Red Emission B, N, S-co-Doped Carbon Dots for Colorimetric and

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Red Emission B, N, S-Co-Doped Carbon Dots for Colorimetric and Fluorescent Dual Mode Detection of Fe3+ Ions in Complex Biological Fluids and Living Cells Yinghua Liu, Wenxiu Duan, Wei Song, Juanjuan Liu, Cuiling Ren, Jiang Wu, Dan Liu, and Hongli Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b15746 • Publication Date (Web): 24 Mar 2017 Downloaded from http://pubs.acs.org on March 26, 2017

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ACS Applied Materials & Interfaces

Red Emission B, N, S-Co-Doped Carbon Dots for Colorimetric and Fluorescent Dual Mode Detection of Fe3+ Ions in Complex Biological Fluids and Living Cells Yinghua Liu

1, 2

, Wenxiu Duan 3, Wei Song

1, 2

, Juanjuan Liu

1, 2

, Cuiling Ren

1, 2,

*,

Jiang Wu 1, 2, Dan Liu 3, Hongli Chen 1, 2 1 College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China 2 Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province, Lanzhou 730000, China 3 School of Life Sciences, University of Science and Technology of China, Hefei 230027, China Abstract: Colorimetric and fluorescent dual mode detection methods have gained much attention in recent years, however, it is still desirable to develop new colorimetric and fluorescent dual mode nanosensors with more simple preparation procedure, low cost and excellent biocompatibility. Herein, a colorimetric and fluorescent nanosensor based on B, N, S-co-doped carbon dots (BNS-CDs) were synthesized by one-step hydrothermal treatment of 2, 5-diaminobenzenesulfonic acid and 4-aminophenylboronic acid hydrochloride. Using this nanosensor, a highly sensitive assay of Fe3+ in the range of 0.3-546 µM with a detection limit of 90 nM was provided by quenching the red emission fluorescence. It is more attractive that Fe3+ can also be visualized by this nanosensor via evident color changes of the solution (from red to blue) under sunlight without the aid of ultraviolet (UV) lamp. Furthermore, the designed nanosensor can be applied for efficient detection of intracellular Fe3+ with excellent biocompatibility and cellular imaging capability, and it holds great promise in biomedical applications. Keywords: Red emission carbon dots; dual mode; colorimetric and Fluorescent; Fe3+ detection; cell imaging

1

ACS Paragon Plus Environment


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INTRODUCTION In recent years, dual mode detection strategy has gained considerable attention, and has been proved to be more efficient than single mode method.1,

2

A number of

dual-mode assays have been developed, such as colorimetric/fluorescent,3 magnetic/fluorescent2,

4

dual

mode

sensing,

magnetic

resonance/computed

tomography and fluorescence/surface enhanced Raman scattering dual mode imaging and so on.5, 6 Among these methods, colorimetric and fluorescent dual mode assay has attained much more attention, and has become an extremely sensitive analytic tool in biomedical applications. Because these dual mode sensors not only provide highly sensitive fluorescence assay, but also facilitate the visualization of target by unaided eyes.7, 8 Till date, most of the colorimetric and fluorescent dual mode sensors are constructed by integrating two optical parts together, including organic molecules,9 metal–organic

frameworks,10

semiconductor

quantum

dots

(QDs),11

metal

nanoclusters (NCs),12 gold nanoparticles13 and so on. For example, Deng et al. developed a novel colorimetric and fluorescent dual mode chemosensor by encapsulating rhodamine B into stimuli-responsive infinite coordination polymers for detecting alcoholic strength in spirit samples.14 Zhu et al. prepared a ratiometric fluorescence nanohybrid by covalently linking green Au NCs to the surface of silica nanoparticles embedded with red emitting QDs, that can be used to determine lead ions visually with the aid of ultraviolet (UV) light.12 These sensors can realize sensitive, selective and convenient detection of the targets, but the complicated construction procedure, low biocompatibility and high cost restrict their widespread applications. Therefore, it is desirable to develop newer colorimetric and fluorescent dual mode nanosensors with more simple preparation procedure, low cost and excellent biocompatibility. Furthermore, red fluorescence emission nanosensors are favorable for their applications, particularly in the biomedical fields, because of the universal blue-auto fluorescence of biological matrix and photodamage of biological tissues caused by ultraviolet excitation light.15 For this reason, many kinds of red emissive nanosensors 2


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have been reported, such as metal clusters,16 quantum dots,17 carbon nanoparticles and so on.18 Among these materials, carbon dots (CDs) have attracted extensive attention due to their superior optical and chemical properties, such as good chemical stability, excellent biocompatibility and simple synthetic routes.19-24 Though red emission CDs are favorable for the applications in biological fields, there are few reports about them. For example, Jiang et al. reported the synthesis of red, green, blue emission CDs using phenylenediamines isomers via a solvothermal method, which can be applied in photoluminescence (PL) bioimaging.15 Ding et al obtained red emission CDs, which needed purification via silica column chromatography, and used it for live mice imaging.25 Their results proved red emissive CDs showed good performance and hold great potential in bioimaging. Based on the above reason, it is useful to develop a colorimetric and fluorescent dual mode nanosensor based on red emission CDs. But to our knowledge, no such work has been reported so far. In the present study, B, N, S-co-doped carbon dots (BNS-CDs) were prepared by a facile hydrothermal method and a colorimetric and fluorescent dual mode nanosensor was constructed for the detection of Fe3+ (Scheme 1). This novel nanosensor had three major advantages. First of all, the preparation procedure was simple, the source materials were cheap and the photostability and water-solubility were excellent. In addition, Fe3+ can be detected selectively with high sensitivity in the range of 0.3-546 µM with a detection limit of 90 nM. The Fe3+ can also be easily visualized via evident color change from red to blue under sunlight without the aid of UV lamp, and the detection limit can reach 0.3 µM. Moreover, it can be used for the efficient detection of intracellular Fe3+ with excellent biocompatibility and cellular imaging capability.

2. EXPERIMENTAL SECTION 2.1 Materials. 2, 5-diaminobenzenesulfonic acid was obtained from Aladdin Chemistry Co., Ltd. (Shanghai, China). 4-aminophenylboronic acid hydrochloride was purchased from Soochiral

Chemistry

Science

&

Technology

Co.,

3


Ltd.

(Suzhou,

China).


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Tris(hydroxymthyl)aminomethane, hydrochloric acid, FeCl3, FeCl2, KCl, NaCl, CuCl2, CoCl2, MnCl2, ZnCl2, NiCl2, HgCl2, AlCl3 and all amino acids were bought from Beijing Chemical Reagent Co. Ltd. (Beijing, China). All reagents were of analytical grade and used directly without any further purification. Deionized (DI) water was used throughout the experiments. Material purification was carried out using 1.0 kDa cutoff membranes (Amicon Ultra-4, Millipore).

2.2 Instrumentation and Characterization. A Hitachi-600 transmission electron microscope (TEM, Hitachi, Japan) was used to obtain transmission electron microscopy (TEM) image of the BNS-CDs. Fourier transform infrared (FT-IR) spectroscopy was recorded on a Nicolet Nexus 670 FTIR spectrometer with KBr pellets. Powder X-ray diffraction (XRD) pattern was performed on a D/max 82400 X-ray powder diffractometer (Rigaku, Japan) by using CuKa (λ=0.154056Å) as the incident radiation. X-ray photoelectron spectroscopy (XPS) measurement was recorded using a PerkinElmer PHI-5702 multifunctional photoelectron spectrometer. UV-Visible absorption spectra were measured by a TU-1901 double beam UV-Vis spectrophotometer (Beijing Purkinje General Instrument Co., Ltd., Beijing, China). Fluorescent (FL) spectra were measured on an RF-5301 spectrofluorophotometer equipped with a Xe lamp using 5/5 nm slit width, and equipped with a 1 cm quartz cell (Shimadzu, Kyoto, Japan). Zeta potentials of the BNS-CDs in different pH solutions were measured by a Zetasizer Nano ZS instrument (Malvern, UK). Fluorescent photographs were taken using a Canon camera (EOS 550) under excitation by a hand-held UV lamp (365 nm). The cellular imaging was carried out using a spinning-disk confocal microscope (Nikon’s inverted Eclipse Ti microscope) and the results of MTT assay were examined by microplate reader (BMG LABTECH, CLARIOstar).

2.3 Synthesis of BNS-CDs. 2,

5-diaminobenzenesulfonic acid

(0.075g)

and

4-aminophenylboronic acid

hydrochloride (0.075g) were dissolved in 30 ml of DI water and the solution was transferred into a teflon autoclave. After being heated at a certain temperature for 4


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some time, the autoclave was cooled down to room temperature naturally. The red suspension was centrifuged at 12000 rpm for 15 min to remove large particles, and the supernatant was kept for dialysis (1000 Da) for 12 h. After removing solvents and further freeze drying, red-brown powder was obtained and stored at 4℃. The obtained CDs samples were re-dispersed in DI water for characterization.

2.4 Calculation of the absolute photoluminescence quantum yield/lifetime. The quantum yield was measured using time resolved and steady state fluorescence spectrometer FLS 920 (Edinburgh). The absolute photoluminescence quantum yield (QY) can be calculated using the following equation.26

QY =

∫L ∫E

solvent

emission

− ∫ Esample

where QY is the absolute quantum yield. Lemission is the photon numbers of FL emission of BNS-CDs, Esample and Esolvent are the photon numbers of excitation light used for excitation BNS-CDs and solvent (DI water), respectively. Time-resolved

FL

spectra

were

obtained

using

a

time-correlated-single-photo-counting (TCSPC) system using FLS 920 fluorescence spectrometer with λex=500 nm and the average lifetimes of the BNS-CDs were calculated.

27

2.5 Detection of Fe3+ using BNS-CDs. Different concentrations of Fe3+ (10 µL each) were separately added into the mixture of 500 µL BNS-CDs solution (5 mg/mL) and 2.5 mL Tris-HCl buffer (pH=7.0). The selectivity of BNS-CDs toward Fe3+ was evaluated by adding 10 µL of other metal ions or amino acids solutions instead of Fe3+ in a similar way. The anti-interference experiments were evaluated by adding Fe3+ to one of other metal ions or amino acids solutions simultaneously in a similar way. To explore the feasibility of the proposed method for analysis of Fe3+ in complex biological samples, human urine and serum samples were obtained from the first affiliated hospital of Lanzhou University and the 5



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authors state that all experiments were performed in compliance with the relevant laws and institutional guidelines. The urine and serum samples were subjected to a 100-fold dilution before analysis. Diluted samples were spiked with various concentrations of standard Fe3+ solution and the FL spectra were recorded under excitation at 500 nm. All experiments were performed at room temperature.

2.6 MTT Assays. The cell viability assessment was carried out using MTT assay. Typically, 100 µL of cells were seeded in a 96-well plate with a density of 2×105 cells per mL and allowed to adhere overnight. After incubation with BNS-CDs for 24 h at 37 ºC in a humidified atmosphere with 5% CO2, 20 µL of MTT (5 mg·mL−1) was added to each well. Three wells with MTT but no cells were used as control. After incubating for another 4 h at 37 °C, the media was removed and 150 µL DMSO was added to each well and shaked for 10 min. Finally, the absorbance was measured at 490 nm using a microplate reader (BMG LABTECH, CLARIOstar). The cell viability was defined as the ratio of the absorbance in the presence of BNS-CDs to that in the absence of BNS-CDs (control). Cell viability = Isample/Icontrol.

2.7 Cellular Imaging. HeLa cells grown on 18×18-mm glass coverslips were first cultured in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS), 100 mg·mL−1 glutamine, 100 mg·mL−1 sodium pyruvate, penicillin (100 units·mL−1), and streptomycin (100 units·mL−1) at 37 °C in a humidified atmosphere of 5% CO2 overnight followed by incubation with 300 µg·mL−1 BNS-CDs for 24 h. Finally, cell imaging was performed with a Nikon’s inverted Eclipse Ti microscope equipped with a 20×0.75 NA objective. For assessing Fe3+ uptake, HeLa cells were incubated with 300 µg·mL−1 BNS-CDs for 24 h as described above, followed by incubation with 100 µM FeCl3 for 6 h at 37 °C.

3. RESULTS AND DISCUSSIONS. 3.1 Optimization of Reaction Conditions. 6


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The experimental conditions including reaction time, temperature and the ratio of reactants were optimized to prepare BNS-CDs with optimal optical properties. The FL intensity of the prepared BNS-CDs was increased when the reaction temperature changed from 160 to 180 °C and then decreased when the reaction temperature further increased to 200 °C (Figure S1a). Thus, the influence of reaction time on the FL intensity of BNS-CDs was studied at 180 °C (Figure S1b). The FL intensity of BNS-CDs increased till 8 h and then decreased with prolonging the carbonization time. Therefore, in the subsequent experiments, the reaction temperature and time were set at 180 °C and 8 h, respectively. As the doping contents played an important role in regulating the FL properties of the prepared CDs, the mass ratio between 4-aminophenylboronic acid hydrochloride (B) and 2, 5-diaminobenzenesulfonic acid (S) in preparing BNS-CDs was adjusted. The ratio of B:S not only influenced the intensity of the prepared CDs but also the emission wavelength (Figure S1c). The carbon dots showed a broad emission peak with red shift if B:S ratio is changed from 10:1 to 1:1. There was no change in emission wavelength if the B:S ratio was smaller than 1:1. In addition, the FL intensity decreased with decreasing of the B:S ratio. Considering the emission wavelength and FL intensity (Figure S1d), 1:1 was chosen as the optimal reagent ratio.

3.2 Characterization of BNS-CDs. Morphology and size distribution of the synthesized BNS-CDs were characterized by TEM. The synthesized BNS-CDs were monodispersed and uniform in size (Figure 1A). Their size distribution agreed well with Gaussian distribution (200 CDs measured), and the statistical diameter was 2.4 ± 0.6 nm (Figure 1B). The crystal structure of BNS-CDs was characterized by high resolution TEM (HRTEM) (inset in Figure 1A), their ambiguous crystal structure may due to their disordered structure. Another evidence was provided by XRD pattern (Figure S2a), which displayed a broad diffraction peak centered at about 25° (0.34 nm), further confirming that the structures of BNS-CDs are composed of small crystalline cores with disordered surface.28 7



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In order to identify the structures and functional groups of BNS-CDs, FT-IR (Figure S2b) and XPS (Figure 2) were performed. As can be seen from Figure S2b, the presence of O-H and N−H bonds were conformed by the peaks located at 3000−3500 cm−1,29 and the peak at 2594 cm-1 was due to -SH vibrations, indicating sulfur was present in BNS-CDs.30 In addition, three peaks at 1724, 1552 and 1403 cm-1 proved the presence of C=O, C=C and C-N bonds respectively.31, 32 The peak at 1230 cm-1 was ascribed to the stretching vibration of C–C and C–S bond.33 The distinct absorption bands at 1195, 1093 and 1027 cm−1 can be attributed to B−O−H bending vibration, C−B stretching mode and B−O−H deformation vibration, respectively.34 The XPS spectrum provided more convincing evidence for elemental contents and surface groups of the prepared BNS-CDs. The full range XPS (Figure S3) spectrum clearly showed five peaks at 166.4, 196.2, 285.3, 399.2 and 531.5 eV, which were attributed to S2p, B1s, C1s, N1s and O1s, respectively. This further corroborate that the prepared BNS-CDs mainly contain sulfur, boron, carbon, nitrogen and oxygen. High-resolution spectrum (Figure 2A) of C1s indicated the presence of C-B (283.5 eV), C-C/C=C (284.7 eV), C−O/C-S bonds (285.8 eV) and C=O (288.2 ev) on the surface of BNS-CDs.34-36 In addition, the spectrum (Figure 2B) of the N1s showed two dominant peaks which were attributed to pyridinic type (399.2 eV) and pyrrolic type (399 .9 eV) N atoms, respectively, which proved the successful adulteration of nitrogen atoms into the BNS-CDs.37 The C−B bond signal appeared at 190.3 eV, while the signal at 195.1 eV revealed the presence of B−O, and 196.4 eV might attributed to B-S bond (Figure 2C).38,

39

The S2p XPS spectrum (Figure 2D)

demonstrates three peaks centered at 165.3, 166.2 and 167.0 eV, which were ascribed to -C-SOx- (x =2, 3, 4) species, respectively.40,

41

The surface components of

BNS-CDs determined by the XPS were in agreement with that of FT-IR results. According to the FT-IR and XPS results, sulfur, boron and nitrogen have co-doped in BNS-CDs, and their related functional groups, including hydroxyl, carboxyl and amino were existed on their surface.

3.3 PL Properties of BNS-CDs. 8


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UV/Vis absorption and FL spectra were used to investigate the optical properties of BNS-CDs. The UV/Vis spectrum exhibited two absorption peaks at 233 and 281 nm, which are attributed to π-π* transition of the aromatic sp2 domains (Figure 3A).28 In addition, there was a broad absorption band around 520 nm which could be adopted to excite BNS-CDs (Inset in Figure 3A).25 The water suspension of BNS-CDs showed an excitation-independent feature that can be attributed to the relative uniform surface state and size distribution.42, 43 As the excitation wavelength varied from 365 to 500 nm (Figure 3B), their maximum emission was all located at ca. 600 nm, the strongest emission intensity (QY=5.44%) was obtained when the excitation wavelength was fixed at 500 nm. Accordingly, red-shift of photoluminescence of CDs might caused by surface functional groups (carboxyl etc.) and doping with other elements (such as nitrogen and sulfur)35, 44-46. In this work, changing the reagent ratio did cause an evident change of the optical properties of BNS-CDs (Figure S1C). So we hypothesize the doped elements played an important role in controlling their FL property. For figuring out the effects of individual dopants (N, S and B), the contents of B, N and S in each sample prepared with different B:S ratio were measured (Table S1) and some control experiments were also carried out (Figure S4). As shown in Table S1, the higher B:S ratio, the higher percentage of B in BNS-CDs. Meanwhile, the contents of S and N showed no obvious tendency. So it was reasonable that the variation of the optical properties of BNS-CDs prepared by different reagents ratio was mainly caused by the change of B content. Additionally, the FL intensity of BNS-CDs (Figure S4, line a) was stronger than that of the CDs prepared by 2, 5-diaminobenzenesulfonic acid and aniline (Figure S4, line b), further confirming doped B can improve the FL performance of BNS-CDs. S free CDs showed a very weak emission (Figure S4, line c), implying S doping can also boost the emission intensity of BNS-CDs. Moreover, effects of nitrogen elements provided by 2, 5-diaminobenzenesulfonic acid and 4-aminophenylboronic acid on regulating the optical property of BNS-CDs were investigated respectively. When 2, 5-diaminobenzenesulfonic acid was replaced by benzenesulfonic acid, the hydrothermal product showed very weak emission signal 9



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with short wavelength (Figure S4, line d), suggesting the nitrogen element provided by 2, 5-diaminobenzenesulfonic acid can greatly enhance the fluorescence of BSN-CDs. CDs prepared by 2, 5-diaminobenzenesulfonic acid and phenylboronic acid showed a strong but wide emission band with short wavelength (Figure S4, line e) compared with that of BNS-CDs. Based on the above results, the strong FL emission may be attributed to the doped B, S and N (provided by 2, 5-diaminobenzenesulfonic acid) atoms could introduce more surface defects, which can reduce nonradiative recombination.47 Meanwhile, the co-doped elements, especially the nitrogen atoms supplied by 4-aminophenylboronic acid, might reduce the band gap. In addition, we speculate this type of N atoms can also make the band gap more uniform, which could be confirmed by the narrow emission peak of BNS-CDs.48 The different performance of nitrogen atoms provided by the two reagents might stem from the distinctive electron affinity of boron and sulfur atoms.49, 50

3.4 Stability of the BNS-CDs. The prepared BNS-CDs (500 µL) was diluted by 2.5 ml Tris-HCl buffer solution (pH=7.0) for FL measurement. The stability of the prepared BNS-CDs under various conditions was studied. The FL intensity of BNS-CDs was almost unchanged under continuous irradiation for 60 min or under NaCl (1.0 M), respectively (Figure 4A and 4B), whereas it decreased slightly when the temperature was increased from 25 to 45 °C (Figure 4C). This implies that BNS-CDs possessed excellent stability for biomedical applications. Subsequently, the FL signals of BNS-CDs at different pH were also recorded and the results are shown in Figure 4D. The FL intensity was increased when the pH was changed from 3.0 to 7.0, followed by decrease in intensity with further increase in pH. In order to study the reason of this phenomenon, zeta-potentials of BNS-CDs in different pH buffer were measured (Table S2). With the pH increased from 3.0 to 9.0, the zeta-potential of BNS-CDs gradually became more negative. Thus, the effect of the pH on the FL intensity of BNS-CDs was resulted from surface charge change due to protonation−deprotonation.51 10


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3.5 Fluorescence sensing of Fe3+ based on the BNS-CDs. Further, the sensing performance of BNS-CDs to Fe3+ was studied. The FL intensity at 595 nm decreased gradually with increasing concentrations of Fe3+ (Figure 5A). The quenching efficiency (F0/F) displayed a good linear relationship with the concentration of Fe3+ in the range of 0.3-546 µM (R2=0.997), where F0 and F were the FL intensity of the BNS-CDs at 595 nm in the absence and presence of Fe3+ respectively, and the detection limit was estimated to be 90 nM (3s/k, in which s was the standard deviation for the blank solution, and k was the slope of the calibration curve) (Figure 5B). Figure 5C showed the photograph of the BNS-CDs solution after adding various concentrations of Fe3+ under UV lamp irradiation (365 nm). The red fluorescence of BNS-CDs was gradually quenched with the increasing concentrations of Fe3+. Furthermore, the detection process can be completed within 1 min (Figure S5), implying that this sensor can be used for real time detection of Fe3+. As the biological system is complex, so the selectivity and competition experiments toward Fe3+ sensing were also carried out. For investigating the selectivity of the BNS-CDs toward Fe3+, the potential interfering substances coexisted in urine and serum samples, including various metal cations (Co2+, Hg2+, Mn2+, Zn2+, Al3+, Ni2+, Cu2+, Fe2+), amino acids (Thr, Try, L-Tyr, Lys, Arg, Glu, Gly, L-Cys, Ala) and GSH were explored. The black bars shown in Figure 5D and 5E represented the FL response of the BNS-CDs towards the interfering substances. Competition experiments were conducted by subsequently adding 500 µM Fe3+ to each solution (red bars, Figure 5D and 5E). The above results indicated that interferents had no obvious influence on the FL intensity of the BNS-CDs except for Fe2+, which can quench the fluorescence slightly.

3.6 Colorimetric Sensing of Fe3+ based on the BNS-CDs. The color of the BNS-CDs solution changed from red to blue after adding different concentrations of Fe3+, and can be observed by un-aided eyes under sunlight (Figure 6). The solution color of BNS-CDs became evident deeper after the addition of 0.3 µM Fe3+, which indicated the colorimetric detection limit of BNS-CDs for Fe3+ was 11



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0.3 µM.

3.7 Possible Mechanism of the Optical Response of BNS-CDs to Fe3+. The quenching of fluorescence in solution by Fe3+ followed a typical Stern-Volmer equation: I0/I=1+Ksv[Fe3+], where I0 is the FL intensity without Fe3+, I is the FL intensity in the presence of Fe3+ and Ksv was the static Stern-Volmer constant. The analysis of the Stern-Volmer plot showed that they followed a linear trend with a Ksv=7.2×103 M-1. Moreover, after the addition of Fe3+, the average FL lifetime of the BNS-CDs was changed from 2.30 to 2.27 ns (Figure 7A). Therefore, judging from the relative large magnitude of the Stern–Volmer constant and nearly constant FL lifetime, the fluorescence quenching provoked by Fe3+ was probably due to static quenching arising from the formation of a stable non-fluorescence complex between surface functional groups of BNS-CDs and Fe3+.31, 52 Meanwhile, the color change of the solution may also stem from the complex interaction between BNS-CDs and Fe3+. To prove this possible explanation, UV/Vis absorption spectra, Zeta-potential and FT-IR were performed. The absorption peaks of the BNS-CDs in the presence of Fe3+ gradually shifted to a longer wavelength (from 520 to 645 nm) in contrast to other metal ions which proves that Fe3+ indeed complexed with BNS-CDs (Figure 7B).53 Hydroxyl, carboxyl and amino groups were on the surface of BNS-CDs (Figure S2b and Figure 2), and carboxyl and hydroxyl groups were in majority because the zeta potential of the prepared CDs in Tris-HCl buffer was -7.18 mv (Table S2). After the addition of Fe3+, the zeta potential value was changed to -10.80 mv. If most Fe3+ was coordinated with the carboxyl or hydroxyl group, this should make the zeta potential more positive.54 Therefore, we deduced that one Fe3+ could coordinate with multiple amino groups, and the positive charge of amino group was shielded partially. Meanwhile, very few carboxyl or hydroxyl may also coordinate with Fe3+. FT-IR was used to further confirm this deduction. Compared with the FT-IR spectrum of BNS-CDs, an evident change was observed when Fe3+ was present (Figure S6), further proving that Fe3+ was selectively coordinated with the amino groups on the surface of BNS-CDs, which was stemming from their stronger complexation 12


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compared with other metal cations.55

3.8 Intracellular Imaging of Fe3+. To evaluate the biomedical applications of BNS-CDs nanosensor, their cytotoxicity to HeLa cells was determined using MTT assay. The cell viability was shown to be more than 93% upon the addition of BNS-CDs over a concentration range of 54-214 µg·mL−1 (Figure S7). Because of their low cytotoxicity and excellent biocompatibility, these nanosensors showed great promise for monitoring Fe3+ in living cells. Hence, cell imaging experiments were carried out to further demonstrates their feasibility in biological applications. After incubating the HeLa cells with BNS-CDs for 6 h at 37 °C, a significant red emission from the intracellular region was observed (Figure 8A). To further investigate their applicability for monitoring intracellular Fe3+ level, exogenous Fe3+ was introduced into the BNS-CDs-pretreated HeLa cells. It was observed that upon supplementing cells with 100 µM Fe3+ in the growth medium for 6 h at 37 °C, as expected, microscope images showed very weak intracellular fluorescence (Figure 8B), which indicated that this nanosensor can be used for detecting intracellular Fe3+ with red fluorescence emission. Thus, BNS-CDs nanosensors can serve as an effective probe for intracellular Fe3+ sensing.

3.9 Detection of Fe3+ in Biological Samples. To further evaluate their practical applications, the BNS-CDs nanosensors were applied for detecting Fe3+ in human urine and serum samples. The urine and serum samples were subjected to a 100-fold dilution before analysis. Diluted samples were spiked with various concentrations of standard Fe3+ solution. As shown in Table S3, good recoveries and high analytical precision were obtained. These results further confirmed the reliability and feasibility of the BNS-CDs-based nanosensors for monitoring Fe3+ in biological samples. Comparing the performance of the prepared BNS-CDs with the previously reported CDs in sensing Fe3+ (Table 1), the sensitivity and selectivity of this sensor was comparable in both fluorescent and colorimetric manner.

4. CONCLUSIONS 13



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BSN-CDs were successfully prepared in one-step hydrothermal treatment of 2, 5-diaminobenzenesulfonic acid and 4-aminophenylboronic acid hydrochloride. The prepared BSN-CDs nanosensors possess excellent optical and chemical properties. Furthermore, a colorimetric and fluorescent dual mode sensing method based on the BSN-CDs for effective and fast sensing of Fe3+ was developed. More significantly, the established nanosensor exhibited good biocompatibility, high selectivity, low cytotoxicity and could be successfully applied for imaging intracellular Fe3+. More in-depth research on the nanosensors which can specifically monitor the disease-related molecular targets is required.

AUTHOR INFORMATION *Corresponding Author Cuiling Ren, Doctor, Tel: 86-931-8912763 Fax: 86-931-8912582 E-mail: [email protected]

ASSOCIATED CONTENT Supporting Information Figure S1-S7, FL signal of BNS-CDs obtained under different carbonization temperature, time, and reagent ratio, XRD pattern, FT-IR spectrum and XPS wide spectrum of BNS-CDs, control experimental results, time-dependent FL intensity of BNS-CDs with the addition of 500 µM Fe3+, FT-IR spectrum of BNS-CDs and their coordination product with Fe3+, cell viability of HeLa cells with different concentrations of BNS-CDs. Table S1-S3, elemental content of BNS-CDs under different reagent ratio (B:S), Zeta-potential of BNS-CDs under different conditions, Fe3+ determination results in urine and human serum samples.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (no. 21207057) and the Fundamental Research Funds for the Central Universities 14


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(lzujbky-2016-43).

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Figures and Tables. Scheme 1. Synthesis of BNS-CDs and its sensing performance for the detection of Fe3+ in colorimetric and fluorescent manner. Figure 1. TEM images (A) and size distribution histograms (B) of the synthesized BNS-CDs. Figure 2. High resolution XPS spectra of C1s (A), N1s (B), B1s (C) and S2p (D) of the prepared BNS-CDs. Figure 3. UV−Vis absorption (A) and FL emission spectra (λex=365~500 nm) (B) of BNS-CDs. Figure 4. FL intensity variation of the BNS-CDs as a function of illumination time (A), concentrations of NaCl (B), temperature (C) and (D) pH. (λex =500 nm). Figure 5. FL emission spectra of the BNS-CDs upon the addition of various concentrations of Fe3+ from 0 to 642 µM (A), Relationship between F0/F and the concentration of Fe3+, where F0 and F were FL intensities of the BNS-CDs at 595 nm in the absence and presence of Fe3+, respectively (B), Photographs of BNS-CDs with the addition of different concentrations of Fe3+ (from left to right: 0, 0.3, 25, 60, 100, 250, 400, 500 µM) under 365 nm UV lamp irradiation (C), FL intensity response (F/F0) of BNS-CDs toward various metal ions (Fe3+: 500 µM; Co2+, Hg2+, Mn2+, Zn2+, Al3+, Ni2+: 5 mM; Cu2+: 2.5 mM; Fe2+: 500 µM; black bars) and the subsequent addition of 500 µM Fe3+ (red bars) (D), FL intensity response (F/F0) of the BNS-CDs toward various amino acids and GSH (Fe3+: 500 µM; Thr, Try, L-Tyr, Lys, Arg, Glu, Gly: 5 mM; GSH, L-Cys: 500 µM; black bars) and the subsequent addition of 500 µM Fe3+ (red bars) (E), where F0 and F were FL intensities of the BNS-CDs at 595 nm in the absence and presence of Fe3+, respectively. Figure 6. Photographs of BNS-CDs solution with the addition of different concentrations of Fe3+ (from left to right: 0, 0.3, 25, 60, 100, 250, 400, 500 µM) under sunlight. Figure 7. Fluorescence decay traces of BNS-CDs in the absence (black curve) and presence (red curve) of Fe3+ (A), UV-Vis absorption spectra of BNS-CDs and after the 22


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addition of different metal cations (B). Figure 8. Fluorescence microscopy images (A, B) and their corresponding bright-field transmission images (C, D) of HeLa cells: (A, C) incubated with 0.3 mg·mL−1 BNS-CDs for 24 h at 37 °C. (B, D) first incubated with 0.3 mg·mL−1 BNS-CDs for 24 h and then incubated with 100 µM FeCl3 for 6 h at 37 °C. Table 1. Comparison of different CDs for the sensing of Fe3+.

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Scheme 1. Synthesis of BNS-CDs and its sensing performance for the detection of Fe3+ in colorimetric and fluorescent manner.

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Figure 1. TEM images (A) and size distribution histograms (B) of the synthesized BNS-CDs.

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Figure 2. High resolution XPS spectra of C1s (A), N1s (B), B1s (C) and S2p (D) of the prepared BNS-CDs.

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Figure 3. UV−Vis absorption (A) and FL emission spectra (λex=365~500 nm) (B) of BNS-CDs.

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Figure 4. FL intensity variation of the BNS-CDs as a function of illumination time (A), concentrations of NaCl (B), temperature (C) and (D) pH. (λex =500 nm).

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Figure 5. FL emission spectra of the BNS-CDs upon the addition of various concentrations of Fe3+ from 0 to 642 µM (A), Relationship between F0/F and the concentration of Fe3+, where F0 and F were FL intensities of the BNS-CDs at 595 nm in the absence and presence of Fe3+, respectively (B), Photographs of BNS-CDs with the addition of different concentrations of Fe3+ (from left to right: 0, 0.3, 25, 60, 100, 250, 400, 500 µM) under 365 nm UV lamp irradiation (C), FL intensity response (F/F0) of BNS-CDs toward various metal ions (Fe3+: 500 µM; Co2+, Hg2+, Mn2+, Zn2+, Al3+, Ni2+: 5 mM; Cu2+: 2.5 mM; Fe2+: 500 µM; black bars) and the subsequent addition of 500 µM Fe3+ (red bars) (D), FL intensity response (F/F0) of the BNS-CDs toward various amino acids and GSH (Fe3+: 500 µM; Thr, Try, L-Tyr, Lys, Arg, Glu, Gly: 5 mM; GSH, L-Cys: 500 µM; black bars) and the subsequent addition of 500 µM Fe3+ (red bars) (E), where F0 and F were FL intensities of the BNS-CDs at 595 nm in the absence and presence of Fe3+, respectively. 29



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Figure 6. Photographs of BNS-CDs solution with the addition of different concentrations of Fe3+ (from left to right: 0, 0.3, 25, 60, 100, 250, 400, 500 µM) under sunlight.

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Figure 7. Fluorescence decay traces of BNS-CDs in the absence (black curve) and presence (red curve) of Fe3+ (A), UV-Vis absorption spectra of BNS-CDs and after the addition of different metal cations (B).

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Figure 8. Fluorescence microscopy images (A, B) and their corresponding bright-field transmission images (C, D) of HeLa cells: (A, C) incubated with 0.3 mg·mL−1 BNS-CDs for 24 h at 37 °C. (B, D) first incubated with 0.3 mg·mL−1 BNS-CDs for 24 h and then incubated with 100 µM FeCl3 for 6 h at 37 °C.

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Table 1. Comparison of different CDs for the sensing of Fe3+. Detection Type of

Detection

UV

Line range

Detection

technique

irridiation

(µM)

limit (µM)

Wavelengt probe

ref

h (nm) CDs

450

Fluorometry

Yes

0.16-1.66

6.05

56

CDs

405

Fluorometry

Yes

1-5&5-50

0.025

57

CDs

450

Fluorometry

Yes

1-10&25-700

0.48

58

N-CDs

411

Fluorometry

Yes

0-100

0.96

59

N-CDs

435

Fluorometry

Yes

0.01-500

0.0025

29

N-CDs

443

Fluorometry

Yes

0.5-2000

0.0136

54

N-CDs

497

Fluorometry

Yes

50-300

10.8

60

S-CDs

440

Fluorometry

Yes

1-500

0.1

61

B-CDs

437

Fluorometry

Yes

0-16

0.242

62

N,S-CDs

449

Fluorometry

Yes

6-200

0.8

31

N,P-CDs

436

Fluorometry

Yes

0.005-0.1

0.0018

53

595

Fluorometry & Colorimetry

Yes & No

0.3-546 & ~

0.09 & 0.3

This work

BNS-CDs

~ No linear range for colorimetric detection

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Red Emission B, N, S-co-Doped Carbon Dots for Colorimetric and

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