltage of 200 kV. The samples for TEM characterization were prepared by coating the given volume of N,S,C-dots dispersion on a carbon-coated copper
N,S,C-dots
were
recorded
by
an
FL
spectrophotometer
(Shi-madzu,
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as-prepared
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grid, and then dried under vacuum at 50 oC for 24h. Fluorescent emission spectra of the
RF-5301PC). The size distribution of particle was measured by using Zetasizer Nano Series
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ed
(Malvern instruments, UK).
Preparation of N,S,C-dots
N,S,C-dots were synthesized via a hydrothermal method using garlic as carbon precursor. In
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a typical synthesis, 2.0 g fresh chopped garlic was added into 10 mL ultrapure water and stirred for 5 min. The mixture was then transferred into a 25 mL Teflon-lined autoclave and heated at 180 oC for 10 h in an oven. The N,S,C-dots solution were collected after removing the carbide slag via centrifugation at 8000 rpm for 15 min. The obtained dark brown solution was further purified by filtration (0.22 μm nitrocellulose filters) and then diluted with DI water for further use.
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Measurement of fluorescence QY The QY of N,S,C-dots was determined according to an established procedure. In brief, quinine sulfate in 0.1M H 2SO4 (QY: 0.54 at 340 nm) was chose as the standard. In order to minimize the re-absorption effects, absorbencies in the 10 mm fluorescence cuvette were
calculated by the following equation:
I AR n2 I R A nR2
(1)
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Q QR
cr
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kept under 0.05 at the excitation wavelength (360 nm). The QY of the N,S,C-dots was
Where Ԛ is the QY, I is the measured integrated emission intensity, n is the refractive
an
index of the solvent, and A refers to the absorbance. The subscript R refers to the
Detection of Fe3+ ion
ed
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corresponding parameter of known fluorescent standard.
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The detection of Fe3+ was carried out in 10 mM NaAc-HAc (pH, 5.5) buffer solution. In a typical assay, 10μL N,S,C-dots (11 mg/mL) dispersion was diluted to 2 mL with NaAc-HAc buffer solution. Then different concentrations of Fe 3+ were added and mixed thoroughly
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before characterization. The fluorescence spectra were recorded after reaction for 5min. The selectivity of Fe3+sensing was confirmed by adding other common metal ions stock solutions (including Al 3+ ,Cd2+, Cu2+, Co2+, Pb2+, Zn2+,Fe2+, Ag+, Mn2+ ,Hg2+ ,K+, Na+, Ca2+ and Ni 2+ ions) instead of Fe 3+ and determining in the same way. In addition, some common anions and organic compounds including Cl-, NO3-, SO42-, CO32-, PO43-, glycine, cysteine, ascorbic acid, ethanol, methanal, and dopamine were selected to explore the effect of these species on the detection of Fe 3+. The concentration of N,S,C-dots and these species were
Page 7 of 32
0.055 mg/mL and 50 µM, respectively. The fluorescence was recorded at the excitation wavelength of 340nm. All experiments were performed at room temperature.
Analysis of Fe3+ ion in environmental waters
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To determine Fe3+ detection function of prepared N,S,C-dots sensor in environmental waters,
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water samples collected from laboratory tap and the Chongde Lake (Southwest University, Chongqing, China) were utilized to replace ultrapure water. Water samples were filtered
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through a 0.22 μm membrane (Millipore) prior to detection. Aliquots (500 μL) of this sample
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solution were spiked with standard Iron ions solutions (final concentration in the range of 2 nM – 100 μM). The spiked samples were then diluted to 2 mL with NaAc-HAc (10 mM, pH
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5.0) containing N,S,C-dots (final concentration 0.055 mg/mL ) and then analyzed by the
ed
above described method.
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Fluorescence Imaging Experiments
RAW264.7 cells were seeded in each well of cover glass-Bottom Dish and cultured at 37 oC for 24 h. The filtered N,S,C-dots (0.5mg/mL) fluorescent suspension (10-30 μL) was mixed
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with the culture medium (1mL) and then introduced into the wells in which the RAW264.7 cells were grown. After an incubation of 24 h, the cells were washed thoroughly three times with PBS (1mL each time) and kept in PBS for the fluorescence imaging. Thereafter, 200 μM Fe3+ was added, incubated for 10 min. The fluorescence imaging of cell was recorded by using a fluorescence microscope (ZX71, Olympus Corp., Tokyo, Japan, λex = 448 nm). The fluorescence intensity was analyzed by using "Image J" software.
Page 8 of 32
Results and discussion Synthesis and characterization of N,S,C-dots The N,S,C-dots were synthesized using garlic as carbon precursor via a simple hydrothermal treatment at 180 oC for 10h. Fig. 1A showed a typical TEM image of the
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products, revealing that the average size of the monodispersed nanoparticles was around 3.6 nm. The corresponding nanoparticle size distribution histogram was obtained by counting
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about 150 C-dots (the inset of Fig. 1). The FTIR spectrum of the N,S,C-dots was given in Fig.
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2A The broad band in the region of 3060 – 3600 cm-1 arouse from stretching vibrations of O–H and N–H and the small band at 2928 cm-1 could be identified as the stretching vibrations
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of C–H bonds. The peak at 1632 cm -1 could be assigned to the stretching vibrations of C-O
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and the bending vibrations of N–H.[16] The band at 1700 cm-1 was attributed to the stretching vibrations of C=O. The peak at 1400 cm -1 could be identified as C–N, N–H, and
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vibrations of C–H.
ed
–COO groups.[15] The broad band around 1067–1209 cm-1 was ascribed to the bending
XPS was used to analyze the elemental composition of the prepared N,S,C-dots. As shown
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in Fig. 2B, the XPS spectra revealed that the N,S,C-dots were mainly composed of carbon (64.33%), nitrogen (4,32%), oxygen (29.84%) and sulfur (0.72%). The high resolution spectrum of C 1s in Fig. 2C exhibited four main peaks. The binding energy peak at 284.5 eV was attributed to the graphitic structure C–C bond, suggesting the great amount of graphite structuresin the obtained N,S,C-dots. The peak at about 285.5 eV could be assigned to C–O, C–N, and C–S, while the peaks around 287.5 eV and 288.6 eV could be ascribed to C=O and C–OOR. The high resolution spectrum of O1s (Figure 2D) displays two peaks at 530.9 and 532.4 eV, which can be attributed to C = O and C–OH/C–O–C groups, respectively.[7g] The Page 9 of 32
N1s spectrum (Fig. 2E) can be divided into two peaks. The main peak with the binding energies of 399.28 eV, which was attributed to the pyridinic N, accounted for 90% of the total peak area. Another small peak at 401.13 eV originated from pyrrolic N.[6] The S2p spectrum in Fig. 2F was mainly consisted of three peaks centered at 163.4, 164.8eV and 167.8 eV respectively, indicating the existing three forms of sulfur element. The former two
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peaks agreed with the –C–S– (S2p,3/2 and S2p,1/2) covalent bond of the thiophene-S due to the
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spin–orbit couplings. 6 The latter one can be ascribed to the –C–SO3– species, such as sulfate
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or sulfonate.[15]
These XPS and FTIR data demonstrated that the as-prepared N,S,C-dots contained
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functional groups with oxygen, nitrogen and sulphur elements, which endow this fluorescent
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material excellent water solubility and facilitate its further modifications and applications.
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Optical properties
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The optical properties of the S,N,C-dots were explored. The UV–vis absorption spectra of the N,S,C-dots dispersed in water showed a strong absorption peak at 282.4 nm, which was similar to the C-dots made from grass [7g]. The emission spectra in Fig. 3 showed that a
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strong emission with the maximum emission wavelength at 428 nm could be observed under excitation at 340 nm. Very bright blue luminescence was seen under the illumination of UV (365 nm) light in aqueous solution (as shown in the inset of Fig. 3). The QY of the N,S,C-dots was calculated to be about 13 % with quinine sulfate as standard, demonstrating the good fluorescence properties. Similar to the common C-dots, the prepared N,S,C-dots exhibited excitation-dependent emission character. As shown in Fig. 4, the fluorescent peak red-shifted with the increase of excitation wavelength from 320nm to 460 nm, and the strongest emission spectrum w as Page 10 of 32
obtained at the excitation wavelength of 340 nm. These behaviors resulted from the different sizes of nano-dots and a distribution of different surface states due to the different organic groups on C-dots’ surface.[17] Then the photostability of the fluorescent N,S,C-dots was studied. As shown in Fig. S1(a),
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the fluorescence intensity did not display significant change after continuous irradiation under a 150 W Xe lamp for several hours, indicating the excellent photostability of the
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fluorescent probe. In addition, the effect of pH on the fluorescence behavior of the
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N,S,C-dots was also explored. As shown in Fig. S1(b), the fluorescence was enhanced with the increase of pH values from 3.0 to 5.5. However, when pH was higher than 6.0, the
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fluorescent intensity decreased dramatically with the increase of pH values. The phenomena
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were similar to those of carbon nanocrystals obtained by electrooxidation of graphite,
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suggesting the N,S,C-dots share the same luminescent origin as the carbon nanocrystals.[20]
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Fluorescence response of the N,S,C-dots to the Fe3+ The UV−vis absorption spectra showed that the N,S,C-dots had an obvious absorption bands centered at 283 nm (Fig. 5A). In aqueous solution, Fe 3+solution displayed a strong absorption
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centered at 300 nm. The absorption of the N,S,C-dots increased evidently upon the addition of 100 μM Fe3+, meanwhile, the absorption peak of Fe 3+ ion disappeared, which indicated the reaction happened between N,S,C-dots and Fe3+ and the formation of N,S.C-dots/Fe complex. Fig. 5B showed the fluorescence spectra of the N,S,C-dots at excitation wavelength of 360 nm. The N,S,C-dots showed strong fluorescence and the fluorescence was quenched drastically upon the addition of 150 μM Fe3+. The quenching efficiency was calculated to be about 76%. Moreover, the quenching efficiency increases with the increase of Fe 3+
Page 11 of 32
concentration (as shown in Fig. S2). In the presence of 600μM Fe3+, the fluorescence of the N,S,C-dots can be quenched about 90 % of original data. The photograph in Fig. 5B inset exhibited that the bright blue fluorescence under UV light (365nm) was quenched obviously upon the addition of Fe 3+ ions.
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To further elucidate the mechanism of fluorescence quenching, the N,S,C-dots/Fe complex was characterized using TEM. As shown in Fig. 5C and 5D, the monodispersed N,S,C-dots
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aggregated seriously in the presence of 100 μM Fe 3+. In addition, the sizes distribution of the
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N,S,C-dots before and after addition of Fe 3+ was determined by using Zetasizer Nano Series (Malvern instruments, UK). As shown in Fig. S3, the average size of the N,S,C-dots was
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about 3.6 nm. After addition of 100 μM Fe3+, the average size of the N,S,C-dots/Fe complex
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was about 900 nm. These results confirmed the aggregation of nanodots.
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The fluorescence Sensing for Fe3+.
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Optimization of analytical conditions
The Fe3+ inducted fluorescence quenching can be designed for Fe 3+ detection. Firstly, the influence of reaction time and pH on sensing ability was optimized to obtain the best
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detection results. As shown in Fig. S4(a), the N,S,C-dots displayed stable fluorescence signal in aqueous solution. After the addition of 100 μM Fe 3+, the fluorescence of the N,S,C-dots was quenched significantly and fluorescence response became stable in 2 min. The fluorescence response towards Fe3+at different pH from 3 to 11 was exhibited in Fig. S4(b). The result showed that the highest fluorescence quenching rate was obtained at pH 5.5. Additionally, the influence of pH on the selectivity of Fe 3+ sensing against other common metal ions (including Al 3+ ,Cd2+, Cu2+, Co2+, Pb2+, Zn2+,Fe2+,Fe3+, Ag+, Mn2+ ,Hg2+ ,K+, Na+, Page 12 of 32
Ca2+ and Ni 2+) was also studied. Fig. 6 showed the fluorescence response of the N,S,C-dots upon the addition of these common metal ions in pH 11.0, 7.0 and 5.5, respectively. When the same concentration of interfering metal ions were added into the N,S,C-dots solution at pH 11.0, most metal ions displayed strong fluorescence response compared to Fe 3+ at pH
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11.0 solution. The results may attribute to the high OH - concentration facilitating the deposition of metal ions on the N,S,C-dots surface. In pH 7.0, most of the interfering metal
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ions showed quite weak fluorescence response in comparison with Fe 3+ except Ag+. When
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the detection was carried out at pH 5.5, the addition of Fe 3+ caused strong fluorescence response and excellent selectivity. The photograph in Fig.6D also indicated that the prepared
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N,S,C-dots showed excellent response to Fe 3+ compared with other common metal ions at pH
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5.5.
According to the XPS results, oxygen is main component of the N,S,C-dots in comparison
ed
to nitrogen and sulfur, and the -OH/-O-C groups are about 72% of oxygen-containing groups. The FTIR data also demonstrated that there were plentiful hydroxyl groups on the surface of
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the N,S,C-dots. Moreover, the electronegativity of oxygen containing groups was higher than that of nitrogen and sulfur containing groups [18]. Thus we suggested that the mechanism of
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the fluorescence quenching was mostly attributed to the binding affinity of hydroxyl group on N,S,C-dots’ surface to Fe 3+. The hydroxyl groups on carbon dots could react with Fe3+ and form complexes due to coordination.[19] The formed C-dots/Fe complexes promoted electron transfer and restrained exciton recombination, which caused significant fluorescence quenching. And the high selectivity of this sensor to Fe 3+ in acid solution also supported the conclusions.
Sensitivity of the sensor in Fe3+ detection Page 13 of 32
Under the above optimized conditions, we evaluated the capability of the design for the quantification of Fe 3+. As shown in Fig. 7A, the fluorescence intensity of the N,S,C-dots decreased gradually with the increase of the concentration of Fe 3+, proving the feasibility of Fe3+ detection. Fig. 7B indicated the relationship between fluorescence response and Fe 3+ concentration. There is a good linear correlation between the quenching efficiency (F 0/F1)
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and the logarithm of Fe 3+ concentration within the range of 2 nM – 3 µM (the inset of Fig.
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6B). The concentration of Fe 3+ could be calculated using the following equation: (2)
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F0/F1 = 0.08197lgC (mol/L) +1.6944 (R 2 = 0.9960)
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Where F0 and F1 are the fluorescent intensities of the N,S,C-dots before and after the addition of Fe3+, and C represents the concentration of Fe3+. The detect limitation of Fe 3+ (n=11) was
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estimated to be 0.22 nM, indicating that the proposed method was more sensitive than other C-dots-based Fe3+ detection methods (as shown in Table S1)[13]. The results demonstrated
ed
that the method was feasible for the detection of trace Fe 3+. In addition, most C-dots based
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Fe3+ sensor did not exhibit their application in real samples (Table S1). In view of the high sensitivity and remarkable photostability of the N,S,C-dots, it is possible to apply the sensor
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for Fe3+ detection in real samples.
Selectivity of the sensor in Fe3+ detection The results in Fig. 6 displays the excellent selectivity of the sensor towards common metal ions in pH 5.5. Besides metal ions, various anions and organic molecules may co-exist with Fe3+ in natural environment. So we explored the effect of some common anions and organic compounds on the determination of Fe3+. As shown in Fig. 8, in the presence of these interfering species including Cl-, NO3-, SO42-, CO32-, PO43-, glycine, cysteine, ascorbic acid, ethanol, methanal, and dopamine, Page 14 of 32
only slight interference was observed. When all these species were mixed with Fe3+ in the same concentration, the signal interference is less than 10%. The results confirmed the good selectivity of the proposed method for Fe3+detection.
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Detection of Fe3+ in environmental samples From Table S1 we can see that no C-dots based Fe3+ sensor exhibits the application in real
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water samples. To verify the utilization of prepared N,S,C-dots sensor for environmental
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Fe3+, the practical applications of this sensing system for Fe3+ detection were investigated
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using environmental water from Lake Chongde and laboratory tap. Lake water and tap water samples were purified by simple filtration with 0.22 μm nitrocellulose filters prior to
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detection. The concentration of Fe 3+ in the real samples was determined by the proposed method. Then different concentration of Fe3+ was added and the fluorescence responses were
ed
recorded. The recoveries were calculated by the following equation: (3)
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Recovery =[(C2 ‒ C1)/ C0]×100%
Where C0 is the concentration of Fe 3+ added into the real samples; C1 and C2 is obtained according to the equation (1), representing the concentration of Fe 3+ in real samples before
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and after adding the standard Fe 3+. The results have been illustrated in Table 1. The concentrations of Fe3+ in tap water and lake water samples were determined to be zero and 11.15 nM, respectivley. The recoveries of all samples were between 96.75 and 102.54%, demonstrating the proposed method has good accuracy. The relative standard deviations (RSD) for all the determination were less than 2.65%, which indicates the excellent precision of the method. These results demonstrate that it is feasible to detect Fe3+ in environmental
Page 15 of 32
waters with the present method. In addition, the simple, low-cost, green preparative strategy would favour the application and development. Application in cell imaging The N,S,C-dots were introduced into the RAW264.7 cells to show their bioimaging
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capabilities using a fluorescent microscopy test in vitro. The results showed that the photoluminescent carbon dots were observed only in the cell membrane and cytoplasmic area
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of the cell, indicating that the obtained carbon dots easily penetrated into the cell [Fig. 9(b)].
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To explored the effect of Fe 3+ on the fluorescence imaging of the cell, 200 μM Fe3+ was
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mixed with the fluorescence-labeled cell and incubated for 10 min at room temperature. As shown in Fig. 9(d), the fluorescence of cell was drastically quenched. The quenching
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efficiency was calculated to be about 80 % by using "image J" software. These results reveal
ed
that the as-prepared N,S,C-dots may be used for bioimaging or Fe3+ detection in cell. Conclusions
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In summary, the novel N,S,C-dots were successfully synthesized by the hydrothermal treatment of garlic for the first time. These nano-dots displayed good water solubility, strong fluorescence and upconversion fluorescence without any further modification. XPS and FTIR
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characterizations demonstrated that the prepared N,S,C-dots have abundant hydroxyl groups on their surface, which resulted in highly sensitive and selective response to Fe 3+. These properties made this nanomaterial quite suitable to work as environmental Fe 3+ sensor. Explorations of Fe 3+ detection in lake water and tape water illustrated the ability of this sensor to work effectively under strong interferential environmental condition. In addition, The cellular uptake demonstrated that the prepared fluorescent N,S,C-dots are promising candidates for fluorescent cellular imaging.
Page 16 of 32
Acknowledgements This work was supported by the National Key Basic Research Project (973 Program 2011CB911002), PhD start-up grant SWU113112, SWU113111 from Southwest University, and Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of
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Education), Wuhan University.
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Notes and references
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Author Biography Dr Chang Ming Li is currently the Professor and Dean of Faculty for Materials and Energy in Southwest University and Director for Chongqing Key Lab for Clean Energy and Advanced Materials, China. He worked with Motorola as a distinguished technical member, Science Advisory Board Associate and senior scientific manager. Later he became Professor, Head of Bioengineering Division and Director of Centre for Advanced Bionanosystems in Nanyang Technological University, Singapore. He received a number of prestigious awards such as Motorola Master Innovator Award in 1999, Nanyang Innovation and Research Award in 2008, China National Talent 1000
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Award (2011), China Overseas Innovation Talent Award (2012) and Australia National University Craig Visiting Professor (2012). His main research interests are nanomaterials, green energies and biosensors/lab-on-a chip. He has published over 400 peer-reviewed journal papers, 8 book chapters and holds 124 patents with ~ 10 000
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citations. He is a Fellow of American Institute of Medicine and Biological Engineering and an RSC Fellow, serving as
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an Editorial Board Member of RSC Advances and a member of the Advisory Board for Nanoscale, RSC.
Dr Bin Wang received his PhD degrees from the Changchun Institute of Applied Chemistry, Chinese Academy of
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Sciences. Currently, he joined Institute of Clean Energy and Advanced Materials of Southwest University as an associate Professor. His research focuses on synthesis novel nanomaterials and application in focuses on
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biosensors and lab-on-chip systems as well.
Dr Bo Weng received her PhD degrees from the University of Wollongong, Australia. Currently, she joined Institute of Clean Energy and Advanced Materials of Southwest University as an associate Professor. His research focuses on
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synthesis novel nanomaterials and application in focuses on biosensors and lab-on-chip systems as well. Yanfen Chen received his Bachelor degrees from Southwest University in 2013. She is currently a Ph.D student in
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Southwest University, China. Her research focuses on synthesis novel nanomaterials and application in focuses on biosensors and lab-on-chip systems as well.
Yuanya Wu received his Bachelor degrees from Southwest University in 2013. She is currently a Ph.D student in
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Southwest University, China. Her research focuses on synthesis novel nanomaterials and preparation of electrochemical biosensors.
FIGURE CAPTIONS
Fig. 1. TEM image of as-prepared N,S,C-dots (inset: size distribution histogram).
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Fig. 2. A: FTIR spectrum of the N,S,C-dots; B: XPS spectra of the N,S,C-dots. C–F: High-resolution C1s, O1s, N1s, and S2p peaks of the N,S,C-dots, respectively.
Fig.3. UV/Vis absorption spectra and emission spectra of the N,S,C-dots at the excitation of 340 nm.
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Inset: Photograph of the N,S,C-dots under illumination of UV light (365 nm).
Fig. 4. The excitation dependent emission of the N,S,C-dots.
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Fig. 5. A: UV-vis absorption spectra of the N,S,C-dots (0.05mg/mL),100µM Fe3+ andN,S,C-dots
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+100 µM Fe3+; B:Fluorescence spectra of the N,S,C-dots in the absence and presence of 150 μM Fe3+ ions (λex, 360 nm). The inset shows the photos of the corresponding solutions illuminated
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under UV light of 365 nm. C and D: The TEM of the N,S,C-dots in the presence of Fe3+.
Fig. 6. The selectivity of the sensor at different pH (A, B, and C) and Fluorescence response of these
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metal ions (D) at pH 5.5 under excitation wavelenth of 365 nm. (The concentration of N,S,C-dots
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and metal ion were 0.055 mg/mL and 50 µM, respectively; the fluorescence was recorded at 428 nm. Excitation wavelength was 340nm.)
Fig. 7.
(A) fluorescence spectra of N,S,C-dots with different concentrations of Fe 3+ ions;
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(B) The dependence of F 0/F1 on the concentration of Fe 3+ ions within the range of 2 nM – 100 μM. Inset: Plot of the fluorescent response (F 0/F1) versus the logarithm of concentrations of Fe3+ ions. (The concentration of the N,S,C-dots was 0.055 mg/mL)
Fig. 8. The selectivity of the sensor towards common anions and organic compounds at pH 5.5. The mixture containing Fe3+, Cl-, NO3-, SO42-, CO32-, PO43-, glycine, cysteine, ascorbic acid, ethanol, methanal, and dopamine. (The concentration of N,S,C-dots was 0.055 mg/mL, the concentrtion of these anions and organic molecules was 50 µM) Page 22 of 32
Fig. 9. The bright field microphotograph (a) and fluorescence imaging (b) of the RAW264.7 cells labeled with the N,S,C-dots at 37 oC for 24 h; The bright field microphotograph (c) and fluorescence imaging (d) of the RAW264.7 cells labeled with the N,S,C-dots in the presence
Recoveries of Fe 3+ ions in lake water and tap water detected by the proposed
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Table 1.
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of 150 Fe3+. (λex: 448 nm).
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method (n=3).
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Fig. 1.
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Fig. 2.
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Fig. 5.
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Table 1.
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S0925-4005(15)30388-9 http://dx.doi.org/doi:10.1016/j.snb.2015.09.081 SNB 19066
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
6-7-2015 4-9-2015 15-9-2015
Please cite this article as: Y. Chen, Y. Wu, B. Weng, B. Wang, C. Li, Facile Synthesis of Nitrogen and Sulfur Co-doped Carbon Dots and Application for Fe(III) Ions Detection and Cell Imaging, Sensors and Actuators B: Chemical (2015), http://dx.doi.org/10.1016/j.snb.2015.09.081 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.
Facile Synthesis of Nitrogen and Sulfur Co-doped Carbon Dots and Application for Fe(III) Ions Detection and Cell Imaging Yanfen Chena, Yuanya Wua, Bo Wenga, Bin Wangab and Changming Lia a.
Institute for Clean Energy & Advanced Materials, Faculty of Materials and Energy, Southwest
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University, Chongqing 400715, China; Chongqing Key Laboratory for Advanced Materials and
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Technologies of Clean Energies, Chongqing 400715, China; Chongqing Engineering Research Center for Rapid diagnosis of Fatal Diseases, Chongqing 400715, China. Tel: 08623-68254947. E-mail:
Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education),
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b.
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[email protected].
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Wuhan University, Wuhan 430072, China.
Page 1 of 32
ABSTRACT
Novel fluorescent nitrogen and sulfur co-doped carbon dots (N,S,C-dots) were synthesized by hydrothermal treatment of garlic. It was confirmed that the prepared fluorescent nanodots were doped with nitrogen and sulfur in the format of pyridinic-N, pyrrolic-N and thiophene-S. The
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N,S,C-dots displayed strong fluorescence with quantum yield of 13 %, and the fluorescence can be efficiently quenched by Fe3+. Based on the results, Fe3+ sensor was successfully developed and
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utilized to detect Fe3+ in environmental waters with excellent sensitivity and repeatability.
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Eventually, the carbon dots were applied for cell imaging, demonstrating their potential towards
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diverse applications.
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Graphical abstract
KEYWORDS: carbon dots ∙ Fe3+ ∙ cell imaging ∙ optical sensor
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Introduction
Carbon dots (C-dots) have attracted tremendous attentions owing to their captivating properties, such as excellent photostability, favorable biocompatibility, low toxicity, and good water solubility.[1] Until now, various methods for C-dots preparation have been reported. Generally, C-dots can be made by etching from large carbon materials (such as graphite, carbon nanotubes, activate carbon, and carbon fibers[2]) or hydrothermal or microwave treatment of organic compounds such as glucose, citric acid, glycerol, and
Page 2 of 32
glycol.[3] C-dots made from both methods have abundant hydroxy, carboxyl or epoxy groups generated by the oxidization of raw materials (bulk carbon materials) or partially carbonized organic carbohydrates which resulted in oligosaccharide and aliphatic chains condensed on the surface of C-dots. These functional groups stabilized the surface energy traps and made
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C-dots emissive.[4] Using similar method, nitrogen-doped C-dots (N,C-dots) can be prepared by chemical of
nitrogenous
propane-1,3-diol
such (TRIS),
as
amino
acid,
chitosan,
ethylenediaminetetraacetic
acid
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2-amino-2-hydroxymethyl-
compounds
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treatment
(EDTA), and diamine-terminated poly-(ethylene glycol).[5] Most N,C-dots exhibited better
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fluorescence properties than C-dots due to the nitrogenous groups on the surface of C-dots.
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Recently, more effective nitrogen and sulfur co-doped C-dots (N,S,C-dots) was reported to be fabricated by hydrothermal treatment of citric acid and l-cysteine, which displays
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excellent fluorescent properties with quantum yields (QY) of 73%.[6] It was suggested that this high fluorescence QY is mainly attributed to the nitrogen-doping changing surface state
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and facilitating a high yield of radiative recombination. In comparison to C-dots and N,C-dots, N,S,C-dots possess more abundant nitrogen and sulfur-containing groups. These
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wide varieties of surface functional groups may bring the fluorescent materials novel performance and more extensive application prospect. Various natural raw materials such as hair, grass, cow manure, solanum tuberosum, apple juice, and punica granatum fruits, etc. are also used as carbon source for the fabrication of C-dots, N,C-dots or N,S,C-dots.[7] Natural materials widely distribute in nature and easily available to replace usual chemicals, which is conducive to the development and application of C-dots. On the other hand, most natural materials possess complex component, so C-dots made from these materials usual possess various surface groups and special performance. Page 3 of 32
Nowadays, C-dots have been widely studied and found to have great potentials in a wide range of applications including bio-imaging, drug delivery, photodynamic therapy, catalysis, and sensing.[1,8] For chemical sensing application, C-dots have been designed for the detection of protein, DNA, dopamine, glucose, ascorbic acid, NO 2 gas[9] and some heavy
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metal ions (such as Hg 2+, Ag+, Pb2+, and Cu2+).[1] For example, Yan and co-workers synthesized two types of amino-functionalized C-dots using citric acid with 1,2-ethyldiamine
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(C-dots-1) and N-(b-aminoethyl)-g-aminopropyl (C-dots-2). The obtained C-dots possessed
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high QY of 65.5 and 55.4%, respectively, and showed sensitive fluorescence quenching to Hg2+ due to the reaction between carboxyl groups on C-dots surface and Hg 2+ to form non
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fluorescent complexes.[10] Tian et al. built Cu 2+ sensor using C-dots as fluorescent probe with amino-TPEA [N-(2-aminoethyl) -N,N,N' -tris (pyridin -2-ylmethyl) ethane -1,2-diamine]
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modified on C-dots acted as a receptor for Cu 2+ detection. The fabricated sensor was
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successfully applied in intracellular sensing and in vivo imaging of Cu 2+.[11] In addition, C-dots-based Fe3+ sensors have also been reported. Qu and co-workers
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synthesized C-dots by the hydrothermal treatment of dopamine.[12] The hydroquinone groups on the prepared C-dots could be oxidized to the quinone species by Fe 3+ and resulted
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in the fluorescence of C-dots quenching. Based on this mechanism, a sensitive Fe 3+ sensor was fabricated with the detection limit of 0.32 µM; The C-dots synthesized by oxidizing β-cyclodextrin exhibited peroxidase like activity towards H 2O2. Meanwhile, the prepared C-dots could be used for the detection of Fe 3+ with the detect limitation of 0.8 µM.[13] There were also some other C-dots sensors for Fe3+ detection reported to be prepared using graphite, ionic liquids, konjac flour and the mixture of isoleucine and citric acid as carbon precursors.[14] These prepared C-dots illustrated good selectivity to Fe 3+ attributed to the binding affinity of hydroxyl group on C-dots’ surface to Fe 3+. But the possibility of using Page 4 of 32
these specific C-dots in environmental Fe 3+ detection is still in doubt and needs further investigations. Garlic was often regarded as world's healthiest food as it contains allicin, which can help to improve the body’s iron metabolism and protect human beings from both cardiovascular
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diseases and cancer. Also, abundant carbon, nitrogen, sulfur and oxygen elements from crude protein, amino acid, allicin, niacin, lipid, carbohydrates, citral and vitamin in garlic made
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C-dots fabricated from garlic easy to be functionalized. In addition, garlic is quite common in
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everyday life, low cost, thus quite suitable for large-scale fabrication of C-dots.
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Meanwhile, as the increasing amount of Fe3+ in environmental water became great threats for human health, the needs to develop sensitive, selective and inexpensive sensor for Fe 3+
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detection aroused. In the present work, an easy, low cost method was developed to fabricate N,S,C-dots via one-step hydrothermal treatment of garlic to work as environmental Fe 3+
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sensor. The prepared fluorescent materials demonstrated strong fluorescence with QY of
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13 % and excellent selectivity and sensitivity to Fe 3+ in acid solution. The detection limit for Fe3+ could achieve as low as 0.22 nM, which made this material a suitable candidate for environmental Fe3+ detection. The results from the measurement of Fe 3+ in lake water and tap
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water verified our assumption and manifested their potential applications in practi cal applications. Meanwhile, the carbon dots were employed for cell imaging, demonstrating their potential in biomedical fields.
Experimental section Materials and Instrumentation.
Page 5 of 32
All the chemicals in this work were used as purchased from Sigma-Aldrich. The garlic was purchased from local supermarket. Deionized water (18.2 MU) was used throughout for all the experiments. UV−vis spectra were characterized by Shimadzu UV-2550 spectrophotometer with one
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pair of 10mm quartz cell. Fourier Transform infrared (FTIR) spectrums were performed on an FTIR spectrophotometer (Thermo-Fisher, Nicolet 6700) in the range of 400−4000 cm −1.
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Transmission electron microscopy (TEM, JEOL, JEM-2100) measurements were carried out
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using an accelerating voltage of 200 kV. The samples for TEM characterization were prepared by coating the given volume of N,S,C-dots dispersion on a carbon-coated copper
N,S,C-dots
were
recorded
by
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FL
spectrophotometer
(Shi-madzu,
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as-prepared
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grid, and then dried under vacuum at 50 oC for 24h. Fluorescent emission spectra of the
RF-5301PC). The size distribution of particle was measured by using Zetasizer Nano Series
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(Malvern instruments, UK).
Preparation of N,S,C-dots
N,S,C-dots were synthesized via a hydrothermal method using garlic as carbon precursor. In
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a typical synthesis, 2.0 g fresh chopped garlic was added into 10 mL ultrapure water and stirred for 5 min. The mixture was then transferred into a 25 mL Teflon-lined autoclave and heated at 180 oC for 10 h in an oven. The N,S,C-dots solution were collected after removing the carbide slag via centrifugation at 8000 rpm for 15 min. The obtained dark brown solution was further purified by filtration (0.22 μm nitrocellulose filters) and then diluted with DI water for further use.
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Measurement of fluorescence QY The QY of N,S,C-dots was determined according to an established procedure. In brief, quinine sulfate in 0.1M H 2SO4 (QY: 0.54 at 340 nm) was chose as the standard. In order to minimize the re-absorption effects, absorbencies in the 10 mm fluorescence cuvette were
calculated by the following equation:
I AR n2 I R A nR2
(1)
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Q QR
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kept under 0.05 at the excitation wavelength (360 nm). The QY of the N,S,C-dots was
Where Ԛ is the QY, I is the measured integrated emission intensity, n is the refractive
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index of the solvent, and A refers to the absorbance. The subscript R refers to the
Detection of Fe3+ ion
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corresponding parameter of known fluorescent standard.
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The detection of Fe3+ was carried out in 10 mM NaAc-HAc (pH, 5.5) buffer solution. In a typical assay, 10μL N,S,C-dots (11 mg/mL) dispersion was diluted to 2 mL with NaAc-HAc buffer solution. Then different concentrations of Fe 3+ were added and mixed thoroughly
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before characterization. The fluorescence spectra were recorded after reaction for 5min. The selectivity of Fe3+sensing was confirmed by adding other common metal ions stock solutions (including Al 3+ ,Cd2+, Cu2+, Co2+, Pb2+, Zn2+,Fe2+, Ag+, Mn2+ ,Hg2+ ,K+, Na+, Ca2+ and Ni 2+ ions) instead of Fe 3+ and determining in the same way. In addition, some common anions and organic compounds including Cl-, NO3-, SO42-, CO32-, PO43-, glycine, cysteine, ascorbic acid, ethanol, methanal, and dopamine were selected to explore the effect of these species on the detection of Fe 3+. The concentration of N,S,C-dots and these species were
Page 7 of 32
0.055 mg/mL and 50 µM, respectively. The fluorescence was recorded at the excitation wavelength of 340nm. All experiments were performed at room temperature.
Analysis of Fe3+ ion in environmental waters
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To determine Fe3+ detection function of prepared N,S,C-dots sensor in environmental waters,
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water samples collected from laboratory tap and the Chongde Lake (Southwest University, Chongqing, China) were utilized to replace ultrapure water. Water samples were filtered
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through a 0.22 μm membrane (Millipore) prior to detection. Aliquots (500 μL) of this sample
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solution were spiked with standard Iron ions solutions (final concentration in the range of 2 nM – 100 μM). The spiked samples were then diluted to 2 mL with NaAc-HAc (10 mM, pH
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5.0) containing N,S,C-dots (final concentration 0.055 mg/mL ) and then analyzed by the
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above described method.
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Fluorescence Imaging Experiments
RAW264.7 cells were seeded in each well of cover glass-Bottom Dish and cultured at 37 oC for 24 h. The filtered N,S,C-dots (0.5mg/mL) fluorescent suspension (10-30 μL) was mixed
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with the culture medium (1mL) and then introduced into the wells in which the RAW264.7 cells were grown. After an incubation of 24 h, the cells were washed thoroughly three times with PBS (1mL each time) and kept in PBS for the fluorescence imaging. Thereafter, 200 μM Fe3+ was added, incubated for 10 min. The fluorescence imaging of cell was recorded by using a fluorescence microscope (ZX71, Olympus Corp., Tokyo, Japan, λex = 448 nm). The fluorescence intensity was analyzed by using "Image J" software.
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Results and discussion Synthesis and characterization of N,S,C-dots The N,S,C-dots were synthesized using garlic as carbon precursor via a simple hydrothermal treatment at 180 oC for 10h. Fig. 1A showed a typical TEM image of the
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products, revealing that the average size of the monodispersed nanoparticles was around 3.6 nm. The corresponding nanoparticle size distribution histogram was obtained by counting
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about 150 C-dots (the inset of Fig. 1). The FTIR spectrum of the N,S,C-dots was given in Fig.
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2A The broad band in the region of 3060 – 3600 cm-1 arouse from stretching vibrations of O–H and N–H and the small band at 2928 cm-1 could be identified as the stretching vibrations
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of C–H bonds. The peak at 1632 cm -1 could be assigned to the stretching vibrations of C-O
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and the bending vibrations of N–H.[16] The band at 1700 cm-1 was attributed to the stretching vibrations of C=O. The peak at 1400 cm -1 could be identified as C–N, N–H, and
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vibrations of C–H.
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–COO groups.[15] The broad band around 1067–1209 cm-1 was ascribed to the bending
XPS was used to analyze the elemental composition of the prepared N,S,C-dots. As shown
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in Fig. 2B, the XPS spectra revealed that the N,S,C-dots were mainly composed of carbon (64.33%), nitrogen (4,32%), oxygen (29.84%) and sulfur (0.72%). The high resolution spectrum of C 1s in Fig. 2C exhibited four main peaks. The binding energy peak at 284.5 eV was attributed to the graphitic structure C–C bond, suggesting the great amount of graphite structuresin the obtained N,S,C-dots. The peak at about 285.5 eV could be assigned to C–O, C–N, and C–S, while the peaks around 287.5 eV and 288.6 eV could be ascribed to C=O and C–OOR. The high resolution spectrum of O1s (Figure 2D) displays two peaks at 530.9 and 532.4 eV, which can be attributed to C = O and C–OH/C–O–C groups, respectively.[7g] The Page 9 of 32
N1s spectrum (Fig. 2E) can be divided into two peaks. The main peak with the binding energies of 399.28 eV, which was attributed to the pyridinic N, accounted for 90% of the total peak area. Another small peak at 401.13 eV originated from pyrrolic N.[6] The S2p spectrum in Fig. 2F was mainly consisted of three peaks centered at 163.4, 164.8eV and 167.8 eV respectively, indicating the existing three forms of sulfur element. The former two
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peaks agreed with the –C–S– (S2p,3/2 and S2p,1/2) covalent bond of the thiophene-S due to the
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spin–orbit couplings. 6 The latter one can be ascribed to the –C–SO3– species, such as sulfate
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or sulfonate.[15]
These XPS and FTIR data demonstrated that the as-prepared N,S,C-dots contained
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functional groups with oxygen, nitrogen and sulphur elements, which endow this fluorescent
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material excellent water solubility and facilitate its further modifications and applications.
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Optical properties
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The optical properties of the S,N,C-dots were explored. The UV–vis absorption spectra of the N,S,C-dots dispersed in water showed a strong absorption peak at 282.4 nm, which was similar to the C-dots made from grass [7g]. The emission spectra in Fig. 3 showed that a
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strong emission with the maximum emission wavelength at 428 nm could be observed under excitation at 340 nm. Very bright blue luminescence was seen under the illumination of UV (365 nm) light in aqueous solution (as shown in the inset of Fig. 3). The QY of the N,S,C-dots was calculated to be about 13 % with quinine sulfate as standard, demonstrating the good fluorescence properties. Similar to the common C-dots, the prepared N,S,C-dots exhibited excitation-dependent emission character. As shown in Fig. 4, the fluorescent peak red-shifted with the increase of excitation wavelength from 320nm to 460 nm, and the strongest emission spectrum w as Page 10 of 32
obtained at the excitation wavelength of 340 nm. These behaviors resulted from the different sizes of nano-dots and a distribution of different surface states due to the different organic groups on C-dots’ surface.[17] Then the photostability of the fluorescent N,S,C-dots was studied. As shown in Fig. S1(a),
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the fluorescence intensity did not display significant change after continuous irradiation under a 150 W Xe lamp for several hours, indicating the excellent photostability of the
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fluorescent probe. In addition, the effect of pH on the fluorescence behavior of the
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N,S,C-dots was also explored. As shown in Fig. S1(b), the fluorescence was enhanced with the increase of pH values from 3.0 to 5.5. However, when pH was higher than 6.0, the
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fluorescent intensity decreased dramatically with the increase of pH values. The phenomena
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were similar to those of carbon nanocrystals obtained by electrooxidation of graphite,
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suggesting the N,S,C-dots share the same luminescent origin as the carbon nanocrystals.[20]
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Fluorescence response of the N,S,C-dots to the Fe3+ The UV−vis absorption spectra showed that the N,S,C-dots had an obvious absorption bands centered at 283 nm (Fig. 5A). In aqueous solution, Fe 3+solution displayed a strong absorption
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centered at 300 nm. The absorption of the N,S,C-dots increased evidently upon the addition of 100 μM Fe3+, meanwhile, the absorption peak of Fe 3+ ion disappeared, which indicated the reaction happened between N,S,C-dots and Fe3+ and the formation of N,S.C-dots/Fe complex. Fig. 5B showed the fluorescence spectra of the N,S,C-dots at excitation wavelength of 360 nm. The N,S,C-dots showed strong fluorescence and the fluorescence was quenched drastically upon the addition of 150 μM Fe3+. The quenching efficiency was calculated to be about 76%. Moreover, the quenching efficiency increases with the increase of Fe 3+
Page 11 of 32
concentration (as shown in Fig. S2). In the presence of 600μM Fe3+, the fluorescence of the N,S,C-dots can be quenched about 90 % of original data. The photograph in Fig. 5B inset exhibited that the bright blue fluorescence under UV light (365nm) was quenched obviously upon the addition of Fe 3+ ions.
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To further elucidate the mechanism of fluorescence quenching, the N,S,C-dots/Fe complex was characterized using TEM. As shown in Fig. 5C and 5D, the monodispersed N,S,C-dots
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aggregated seriously in the presence of 100 μM Fe 3+. In addition, the sizes distribution of the
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N,S,C-dots before and after addition of Fe 3+ was determined by using Zetasizer Nano Series (Malvern instruments, UK). As shown in Fig. S3, the average size of the N,S,C-dots was
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about 3.6 nm. After addition of 100 μM Fe3+, the average size of the N,S,C-dots/Fe complex
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was about 900 nm. These results confirmed the aggregation of nanodots.
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The fluorescence Sensing for Fe3+.
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Optimization of analytical conditions
The Fe3+ inducted fluorescence quenching can be designed for Fe 3+ detection. Firstly, the influence of reaction time and pH on sensing ability was optimized to obtain the best
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detection results. As shown in Fig. S4(a), the N,S,C-dots displayed stable fluorescence signal in aqueous solution. After the addition of 100 μM Fe 3+, the fluorescence of the N,S,C-dots was quenched significantly and fluorescence response became stable in 2 min. The fluorescence response towards Fe3+at different pH from 3 to 11 was exhibited in Fig. S4(b). The result showed that the highest fluorescence quenching rate was obtained at pH 5.5. Additionally, the influence of pH on the selectivity of Fe 3+ sensing against other common metal ions (including Al 3+ ,Cd2+, Cu2+, Co2+, Pb2+, Zn2+,Fe2+,Fe3+, Ag+, Mn2+ ,Hg2+ ,K+, Na+, Page 12 of 32
Ca2+ and Ni 2+) was also studied. Fig. 6 showed the fluorescence response of the N,S,C-dots upon the addition of these common metal ions in pH 11.0, 7.0 and 5.5, respectively. When the same concentration of interfering metal ions were added into the N,S,C-dots solution at pH 11.0, most metal ions displayed strong fluorescence response compared to Fe 3+ at pH
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11.0 solution. The results may attribute to the high OH - concentration facilitating the deposition of metal ions on the N,S,C-dots surface. In pH 7.0, most of the interfering metal
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ions showed quite weak fluorescence response in comparison with Fe 3+ except Ag+. When
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the detection was carried out at pH 5.5, the addition of Fe 3+ caused strong fluorescence response and excellent selectivity. The photograph in Fig.6D also indicated that the prepared
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N,S,C-dots showed excellent response to Fe 3+ compared with other common metal ions at pH
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5.5.
According to the XPS results, oxygen is main component of the N,S,C-dots in comparison
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to nitrogen and sulfur, and the -OH/-O-C groups are about 72% of oxygen-containing groups. The FTIR data also demonstrated that there were plentiful hydroxyl groups on the surface of
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the N,S,C-dots. Moreover, the electronegativity of oxygen containing groups was higher than that of nitrogen and sulfur containing groups [18]. Thus we suggested that the mechanism of
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the fluorescence quenching was mostly attributed to the binding affinity of hydroxyl group on N,S,C-dots’ surface to Fe 3+. The hydroxyl groups on carbon dots could react with Fe3+ and form complexes due to coordination.[19] The formed C-dots/Fe complexes promoted electron transfer and restrained exciton recombination, which caused significant fluorescence quenching. And the high selectivity of this sensor to Fe 3+ in acid solution also supported the conclusions.
Sensitivity of the sensor in Fe3+ detection Page 13 of 32
Under the above optimized conditions, we evaluated the capability of the design for the quantification of Fe 3+. As shown in Fig. 7A, the fluorescence intensity of the N,S,C-dots decreased gradually with the increase of the concentration of Fe 3+, proving the feasibility of Fe3+ detection. Fig. 7B indicated the relationship between fluorescence response and Fe 3+ concentration. There is a good linear correlation between the quenching efficiency (F 0/F1)
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and the logarithm of Fe 3+ concentration within the range of 2 nM – 3 µM (the inset of Fig.
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6B). The concentration of Fe 3+ could be calculated using the following equation: (2)
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F0/F1 = 0.08197lgC (mol/L) +1.6944 (R 2 = 0.9960)
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Where F0 and F1 are the fluorescent intensities of the N,S,C-dots before and after the addition of Fe3+, and C represents the concentration of Fe3+. The detect limitation of Fe 3+ (n=11) was
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estimated to be 0.22 nM, indicating that the proposed method was more sensitive than other C-dots-based Fe3+ detection methods (as shown in Table S1)[13]. The results demonstrated
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that the method was feasible for the detection of trace Fe 3+. In addition, most C-dots based
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Fe3+ sensor did not exhibit their application in real samples (Table S1). In view of the high sensitivity and remarkable photostability of the N,S,C-dots, it is possible to apply the sensor
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for Fe3+ detection in real samples.
Selectivity of the sensor in Fe3+ detection The results in Fig. 6 displays the excellent selectivity of the sensor towards common metal ions in pH 5.5. Besides metal ions, various anions and organic molecules may co-exist with Fe3+ in natural environment. So we explored the effect of some common anions and organic compounds on the determination of Fe3+. As shown in Fig. 8, in the presence of these interfering species including Cl-, NO3-, SO42-, CO32-, PO43-, glycine, cysteine, ascorbic acid, ethanol, methanal, and dopamine, Page 14 of 32
only slight interference was observed. When all these species were mixed with Fe3+ in the same concentration, the signal interference is less than 10%. The results confirmed the good selectivity of the proposed method for Fe3+detection.
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Detection of Fe3+ in environmental samples From Table S1 we can see that no C-dots based Fe3+ sensor exhibits the application in real
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water samples. To verify the utilization of prepared N,S,C-dots sensor for environmental
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Fe3+, the practical applications of this sensing system for Fe3+ detection were investigated
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using environmental water from Lake Chongde and laboratory tap. Lake water and tap water samples were purified by simple filtration with 0.22 μm nitrocellulose filters prior to
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detection. The concentration of Fe 3+ in the real samples was determined by the proposed method. Then different concentration of Fe3+ was added and the fluorescence responses were
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recorded. The recoveries were calculated by the following equation: (3)
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Recovery =[(C2 ‒ C1)/ C0]×100%
Where C0 is the concentration of Fe 3+ added into the real samples; C1 and C2 is obtained according to the equation (1), representing the concentration of Fe 3+ in real samples before
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and after adding the standard Fe 3+. The results have been illustrated in Table 1. The concentrations of Fe3+ in tap water and lake water samples were determined to be zero and 11.15 nM, respectivley. The recoveries of all samples were between 96.75 and 102.54%, demonstrating the proposed method has good accuracy. The relative standard deviations (RSD) for all the determination were less than 2.65%, which indicates the excellent precision of the method. These results demonstrate that it is feasible to detect Fe3+ in environmental
Page 15 of 32
waters with the present method. In addition, the simple, low-cost, green preparative strategy would favour the application and development. Application in cell imaging The N,S,C-dots were introduced into the RAW264.7 cells to show their bioimaging
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capabilities using a fluorescent microscopy test in vitro. The results showed that the photoluminescent carbon dots were observed only in the cell membrane and cytoplasmic area
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of the cell, indicating that the obtained carbon dots easily penetrated into the cell [Fig. 9(b)].
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To explored the effect of Fe 3+ on the fluorescence imaging of the cell, 200 μM Fe3+ was
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mixed with the fluorescence-labeled cell and incubated for 10 min at room temperature. As shown in Fig. 9(d), the fluorescence of cell was drastically quenched. The quenching
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efficiency was calculated to be about 80 % by using "image J" software. These results reveal
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that the as-prepared N,S,C-dots may be used for bioimaging or Fe3+ detection in cell. Conclusions
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In summary, the novel N,S,C-dots were successfully synthesized by the hydrothermal treatment of garlic for the first time. These nano-dots displayed good water solubility, strong fluorescence and upconversion fluorescence without any further modification. XPS and FTIR
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characterizations demonstrated that the prepared N,S,C-dots have abundant hydroxyl groups on their surface, which resulted in highly sensitive and selective response to Fe 3+. These properties made this nanomaterial quite suitable to work as environmental Fe 3+ sensor. Explorations of Fe 3+ detection in lake water and tape water illustrated the ability of this sensor to work effectively under strong interferential environmental condition. In addition, The cellular uptake demonstrated that the prepared fluorescent N,S,C-dots are promising candidates for fluorescent cellular imaging.
Page 16 of 32
Acknowledgements This work was supported by the National Key Basic Research Project (973 Program 2011CB911002), PhD start-up grant SWU113112, SWU113111 from Southwest University, and Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of
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Education), Wuhan University.
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Notes and references
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6 Y. Q. Dong, H. C. Pang, H. B. Yang, C. X. Guo, J. W. Shao, Y. W. Chi, C. M. Li, and T. Yu,
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Angew. Chem. Int. Ed. 2013, 52, 7800 .
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Asiri , A. O. Al-Youbi , and X.P. Sun, Adv. Mater. 2012, 24, 2037. 8 S. N. Baker and G. A. Baker, Angew. Chem. Int. Ed. 2010, 49, 6726. 9 (a) L. Xu, C. Q. Zhao, W. L. Wei, J. S. Ren, D. Miyoshi, N. Sugimoto and X. G. Qu, Analyst, 2012, 137, 5483; (b) Z. S. Qian, X. Y. Shan, L. J. Chai, J. J. Ma, J. R. Chen, H. Feng, Biosens. Bioelectron., 2014, 60, 64; (c) H. L. Li, Y. W. Zhang, L. Wang, J. Q. Tian and X. P. Sun, Chem. Commun., 2011, 47, 961; (d) S. Liu, J. Q. Tian, L. Wang, Y. L. Luo and X. P.
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Author Biography Dr Chang Ming Li is currently the Professor and Dean of Faculty for Materials and Energy in Southwest University and Director for Chongqing Key Lab for Clean Energy and Advanced Materials, China. He worked with Motorola as a distinguished technical member, Science Advisory Board Associate and senior scientific manager. Later he became Professor, Head of Bioengineering Division and Director of Centre for Advanced Bionanosystems in Nanyang Technological University, Singapore. He received a number of prestigious awards such as Motorola Master Innovator Award in 1999, Nanyang Innovation and Research Award in 2008, China National Talent 1000
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Award (2011), China Overseas Innovation Talent Award (2012) and Australia National University Craig Visiting Professor (2012). His main research interests are nanomaterials, green energies and biosensors/lab-on-a chip. He has published over 400 peer-reviewed journal papers, 8 book chapters and holds 124 patents with ~ 10 000
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citations. He is a Fellow of American Institute of Medicine and Biological Engineering and an RSC Fellow, serving as
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an Editorial Board Member of RSC Advances and a member of the Advisory Board for Nanoscale, RSC.
Dr Bin Wang received his PhD degrees from the Changchun Institute of Applied Chemistry, Chinese Academy of
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Sciences. Currently, he joined Institute of Clean Energy and Advanced Materials of Southwest University as an associate Professor. His research focuses on synthesis novel nanomaterials and application in focuses on
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biosensors and lab-on-chip systems as well.
Dr Bo Weng received her PhD degrees from the University of Wollongong, Australia. Currently, she joined Institute of Clean Energy and Advanced Materials of Southwest University as an associate Professor. His research focuses on
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synthesis novel nanomaterials and application in focuses on biosensors and lab-on-chip systems as well. Yanfen Chen received his Bachelor degrees from Southwest University in 2013. She is currently a Ph.D student in
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Southwest University, China. Her research focuses on synthesis novel nanomaterials and application in focuses on biosensors and lab-on-chip systems as well.
Yuanya Wu received his Bachelor degrees from Southwest University in 2013. She is currently a Ph.D student in
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Southwest University, China. Her research focuses on synthesis novel nanomaterials and preparation of electrochemical biosensors.
FIGURE CAPTIONS
Fig. 1. TEM image of as-prepared N,S,C-dots (inset: size distribution histogram).
Page 21 of 32
Fig. 2. A: FTIR spectrum of the N,S,C-dots; B: XPS spectra of the N,S,C-dots. C–F: High-resolution C1s, O1s, N1s, and S2p peaks of the N,S,C-dots, respectively.
Fig.3. UV/Vis absorption spectra and emission spectra of the N,S,C-dots at the excitation of 340 nm.
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Inset: Photograph of the N,S,C-dots under illumination of UV light (365 nm).
Fig. 4. The excitation dependent emission of the N,S,C-dots.
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Fig. 5. A: UV-vis absorption spectra of the N,S,C-dots (0.05mg/mL),100µM Fe3+ andN,S,C-dots
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+100 µM Fe3+; B:Fluorescence spectra of the N,S,C-dots in the absence and presence of 150 μM Fe3+ ions (λex, 360 nm). The inset shows the photos of the corresponding solutions illuminated
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under UV light of 365 nm. C and D: The TEM of the N,S,C-dots in the presence of Fe3+.
Fig. 6. The selectivity of the sensor at different pH (A, B, and C) and Fluorescence response of these
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metal ions (D) at pH 5.5 under excitation wavelenth of 365 nm. (The concentration of N,S,C-dots
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and metal ion were 0.055 mg/mL and 50 µM, respectively; the fluorescence was recorded at 428 nm. Excitation wavelength was 340nm.)
Fig. 7.
(A) fluorescence spectra of N,S,C-dots with different concentrations of Fe 3+ ions;
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(B) The dependence of F 0/F1 on the concentration of Fe 3+ ions within the range of 2 nM – 100 μM. Inset: Plot of the fluorescent response (F 0/F1) versus the logarithm of concentrations of Fe3+ ions. (The concentration of the N,S,C-dots was 0.055 mg/mL)
Fig. 8. The selectivity of the sensor towards common anions and organic compounds at pH 5.5. The mixture containing Fe3+, Cl-, NO3-, SO42-, CO32-, PO43-, glycine, cysteine, ascorbic acid, ethanol, methanal, and dopamine. (The concentration of N,S,C-dots was 0.055 mg/mL, the concentrtion of these anions and organic molecules was 50 µM) Page 22 of 32
Fig. 9. The bright field microphotograph (a) and fluorescence imaging (b) of the RAW264.7 cells labeled with the N,S,C-dots at 37 oC for 24 h; The bright field microphotograph (c) and fluorescence imaging (d) of the RAW264.7 cells labeled with the N,S,C-dots in the presence
Recoveries of Fe 3+ ions in lake water and tap water detected by the proposed
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Table 1.
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of 150 Fe3+. (λex: 448 nm).
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method (n=3).
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Fig. 1.
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Fig. 5.
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Table 1.
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