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Deep eutectic solvent assisted preparation of nitrogen/chloride doped carbon dots for intracellular biological sensing and live cell imaging Ning Wang, An-Qi Zheng, Xun Liu, Junjie Chen, Ting Yang, Ming-Li Chen, and Jian-Hua Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b00947 • Publication Date (Web): 09 Feb 2018 Downloaded from http://pubs.acs.org on February 10, 2018

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ACS Applied Materials & Interfaces

Deep eutectic solvent assisted preparation of nitrogen/chloride doped carbon dots for intracellular biological sensing and live cell imaging

Ning Wang‡, An-Qi Zheng‡, Xun Liu, Jun-Jie Chen, Ting Yang, Ming-Li Chen* and Jian-Hua Wang* Research Center for Analytical Sciences, Department of Chemistry, College of Sciences, Northeastern University, Shenyang 110819, China ABSTRACT: A novel approach for the preparation of dual-functional carbon dots, i.e., nitrogen and chloride doped carbon dots, shortly as N/Cl-CDs, is developed with the assistance of a choline chloride-glycerine deep eutectic solvent (DES). The carbon source is provided by urea and the DES serves as a solvent for controlling the preparation of CDs in the absence of water. The dual-element doped carbon dots are oxygen-rich with hydroxyl and amine groups. They exhibit an average particle size of ca. 3.88 nm, and give rise to strong and pH-sensitive fluorescent emission at λex/λem=340/430 nm with a quantum yield of 16.15±1.36%. It is particularly interesting to see that the fluorescence of N/Cl-CDs remains stable in high salinity matrix, providing vast potentials for treating real biological sample matrixes with high salinity. The N/Cl-CDs provide an optical probe for intracellular pH sensing and multicolor imaging in HeLa cells. In addition, the N/Cl-CDs show obvious fluorescence response to cytochrome c (cyt-c) with a detection limit of 3.6 mg L-1 (ca. 0.29 µmol L-1) within in a range of 10-500 mg L-1, providing potentials for fluorescence detection of cyt-c as well as facilitating intracellular cyt-c imaging. KEYWORDS: deep eutectic solvents, dual-functional carbon dots, nitrogen/chloride doping, intracellular sensing, live cell imaging

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1. INTRODUCTION Deep eutectic solvents (DES) are generally eutectics formed between two naturally occurring biocompatible components and via the interaction between the ammonium salt and the hydrogen-bond donor.1 A series of studies on deep eutectic liquid systems have been directed to choline chloride, e.g., choline chloride-urea,2,3 choline chloride-alcohols4,5 and choline chloride-carboxylic acid,6 which have emerged as important solvents for synthetic chemistry,3,7 catalyst,8,9 separation10 and electrochemisty.5 DES are highly structured supramolecular solvents that show promising potentials for shape-controlled synthesis of nanoparticles,2,6,11 due to their attractive features of low freezing point, outstanding capacity for dissolution, favorable biocompatibility as well as low toxicity. In biological systems, intracellular pH and the level of cytochrome c (cyt-c) play vital roles in the various physiological and pathological processes, e.g., regulation of cell behaviors including protein structures or functions, metabolism, migration, transformation and apotosis.12 Thus the measurement of intracellular pH value as well as cyt-c variation and its distribution in living cells have great significance for better understanding of the cellular functions in addition to providing pivotal assistance for early diagnosis of diseases.13 A number of analytical protocols and techniques have been developed for intracellular pH sensing.14-16 Among those approaches, optical probes based on pH-induced fluctuations in the fluorescence intensity are attracting extensive attentions in pH sensing attributed to the advantages of excellent spatial and temporal resolution, rapid response, high signal-to noise ratio, non-invasiveness and favorable sensitivity.12,17,18 In particular, quite a few optical probes based on organic dyes,19 functional quantum dots13, 17 and fluorescent proteins15 have been widely used for monitor local pH values in cell interior. However, in practical intracellular pH 2


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sensing, the fluorescence intensity change might be influenced by a lot of parameters, including the bleaching of dyes, the cytotoxicity of the probe itself and the binding of macromolecules in the biological systems. cyt-c plays an important physiological role not only in oxidative phosphorylation and apoptosis process, but it is also a marker for acute liver failure.20 Therefore, the measurement of cyt-c in biological systems, particularly in cell interiors, is highly important for better understanding the cell apoptosis.21 So far, a variety of methods have been developed for cyt-c detection based on electrochemiluminescence,22,23 immunosorbent assay,20 liquid chromatography,24 flow cytometry25 and electrochemistry.26,27 However, at the present it is still highly challenging when handling complex real biological systems by using these analytical techniques. For the purpose of overcoming the above mentioned limitations in the sensing of intracellular pH and cyt-c level, eco-friendly multi-functional carbon dots with multicolor bioimaging responsive features are highly desired. In the present work, we report the preparation of novel nitrogen and chloride doped dual-functional carbon dots (N/Cl-CDs) in a choline chloride-glycerine deep eutectic solvent with urea as carbon source. The dual-element doped carbon dots contain plenty of hydroxyl and amine groups. They exhibit low cytotoxicity and provide extraordinary photostability. In addition, the multiple functional groups in N/Cl-CDs facilitate their multicolor emission feature, offering a selective probe for the sensing of both intracellular pH and cyt-c level by cell imaging.

2. EXPERIMENTAL SECTION 2.1 Preparation of nitrogen/chloride doped carbon dots (N/Cl-CDs). A choline chloride-glycerine deep eutectic solvent is first prepared by the following procedure. 3



43 g of choline chloride and 22.7 g of glycerine (with a molar ratio of 2.5:2) were dried at 60oC for 24 h in an oven. They were then mixed and stirred at 100oC for 2 h until a uniform liquid eutectics mixture was obtained, that is the choline chloride-glycerine deep eutectic solvent. 0.15 g of urea was taken to mix in a beaker with 5 mL of choline chloride-glycerine deep eutectic solvent under stirring until the urea was thoroughly dissolved. The mixture was allowed to undergo further reaction in a microwave oven at 720 W for 5 min. After cooling down to room temperature, brown color solid product was obtained. Afterwards, 5 mL of deionized water was added followed by ultrasonic oscillation for 5 min, to disperse the large or agglomerated particles which were collected by centrifugation at 13000 rpm for 10 min. Finally, the obtained N/Cl-CDs were further purified with dialysis for 72 h by using cellulose ester membrane (MWCO: 100-500). For the purpose of comparison, nitrogen doped carbon dots (N-CDs) were prepared with choline instead of choline chloride by following the same procedures. 2.2 Fluorescence property of N/Cl-CDs in saline matrix. Various concentrations of sodium chloride within a range of 0-1.2 mol L-1 were used to investigate the dependence of fluorescence property and its stability on the variation of ionic strength in the reaction medium. In addition, for the purpose of evaluating the interfering effects of various species in biological sample matrixes, the influence of commonly encountered coexisting cationic and anionic species, e.g., K+, Ca2+, Na+, Mg2+, Al3+, Co2+, Cu2+, Fe2+, SO42-, Cl-, H2PO4-, NO3-, amino acids, e.g., glutamic, serine, valine, isoleucine, proline, alanine, threonine and phenylalanine, as well as protein species, e.g., HSA, lysozyme, ovalbumin, γ-globulin from bovine, H2O2 and dexamethasone were thoroughly investigated. For all the studies, the fluorescence was recorded at the maximum excitation/emission wavelengths, i.e., at λex/λem 340/430 nm. 4


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2.3 pH-sensitive feature and fluorescent sensing of cyt-c. The pH-sensitive fluorescent feature of the nitrogen/chloride doped carbon dots was investigated by recording the fluorescence spectra of N/Cl-CDs at 0.5 mg mL-1 by varying the pH values of the medium within a range of pH 5.02-10.00 regulated with BR buffers. In addition, pH sensing in the cell lysates and surface water samples were performed to further demonstrate its practical applications. The cell lysates were obtained by treating HepG2 and A549 cells under ultrasonic oscillation for 20 min followed by filtration through a 0.2 µm filter membrane. The surface water samples were filtered through a 0.2 µm filter membrane for pH sensing. For cyt-c sensing, cyt-c solutions (300 µL, 10-1000 mg L-1) were mixed with 100 µL of N/Cl-CDs solution (2 mg mL-1) giving rise to an ultimate concentration of N/Cl-CDs of 0.5 mg mL-1. The fluorescence spectra were then recorded with λex/λem at 340/430 nm. For intracellular sensing of cyt-c, HeLa cells were first incubated with 0.5 mg mL-1 of N/Cl-CDs, and then mixed with dexamethasone of 100 µg L-1 to accelerate the release of cyt-c from mitochondria. Afterwards the apoptosis induced by cyt-c was identified with confocal fluorescence imaging. 2.4 Cytotoxicity test and cellular imaging. HeLa cells were first cultured in DMEM medium in 100 units mL-1 penicillin, 100 mg mL-1 streptomycin and 10% fetal bovine serum (FBS), and were then employed for in vitro cytotoxicity assay and cell fluorescence imaging. Thereafter, HeLa cells were added into a 96-well plate by incubating for 24 h in the presence of N/Cl-CDs at various concentration levels, e.g., 0.01-5 mg mL-1. After a further incubation time of 20 h at 37oC in 5% CO2, 15 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was added into each well and the cells were incubated for additional 4 h. After removing the supernatant, 200 µL of DMSO was added into each well, and the absorbance was measured at 490 5



nm with an ELISA plate reader. For in vitro imaging investigations, the HeLa cells were washed for three times with a PBS buffer (10 mmol L-1, pH 7.4), and then incubated in a medium containing 0.5 mg mL-1 of N/Cl-CDs which was filtered through a 0.2 µm sterilized filter membrane before use. After incubating for 1, 3, 5, 7 and 9 h, the HeLa cells were washed with PBS buffer (10 mmol L-1, pH 7.4) and the free N/Cl-CDs were removed followed by in vitro imaging. For the uptake of N/Cl-CDs by HeLa cells, the cells in three dishes were treated in the presence of 0.5 mg mL-1 of N/Cl-CDs as described herein: the first dish was incubated at 37oC for 5 h, the second at 4oC for 5 h, and the third was first treated with 2-deoxy-D-glucose (50 mmol L-1) at 37oC for 45 min, followed by incubation at 4oC for 5 h. 2-deoxy-D-glucose was used for disturbing the production of adenosine triphosphate in the cells to block the endocytic pathway. The cells were afterwards washed with PBS buffer (10 mmol L-1, pH 7.4) for three times. Thereafter the fluorescence images were recorded with a confocal fluorescent microscope with laser excitation at λex 405, 488, 559 nm, and λem 425-475, 500-535, 575-675 nm to demonstrate the uptake pathway of N/Cl-CDs. For intracellular pH sensing, HeLa cells were firstly incubated with 0.5 mg mL-1 of N/Cl-CDs for 5 h. Afterwards the medium was removed and the cells were treated with nigericin (5 µg mL-1) for 10 min in a HEPES buffer at various pH values (pH 5.37, 6.38, 7.38, 8.34 and 9.57). Nigericin is used to alter the permeability of cell membranes and improve proton (H+) balance in the interior and exterior of HeLa cells, and thus to maintain an equal pH therein. The cells were washed for three times with PBS buffer (10 mmol L-1, pH 7.4). Thereafter the fluorescence images were recorded with a confocal fluorescent microscope at λex 405, 488, 559 nm, and λem at 425-475, 6


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500-535, 575-675 nm to evaluate the pH-fluorescence relationship. For intracellular fluorescence sensing of cyt-c, the HeLa cells were first incubated with 0.5 mg mL-1 of N/Cl-CDs for 5 h, and then the culture medium was removed. The HeLa cells after uptaking N/Cl-CDs were further cultured in a fresh medium in the presence of 100 µg L-1 dexamethasone to accelerate the release of cytc-c from mitochondria. Afterwards the apoptosis induced by cyt-c was identified with confocal fluorescence imaging at the same excitation/emission wavelength as those for intracellular imaging. 3. RESULTS AND DISCUSSION 3.1 Characterizations. High resolution transmission electron microscopy (HRTEM) image as illustrated in Figure 1a indicated that the prepared N/Cl-CDs were spherical and well dispersed without agglomeration in the aqueous medium. As shown in Figure 1b, the diameter of N/Cl-CDs was estimated at 1.35-6.95 nm, and the average particle size distribution was centered at ca. 3.88 nm, which was well consistent with the size detected by using dynamic light scattering (DLS) as shown in Figure S1,where the diameter of N/Cl-CDs was derived to be within 2.69-6.51 nm, with an average particle size distribution centered at ca.3.79 nm.

Figure 1. HRTEM image (a) and particle size distribution (b) of the nitrogen/chloride doped carbon dots (N/Cl-CDs). 7



Atomic force microscopy (AFM) image in Figure 2 illustrated that the height distribution was found in the range of 1-4 nm. This observation was well consistent with that achieved in the HRTEM image, i.e., an average particle size distribution centered at ca. 3.88 nm.

Figure 2. AFM image (a) and particle height distribution (b) of the nitrogen/chloride doped carbon dots (N/Cl-CDs)

X-ray photoelectron spectra (XPS) were performed to investigate the surface state of the nitrogen/chloride doped carbon dots (N/Cl-CDs) as shown in Figure S2. It is seen that four typical peaks for C1s (284.7 eV), N1s (401.3 eV), O1s (532.0 eV) and Cl2p (196.9 eV) were identified in the full scan spectrum, and elemental analysis resulted in the composition of 65.47 wt% for C, 8.17 wt% for N, 17.43 wt% for O and 8.93 wt% for Cl. This result clearly illustrated the doping of N and Cl on CDs surface. The peak of C1s was further deconvoluted into four peaks for C-C, C-N, C-O (C-Cl) and C=O bonds at 284.6 eV, 285.8 eV, 286.6 eV and 287.8 eV, respectively, as indicated in the high resolution C1s XPS spectra (Figure 3a).28 Similarly, N1s spectra contained two peaks (Figure 3b), attributed to C-N (399.4 eV) and N-H (401.4 eV), respectively. The two main bands at 531.3 eV and 532.2 eV in the O1s spectra (Figure 3c) could be identified as C=O and C-OH. Cl2p spectra (Figure 3d) were confirmed to contain two main bands at 196.9 eV and 198.5 eV, which were ascribed to chlorine in the two groups, e.g., C-Cl and N-Cl, respectively.29,30 8


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Figure 3. High resolution XPS spectra of the nitrogen/chloride doped carbon dots (N/Cl-CDs). C1s (a), N1s (b), O1s (c) and Cl2p (d).

FT-IR spectra of N/Cl-CDs were illustrated in Figure S3 with those of urea and DES for comparison. It is indicated that the broad band in the range of 3160-3540 cm-1 was ascribed to the O-H and N-H stretching vibrations of amine groups in the structure of N/Cl-CDs. The absorptions at 2880 and 2950 cm-1 were attributed to the C-H stretching vibrations, while those appeared at 1680 and 1630 cm-131 were assigned to the stretching vibrations of the CO-NH group. In addition, the absorption band at 1460 cm-1 was due to the typical stretching vibration mode of C-N, and the absorptions at 1040 to 1100 cm-1 were associated with the C-O bending vibrations.32 It was seen that the characteristic absorption bands in both urea and DES structure were identified in the FT-IR spectrum for N/Cl-CDs. Figure S4a showed an obvious absorption at 275 nm in the UV-vis adsorption spectra of the nitrogen/chloride doped carbon dots (N/Cl-CDs). This absorption could 9



be assigned to the n-π* transition of the C=O band. The fluorescence spectra in Figure S4b illustrated a wavelength-dependent emission feature, and the emission wavelength shifted from 420 nm to 480 nm by varying the excitation wavelength from 320 nm to 420 nm. A maximum fluorescence emission was encountered at λex/λem=340/430 nm. This result clearly indicated that the present N/Cl-CDs could be used for multicolor imaging with excitation at various wavelengths. A quantum yield (QY) of 16.15±1.36% was derived for the nitrogen/chloride doped CDs by using quinine sulfate as the standard fluorophore with a quantum yield of Φ std 0.55.33 3.2 Fluorescence stability of N/Cl-CDs in high salinity matrix. For the purpose of evaluating the effect of ionic strength on the fluorescence property of N/Cl-CDs and its stability in saline matrix, the fluorescence spectra of N/Cl-CDs solution (0.5 mg mL-1) were recorded in the presence of various concentration levels of NaCl (0-1.2 mol L-1), and the results were given in Figure S5a. It was seen that virtually no effect on the fluorescence of N/Cl-CDs was observed within the NaCl concentration range studied. That is to say the fluorescence of N/Cl-CDs remains stable in the presence of high salinity sample matrix, and this feature is particularly important for treating real biological sample matrixes, where high salinity is frequently encountered. In addition, Figure S5b illustrated favorable stability of the fluorescence of N/Cl-CDs, where no obvious variation was observed after illumination for 2 h. 3.3 pH-sensitive fluorescence feature of N/Cl-CDs. It was previously reported that carbon nanoparticles derived from PEI were pH-sensitive due to the response of amide groups in an acidic medium.34 The experimental results indicated that the nitrogen/chloride doped carbon dots prepared in DES is highly pH-sensitive. As showed in Figure 4a, a significant decrement of the fluorescence emission intensity was recorded by varying pH value of the medium within a range of pH 5.02-10.00 as 10


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adjusted by a BR buffer solution. Figure 4b illustrated a linear relationship between fluorescence intensity of N/Cl-CDs and pH value of the medium, as expressed by a regression equation of F=-258.2pH+4579 with a correlation coefficient of 0.9938.

Figure 4. The dependence of fluorescence spectra for the nitrogen/chloride doped carbon dots on pH values within a range of pH 5.02-10.00 (a). A linear relationship by plotting the fluorescence intensity of the nitrogen/chloride doped carbon dots versus pH value of the medium (b).

Previous studies have demonstrated that the pH-sensitive feature of nanoparticles is associated with various functional groups on their surfaces, e.g., amide, carbonyl or amino groups.35-37 In the present work, the strongly pH dependent photoluminescence emission indicated the presence of a large number of acidic or basic functional groups in the structure of N/Cl-CDs. The surface charge property or zeta potential for the N/Cl-CDs was investigated by varying pH value of the medium. Table 1 illustrated zeta potentials for the N/Cl-CDs solution (0.5 mg mL-1) derived at pH 5.37, pH 6.38, pH 7.38, pH 8.34 and pH 9.57. It is seen that a minor change on pH value resulted in an obvious variation on zeta potential. This indicated that the surface of N/Cl-CDs was highly sensitive to the acidity of solution, and this observation is consistent with that achieved from the surface structure analysis results. In general, the observations herein showed that amino and hydroxyl groups were the main functional groups on the surface of N/Cl-CDs which make them ease of protonation and deprotonation.38 In 11



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addition, Cl doped carbon dots tend to form hydrogen bonds, providing a further driving force for the sensitive detection of pH value. The relationship between fluorescence intensity and pH value as described in Figure S6, i.e., F=-151.4pH+2918, demonstrated that the N/Cl-CDs provide a much higher sensitivity for pH sensing with respect to that with N-CDs as the sensing element. Table 1. Zeta potentials of the nitrogen/chloride doped carbon dots (N/Cl-CDs) derived at various pH values. pH 5.37 6.38 7.38 8.34 9.57

Zeta potential (mV) 11.70±1.09 8.25±1.10 6.47±0.74 3.94±0.12 1.22±0.34

The practical applications with N/Cl-CDs as a fluorescence probe for pH sensing have been preliminarily demonstrated with various sample matrixes including cell lysates and surface water samples. The sensing results were displayed in Table 2. It could be seen that the corresponding pH values derived by the N/Cl-CDs probe were comparable with those obtained by using a pH meter. Table 2. pH sensing in cell lysates and surface water samples with the nitrogen/chloride doped carbon dots (N/Cl-CDs) as probe with excitation/emission at λex/λem=340/430 nm. Sample medium HepG2 cell lysate A549 cell lysate River water Seawater Tap water

N/Cl-CDs probe 7.24±0.01 6.96±0.05 7.64±0.05 7.67±0.04 6.74±0.07

pH meter 7.19±0.02 6.99±0.07 7.68±0.03 7.46±0.14 6.87±0.16

3.4 Cyt-c sensing performance. The experiments indicated that the fluorescence of N/Cl-CDs solution at λex/λem 340/430 nm exhibits an obvious response in the presence of cyt-c, that is, a significant decrease on the fluorescence intensity was 12


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observed by the addition of cyt-c into the N/Cl-CDs solution (Figure 5a). By mixing 100 µL of N/Cl-CDs solution (2 mg mL-1) with 300 µL of cyt-c solution of various concentrations (10-1000 mg L-1) under continuous shaking for 10 min, a maximum fluorescence was observed and a linear calibration was obtained by plotting the net fluorescence decrease (F-F0) versus cyt-c concentration (Figure 5b), where F and F0 represent the fluorescence intensity of the N/Cl-CDs solution in the absence and presence of cyt-c, respectively. The present approach provided a favorable sensitivity for cyt-c sensing with a limit of detection of 3.6 mg L-1 (0.29 µmol L-1). As a comparison, a limit of detection of 0.41 µmol L-1 was reported with CdTe quantum dots as a fluorescence probe,39 and a limit of detection of 0.5±0.03 µmol L-1 was achieved with carbon nanotubes incorporated polypyrrole as the sensing probe.40

Figure 5. Fluorescence spectra of the nitrogen/chloride doped carbon dots (N/Cl-CDs) at various concentration levels of cyt-c (a) within a range of 10-1000 mg L-1, and the linear relationship by plotting F0-F versus cyt-c concentration (b).

It is known that the release of cyt-c is an important step in apoptosis. The oxidized form of cyt-c (Fe3+) could induce caspase activation via the apoptosome, while this phenomenon was not observed in the reduced form (Fe2+) of cyt-c.41 In the present study, the decrease of fluorescence of N/Cl-CDs in the presence of cyt-c might be due to the fact that plenty of functional groups on the surface of N/Cl-CDs, e.g., amino and hydroxyl groups, could transform cyt-c (Fe3+) to its reduced form (Fe2+). For the

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purpose of comparison, the fluorescence intensity of N-CDs solution also exhibits a response to cyt-c, as shown in Figure S7, where a linear calibration curve was obtained by plotting the net fluorescence decrease (F-F0) versus cyt-c concentration. However, the N-CDs system provided a lower sensitivity with respect to that achieved with N/Cl-CDs as the sensing element. That is due to the fact that chloride doped N/Cl-CDs exhibited high separation efficiency of photo-induced electrons and holes,30,42 which further promoted the transform of oxidized form of cyt-c (Fe3+) to its reduced form (Fe2+). 3.5 The potential interfering effects. To evaluate the selectivity of N/Cl-CDs as a sensing probe for cyt-c, the interfering effects of various commonly encountered cationic and anionic species at a certain concentration level (10 mmol L-1). More details were given in the experimental section. The results indicated that these species caused no obvious influence on the sensing of cyt-c (Figure S8a). In addition, 10 mmol L-1 of various amino acids, 1.0 mmol L-1 of H2O2, 100 µg L-1 of dexamethasone, and 1000 mg L-1 of various protein species caused no obvious variation on the fluorescence intensity of N/Cl-CDs (Figure S8b). 3.6 Cytotoxicity test and the suitability for cell imaging by N/Cl-CDs. Cellular cytotoxicity of the N/Cl-CDs was evaluated by a MTT assay and the results were given in Figure S9. It is seen that N/Cl-CDs were of low cytotoxicity producing a high cell viability of ca. 96% after incubating with 1.0 mg mL-1 of N/Cl-CDs for 24 h. Moreover, a cell viability of ca. 90% was still maintained at a N/Cl-CDs concentration of 5.0 mg mL-1. The favorable biocompatibility and low cytotoxicity of N/Cl-CDs is particularly promising for the ensuing cell imaging studies. Figure 6 illustrated the cell imaging results after incubating HeLa cells with 0.5 mg mL-1 of N/Cl-CDs for 1, 3, 5, 7 and 9 h. HeLa cells emitted strong blue, green and red 14


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fluorescence under excitation at 405, 488 and 559 nm. It is obvious that the fluorescence intensity of HeLa cells was increased with the increase of incubation time up to 5 h, this is probably due to gradual accumulation of N/Cl-CDs into the cytoplasm, demonstrating the favorable membrane permeability of N/Cl-CDs. Afterwards, a decrease of fluorescence intensity was observed by further increasing the incubation time, which might be due to the excretion of N/Cl-CDs out of the cells by metabolism. An incubation time of 5 h was thus selected for further experiments.

Figure 6. Confocal fluorescence images of HeLa cells after culturing with 0.5 mg mL-1 of the nitrogen/chloride doped carbon dots (N/Cl-CDs) for 1, 3, 5, 7 and 9 h. The first column was bright field image, while the 2-5 columns showed the images with excitation wavelengths at 405, 488, 559 nm and the merging images. The uptake of N/Cl-CDs by HeLa cells is an energy dependent endocytosis process, which can be proved by incubating the cells at a low temperature (4oC) or in an adenosine triphosphate depleted environment.43 It is known that the treatment with 15



2-deoxy-D-glucose can disturb the production of adenosine triphosphate in the cells and thus block the endocytic pathway. The experimental results indicated that in comparison with the observation at an incubation temperature of 37°C, a much lower fluorescence brightness was found when incubating HeLa cells at 4oC or pretreating with 2-deoxy-D-glucose at 37°C followed by incubating at 4oC (Figure S10). It was further observed that an obvious reduction on the mean optical density (calculated by bio-analysis software ImageJ) was encountered for the latter cases (Figure S11). These observations clearly demonstrated that the entry of N/Cl-CDs into HeLa cells was mainly mediated through an energy-dependent endocytosis pathway. Intracellular pH sensing. In order to demonstrate the application of N/Cl-CDs for intracellular pH sensing, live HeLa cells were cultured with 0.5 mg mL-1 of N/Cl-CDs. After uptaking N/Cl-CDs, the cells were further treated with nigericin (5 µg mL-1) for 10 min in a HEPES buffer at pH 5.37, 6.38, 7.38, 8.34 and 9.57, followed by conducting confocal fluorescence imaging (Figure 7). As described in the Experimental section, nigericin alter the permeability of cell membranes and improve proton balance in the interior and exterior of HeLa cells, for the purpose of ensuing an equal pH inside the cells and in the HEPES buffer medium. It is clearly seen that after incubating HeLa cells in a HEPES buffer at pH 5.37, brightest fluorescence images were obtained by exciting at all the three excitation wavelengths with respect to those observed by culturing HeLa cells at the other pH values. With the increase of pH value in the culture medium, gradual decreases of the brightness or the intensity of fluorescence images were obtained. These observations illustrated a close correlation of the fluorescence images intensity with pH value in the culture medium, which demonstrated N/Cl-CDs as a promising probe for the potential application in intracellular pH sensing. 16


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Figure 7. Confocal fluorescence images of HeLa cells after culturing with 0.5 mg mL-1 of the nitrogen/chloride doped carbon dots (N/Cl-CDs) in a HEPES buffer medium at various pH values (pH 5.37, 6.38, 7.38, 8.34, 9.57). The first column was bright field image, while the 2-5 columns showed the images with excitation wavelengths at 405, 488, 559 nm and the merging images.

Intracellular cyt-c sensing. It is known that dexamethasone promotes the release of cyt-c from mitochondria, and thereafter it leads to cell apoptosis.44 In the present study, N/Cl-CDs and dexamethasone permeate into the interior of HeLa cells during the incubation process, and therein dexamethasone induces the release of cyt-c from mitochondria, which subsequently induces cell apoptosis. The cell apoptosis depends closely on cyt-c concentration, and the variation of cyt-c concentration is strictly correlated with the fluorescence intensity of N/Cl-CDs. In this respect, N/Cl-CDs provide a promising probe for the imaging of cyt-c level in HeLa cells and the

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corresponding cell apoptosis. In practice, HeLa cells were first incubated with 0.5 mg mL-1 of N/Cl-CDs for 5 h, followed by a further incubation in the absence and presence of dexamethasone (100 µg mL-1) for various times, e.g., 0, 8, 16, 24 min. The dexamethasone induced cyt-c release from mitochondria, and the process of apoptosis induced by cyt-c was monitored by confocal fluorescence imaging (Figure 8). It is seen that virtually no variation of the brightness or fluorescence intensity was observed in the absence of dexamethasone (Figure 8a), where no release of cyt-c was involved corresponding to a very low level of HeLa cell apoptosis. On the contrast, the release of cyt-c and the induced HeLa cell apoptosis were significantly promoted in the presence of dexamethasone, coresponding to remarkable decrease of the brightness in the fluorescence cell images. In addition, Figure 8b clearly showed the promotion of cyt-c release and cell apoptosis with the increase of incubation time.

The above

observations demonstrated that N/Cl-CDs provide a promising probe for intracellular cyt-c sensing, and it might be further employed for monitoring cell apoptosis.

Figure 8. Confocal fluorescence images of HeLa cells first incubated by 0.5 mg mL-1 N/Cl-CDs for 5 h followed by a further incubation for 0, 8, 16, 24 min in the absence of dexamethasone (a) and in the presence of 100 µg mL-1 dexamethasone (b). The first column was bright field image, while the 2-5 columns illustrated the images with excitation wavelengths at 405, 488, 559 nm and the merging images.

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4. CONCLUSIONS We described the preparation of novel nitrogen and chloride doped dual-functional carbon dots (N/Cl-CDs) in a choline chloride-glycerine deep eutectic solvent. The hydroxyl and amine groups endow the dual-element doped carbon dots promising pH-sensitive feature, and cyt-c responsive fluorescence property. In addition, the fluorescence of the doped carbon dots remains extremely stable in high saline matrix, which is particularly important for applications in real biological samples. The carbon dots were demonstrated to be a suitable probe for selective sensing of intracellular pH and cyt-c level by cell imaging. This study provides a useful approach for developing unique carbon dots for facilitating specific biological sensing applications. ASSOCIATED CONTENT Supporting Information The supporting Information: Experimental Section; Chemical and Reagents; Instrumentations; Supplementary Figures S1-S11. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (M. Chen). *E-mail: [email protected] (J. Wang). ORCID Mingli Chen: 0000-0001-8536-8864 Jianhua Wang: 0000-0003-2175-3610 Notes The authors declare no conflict of interest. Author Contribution ‡

N. Wang and A.-Q. Zheng contribute equally.

ACKNOWLEDGMENTS The authors appreciate for financial supports from the Natural Science Foundation of China (21675019, 21727811), and Fundamental Research Funds for the Central Universities (N160302001, N150502001). 19



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Table of contents

25


Chloride

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