Large Emission Red-Shift of Carbon Dots by Fluorine Doping and

Subscriber access provided by READING UNIV

Article

Large Emission Red-Shift of Carbon Dots by Fluorine Doping and Their Applications for Red Cell Imaging and Sensitive Intracellular Ag Detection +

Gancheng Zuo, Aming Xie, Junjian Li, Ting Su, Xihao Pan, and Wei Dong J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.7b10179 • Publication Date (Web): 11 Nov 2017 Downloaded from http://pubs.acs.org on November 13, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

The Journal of Physical Chemistry C is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Large Emission Red-Shift of Carbon Dots by Fluorine Doping and Their Applications for Red Cell Imaging and Sensitive Intracellular Ag+ Detection Gancheng Zuo†, Aming Xie*, §, Junjian Li†, Ting Su†, Xihao Pan†, Wei Dong*, † †

School of Chemical Engineering, Nanjing University of Science & Technology,

Nanjing 210094, P. R. China §

School of Mechanical Engineering, Nanjing University of Science & Technology,

Nanjing 210094, P. R. China ABSTRACT: Heteroatom doping is one of the most effective routes to adjust the physicochemical and optical properties of carbon dots (CDs). However, fluorine (F) doped CDs have been barely achieved. In this work, a F-doping strategy was proposed and adopted to modulate optical properties of CDs. A kind of F-doped CDs was synthesized by a solvothermal process using aromatic F bearing moiety as the F source, and shows much longer maximum emissions (up to 600 nm, red fluorescence) than that of undoped CDs, indicating large emission red-shift effect by F-doping. In addition, the F-doped CDs have remarkable water-solubility, high biocompatibility, as well as excellent stability even under broad pH range, ionic strengths and light illumination, thus can be used as a novel probe for the highly efficient cell imaging of various normal cells and cancer cells. The F-doped CDs can selectively bind to Ag+. It therefore makes the F-doped CDs be a highly sensitive probe for the detection of Ag+ under both aqueous solution and various biological systems. It is indicated the huge potential of this F-doping strategy in the rational design of high-performance CDs, as well as in applications of clinical diagnosis and ion detection.

1. INTRODUCTION Comparing to conventional semiconductor quantum dots that of high toxicity and poor solubility under physiological condition, carbon dots (CDs) have a number of advantages, such as excellent photostability, high biocompatibility, high photoluminescence tunability, chemical inertness, and has displayed huge potential in a broad range of applications, such as chemical sensing,1-3 optoelectronic devices,4-6 theranostics7-10 and bioimaging.11-12 Despite many kinds of CDs have been reported up to now, most of them intensely emit only at blue- to green- light

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

regions under excitation of short-wavelengths light.1-16 These drawbacks limit their application scope, especially in the field of bioimaging, due to the well-known blue auto-fluorescence in biological system and meanwhile significant photodamage of short-wavelengths excitation light. In addition, some reported CDs are restricted to widely use just because of their low water dispersibility, although they have long emission wavelengths, even red emission.13,16 Hence, it is highly important to achieve high-efficient CDs that not only emit at light with wavelength longer than that of green-light, particularly red light, but also possess excellent aqueous dispersibility.17-20 Heteroatom doping is one of the most frequently used routes to improve and tune fluorescence properties of CDs.21 Recently, many studies demonstrated that types of heteroatom have significant effects on the emission property of CDs. For instance, nitrogen (N)-doped CDs furnish red emission fluorescence when using p-phenylenediamine as the N source.13,17,18 If combining of phosphorus (P) and N, the fluorescence properties of the N,P-co-doped CDs do not improve obviously, although their aqueous dispersibility increase evidently.22-24 Sulfur (S)-doping is usually to promote the ion detection sensitivity.25-28 But strong red-emissive light can be observed under the excitation wavelength of 543 nm, if the S element is restrained to the ring of polythiophene.19 In addition, a kind of (boron) B, N, S-co-doped CDs have been demonstrated high-efficient red emission and their maximal emission wavelength are around 600 nm.29 Although many heteroatoms including N, P, S, B and their combinations doped CDs have been reported, there is also strong demand for the development of high-efficient CDs. Consequently, it is still highly desirable to explore other heteroatom doping system. Fluorine (F) is one of the most common elements in the world. Despite the absence of F in biological systems, the graft of F-containing moieties can increase the therapeutic efficacy of numerous drugs,30 improve the chemical stability of proteins,31 and enhance phase-separation tendency in both polar and non-polar environments.32,33 Fluorination decreases surface energy of most polymers, and hence makes these fluorinated polymers prefer to associate with each other even at low concentrations,34 facilitating synergistic effect. Fluorinated polymers have high affinity to cell membrane35 and thus can be easy to across the lipid bilayer of cell membrane, as well as the endosome/lysosome membrane,36 furnishing high-efficiency of DNA transfection.37-39 More importantly, F has the maximal electronegativity. F moiety strongly absorbs adjacent electrons and increases the separation extent of positive and negative charges.40 As a result, both


Page 2 of 18

Page 3 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the excitation and emission wavelengths may be obviously red shifted.41 Therefore, if using F element to modify CDs, the fluorescence properties including excitation/emission wavelengths and behaviors in biological systems may be significantly tuned, but this task has rarely been achieved so far.42 Herein, we propose a F-doping strategy to dramatically prolong the emission wavelength of CDs. We synthesize a kind of F-doped CDs (F-CDs) via a one-pot solvothermal process using 1,2-diamino-4,5-difluorobenzene as the fluorine source and tartaric acid to improve aqueous solubility. The emission wavelength of F-CDs shows a red shift around 50 nm compared to that of undoped CDs. The F-CDs emit intense yellow fluorescence under excitation wavelength of 480 nm while red light excited at 540 nm. In addition, the F-CDs furnish highly sensitive intracellular Ag+ detection as well as high efficiency of red cell imaging in various cell systems.

2. EXPERIMENTAL SECTION 2.1. Chemical and Materials. o-Phenylenediamine, 1,2-diamino-4,5-difluorobenzene, tartaric acid, AgNO3, AlCl3·6H2O, FeCl3·6H2O, Ba(NO3)2, CoCl3·6H2O, CrCl3·6H2O, CuCl2·2H2O, Pb(NO3)2, MnSO4·H2O, MgCl2·6H2O, ZnCl2, CaCl2, KCl, NaCl, NiCl2·6H2O and HgCl2 were purchased from Energy Chemical (Shanghai, China). Dulbecco’s Modified Eagle’s Medium (DMEM), Roswell Park Memorial Institute 1640 (RPMI 1640) Medium, penicillin, streptomycin and fetal bovine serum (FBS) were obtained from GIBCO. Methyl tetrazolium (MTT) were purchased from Sigma-Aldrich. All chemicals were analytical-grade and used as received without further purification unless indicated. 2.2. Instrumentations. F-CDs were observed on a high-resolution transmission electron microscope (HR-TEM, Tecnai G2 F20, FEI). Fourier transform infrared (FT-IR) spectra were recorded on a Bruker TENSOR-27 spectrometer. The 19F nuclear magnetic resonance (19F NMR) spectra of F-CDs was taken on a 500 MHz Avance-Ⅱ spectrometer from Bruker using D2O as the solvent. X-ray photoelectron spectrum (XPS) was carried out in a Thermo Scientific ESCALAB 250Xi X-ray photoelectron spectrometer equipped with a monochromatic Al Ka X-ray source (1486.6 eV). UV-vis absorbance spectra were measured with a UV-3010 spectrophotometer (Hitachi, Japan). The photoluminescence (PL) spectra of F-CDs and CDs were recorded on a fluorescence



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

spectrometer (F-2500, Hitachi, Japan). The images of cells were visualized and photographed by a fluorescence microscope (Nikon, Japan). Atomic force microscopic (AFM) images were observed from a Bruker Diension Icon system. Raman spectroscopy was carried out using a Renishaw inVia Raman Microscope equipped with a 633 nm laser. The crystal structures were identified using an X-ray diffractometer (XRD, D/MAX TTRIII, Rigaku) using Cu Ka (l = 1.54 Å) radiation. 2.3. Synthesis of F-CDs. Firstly, o-phenylenediamine (0.36 g) or 4,5-difluoro-1,2-benzenediamine (0.48 g) was dissolved in 15 mL of ethanol. Tartaric acid (0.50 g) was dissolved into another 15 mL of ethanol. Then, the two solutions were mixed and transferred to a teflon-lined stainless-steel autoclave. After heating at 180 oC for 8 h and naturally cooling to room temperature, undoped CDs and F-CDs were purified via silica column chromatography using a mixture of methylene chloride and methanol as the eluent, and dried by a freeze-drying process. 2.4. Determination of Quantum Yield (QY). The QYs of undoped CDs and F-CDs were determined by a relative method.43-45 Specially, rhodamine 6G (QY = 95% in ethanol) for the emission range of 480-560 nm (for undoped CDs and F-CDs herein), and rhodamine B (QY = 56% in ethanol) for emission range of 580-610 nm (for F-CDs herein). The relative fluorescence QY (Φ) of CDs was calculated using the equation: =

The optical density was measured using a UV spectrophotometer and fluorescence spectrometer. Where Φ is fluorescence QY, I is the integrated fluorescence intensity, n is the refractive index of solvent, and A is the optical density (absorption). The subscript R refers to the reference of Fluorescein. 2.5. Cell Culture. HEK 293T (a human embryonic kidney cell line, and contains the SV40 T-antigen, ATCC), NIH 3T3 (mouse embryo fibroblast, ATCC), COS-7 (a fibroblast-like cell line derived from monkey kidney, ATCC) and HepG2 (a human hepatoma cell line, ATCC) were cultured in DMEM supplied with penicillin (100 units mL-1), streptomycin (100 µg mL-1) and 10% FBS at 37 ℃ under a humidified 5% CO2 atmosphere. B16F10 (a murine melanoma cell line) and A549 (a human alveolar adenocarcinoma cell line, ATCC) were cultured in RPMI 1640 supplied with


Page 4 of 18

Page 5 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


penicillin (100 units mL-1), streptomycin (100 µg mL-1) and 10% FBS at 37 ℃ under a humidified 5% CO2 humidified atmosphere. 2.6. Cytotoxicity Assay. The cells were seeded in a 96-well plate at a density of 1 × 104 cells per well for 20-24 h, followed by the incubation of CDs (10, 20, 40, 60, 80, 100, 150, and 200 µg mL-1) with the cells for 24 h. Then, 50 µL of 1 × MTT solution was added to each well. The cells were cultured for another 4 h, followed by removal of the culture medium with MTT. Then 150 µL of DMSO was added to each well and shaken for 15 min. The absorbance of each well was measured at 550 nm. The cell viability was estimated according to the equation: Cell viability % = / Where ODs is obtained in the presence of CDs and ODu is obtained in the absence of CDs. 2.7. Cell Imaging. The cells were cultured in a 6-well plate at a density of 2 × 105 cells per well for 18-24 h before bioimaging, followed by the incubation of F-CDs or CDs (10, 20, 40, 60, 80, and 100 µg mL-1) with cells for 4 h. Next, the treated cells were rinsed with phosphate buffered solution (PBS) buffer (0.01 M，pH = 7.4) three times. The images of the cells were immediately visualized and photographed at ambient temperature by a fluorescence microscope (Nikon, Japan). 2.8. Detection of Ag+ in Aqueous Buff ffer and in Cell. All PL spectra of samples were performed in solution (10 mM PBS buffer, pH=7.4) on fluorescence spectrometer experiments. In the PL spectra of the F-CDs (10 µg mL-1) were recorded upon the addition of 4 mM of other salts (Ag+, Al3+, Fe3+, Ba2+, Co3+, Cr3+, Cu2+, Pb2+, Mn2+, Mg2+, Zn2+, Ca2+, K+, Na+, Ni2+ and Hg2+). In PL spectra titration experiments, F-CDs (10 µg mL-1) dispersion was added to PBS buffer, followed by the addition of Ag+ standard with various concentrations (from 0 to 200 µM). Before bioimaging, the F-CDs (100 µg mL-1) loaded cells were incubated with 2 mL PBS contains Ag+ (200 µM) for 30 min. The images of the cells were immediately visualized and photographed at ambient temperature by a fluorescence microscope.

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of F-CDs. As the determinant factors for fluorescence emission, electron density and delocalization



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ability of a fluorophore is severely influenced by high electronegative F atom. To enhance the electronic effect on conjugate fluorophore, F atom should be directly connected to sp2 carbon in conjugation system rather than sp3 carbon, because the electron of sp2 moving along conjugate chain is more easily than that of sp3 carbon mobilizing along saturated chain and thus prefer to delocalize. Therefore, we chose 4,5-difluorobenzene-1,2-diamine (1, Scheme 1) as the F source, considering high stability of F atom in aromatic ring may be easily retained under solvothermal condition. Furthermore, the two amino groups will afford various N-bearing moieties, such as positive charge localization, Schiff base fragment, and N-heterocycle. In order to improve aqueous solubility, we chose tartaric acid (2, Scheme 1) as another reactant to provide a large number of carboxy groups. A yellow light emitting F-CDs formed after a solvothermal process at 180 oC (Scheme 1). As a comparison, o-phenylenediamine (3, Scheme 1) was used instead of reactant 1, but only a green light emitting CDs was obtained, which was called undoped CDs.

Scheme 1. The synthesis of F-CDs and undoped CDs. The HR-TEM was adopted to observe morphology of F-CDs. As displayed in Figure 1a, the F-CDs present as small dots with a d-spacing of 0.25 nm. The statistical data indicates that the size of F-CDs is around 5.52 nm, distributing from 0 to 8 nm (Figure 1b). The size of F-CDs is also evidenced from the AFM image and height profile (Figure 1c and d). The morphology and size of undoped CDs is similar to that of F-CDs. The undoped CDs show a d-spacing of 0.23 nm, which


Page 6 of 18

Page 7 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


belongs to typical crystal lattice of graphitic carbon (Figure 1e). The size of undoped CDs is about 5.18 nm (Figure 1f), further evidenced by the AFM image (Figure 1g and h).

Figure 1. (a) HR-TEM image, (b) size statistical analysis, (d) AFM image, and (e) height profile of F-CDs; (e) HR-TEM image, (f) size statistical analysis, (g) AFM image, and (h) height profile of undoped CDs. FT-IR spectrum was used to characterize chemical structure of F-CDs (Figure 2a). A broad band is observed from 2800 cm-1 to 3600 cm-1, demonstrating the presence of N-H and O-H bonds. Considering the strong peak at 1734 cm-1 which is stand for carbonyl group, it is reasonable to infer that a number of carboxy groups should be retained to F-CDs. The peaks at 1608 and 1482 cm-1 are corresponding to the characteristic vibration of conjugate C=C bonds, implying the appearance of aromatic rings. The F-CDs must contain -CH2- moiety, evidenced by the signal at 1362 cm-1. The Raman spectrum of F-CDs shows four main peaks at 620, 750, 1360, and 1470 cm-1 (Figure S1). It may indicate the formation of graphitic carbon or the existence of SP3 carbon. F-CDs belong to amorphous solid, due to the only existence of broad diffraction peak in the XRD pattern (Figure 2b). In addition, 19F NMR spectrum was employed to determine F retention in the prepared F-CDs. As shown in Figure 2c, all the peaks are at chemical shift from -160 to -100, and can be assigned to the signals of F atom binding to aromatic ring. The peaks at -113, -118 and -119 ppm may be attributed to the F atom in expended aromatic ring without electron-withdrawing moiety, while the peaks at -145 and -153 ppm should be corresponded to aromatic ring bearing



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

N-containing electron-donating groups. It is demonstrated that the F atom of reactant 1 should be retained well in the prepared F-CDs.

Figure 2. (a) FT-IR spectrum, (b) XRD pattern, and (c) 19F NMR spectrum of F-CDs. In addition, XPS was used to explore the element composition of F-CDs. It is demonstrated that the prepared F-CDs is mainly composed of C, N, O, and F with the binding energy of 284.6, 397.3, 531.4, and 685.2 eV, attributing to C 1s, N 1s, O 1s, and F 1s, respectively (Figure 3a). The composition analysis of F-CDs shows the elemental composition: C (56.8 %), N (8.3 %), O (28 %), and F (6.9 %), indicating high doping percentage of F. A high-resolution spectrum of C 1s indicates the presence of C-H (283.6 eV), C-C/C-N, (284.9 eV), C−O (285.4 eV), and C-F (287.4 eV) bonds on the surface of F-CDs (Figure 3b).42,46 The spectrum of N 1s shows two dominant peaks which are assigned to bridging N-C or N-H bonds (399.6 eV) and positive charge localization in heterocycles (401.1 eV), respectively (Figure 3c).42 Different form the fluorinated carbon nitride,47 the F 1s high-resolution spectrum of F-CDs can be divided into two peaks with binding energy of 685.7 eV and 686.3 eV, respectively (Figure 3d). This phenomenon should be attributed to the complicated condition, such as electron-donating group, electron-withdraw group. In short, the undoped CDs show a similar morphology, size, chemical structure and crystal form in addition to the doped fluorine element (Figure S2 and S3).


Page 8 of 18

Page 9 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. (a) XPS survey spectrum, (b) C 1s, (c) N 1s, and (d) F 1s XPS spectra of F-CDs. 3.2. The Optical Properties of F-CDs. We have explored the optical properties of the prepared F-CDs, including UV−vis absorption, excitation, fluorescence emission, QY, and stability. As shown in Figure 4a, the F-CDs in aqueous solution have two UV−vis absorption peaks at 246 and 275 nm, respectively. These peaks are corresponded to the π−π* transition of C=N or C=C bonds, leading to negligible contribution on fluorescent signal.23 In addition, a broad absorption band covering the region from 360 and 550 nm can be observed. The maximal absorption peak of this band is at 440 nm and ascribed to n−π* transition of C=O.21,23 Meanwhile, the defect surface states trap the excited state energy.22 As a result, strong fluorescence emits. The optimal excitation and emission wavelength are at 440 and 550 nm (Bright yellow light). The F-CDs show an excitation-dependent feature with emission wavelength from 550 nm to 600 nm (Excitation at wavelength from 360 to 580 nm) (Figure 4b). Similarly, the undoped CDs also display an excitation-dependent feature, emitting at a region from 480 to 550 nm under the excitation wavelength from 360 to 580 nm (Figure 4c). It is obviously observed that the CDs doped by F leads to at least 50 nm of red shift at emission wavelength.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4. (a) UV−vis absorption (black line), fluorescence excitation (red line), and fluorescence emission (blue line) spectra of F-CDs in aqueous solution, Excitation-dependent fluorescence behavior of (b) F-CDs and (c) undoped CDs. The QYs of F-CDs were determined to be 31% at the emission wavelength of 550 nm and 14% at 600 nm, while the QYs of undoped CDs are 28% and 11% at emission wavelength of 500 and 550 nm, respectively. The stability of F-CDs under various conditions was investigated. The F-CDs shows pH dependent fluorescence and displays stable fluorescence in the pH range 5-12 (Figure S4), indicating that it can be used under physiological conditions. No obvious changes of fluorescent intensity can be observed at high ionic strengths (Figure S5). This is very important because of high physical salt concentrations under practical conditions. Furthermore, the F-CDs have long-term storage stability under ambient conditions (Figure S6) and excellent photostability under irradiation of Xe lamp (365 nm) for 1 h (Figure S7). 3.3. The cellular imaging by F-CDs. In spite of superior optical properties of F-CDs, high biocompatibility must be strictly considered as a potential biolabeling reagent. Therefore, the cytotoxicity of F-CDs was first evaluated by the standard MTT assay. The results were summarized in Figure 5. It can be found that over 90 % cell viability was obtained after 24 h incubation of cells with the F-CDs (0−200 µg·mL−1), regardless of normal cells including HEK 293T, NIH3T3 and COS-7 cells, and cancer cells including B16F10, A549 and HepG2 cells. This sufficiently confirms the extreme low cytotoxicity of F-CDs. In fact, the undoped CDs has similar stability and biocompatibility to that of F-CDs.


Page 10 of 18

Page 11 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. Cellular cytotoxicity of F-CDs using standard MTT assay toward (a) normal cells and (b) cancer cells. We chose HEK 293 cell and B16F10 cell as the normal cell and cancer cell, respectively, to investigate the cellular imaging ability of both F-CDs and undoped CDs. Most fluorescence microscopes have three excitation wavelengths (360, 480, and 540 nm), and only can show blue, green and red light. The F-CDs only emits yellow fluorescent light under excitation at 360 and 480 nm, and shows red fluorescence excited at the wavelength of 540 nm (Figure S8). As a result, high-quality of red fluorescent light was observed in both living normal cell and cancer cell (Figure 6a-d). Very differently, only green fluorescent images can be obtained using the undoped CDs (Figure 6e-h, S9). The F-CDs can also be applied to the high efficient cellar imaging for NIH3T3, COS-7, A549 and HepG2 cells (Figure 7). In addition, B16F10 cell was used as the representative to optimize the concentration of F-CDs for cellular imaging (Figure S10). The results indicate that 60 µg mL-1 is the optimal concentration for cellular imaging by F-CDs. The above results confidently demonstrate the huge potential of F-CDs in bioimaging for a broad range of cells.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 6. Cellular imaging of HEK 293 and B16F10 cells by (a-d) F-CDs and (e-h) undoped CDs.

Figure 7. Cellular imaging of (a,b) NIH3T3, (c,d) A549, (e,d) COS-7, and (g,h) HepG2 cells by F-CDs. 3.4. The Ag+ detection by F-CDs. The unique optical properties of F-CDs encouraged us to further investigate its potential sensing applications. After broadly screening, the F-CDs are found to show an intense quenching phenomenon with the addition of 4 mM of Ag+ (Figure 8a). Other metal cations including Ag+, Pb2+, Cd2+, Cr3+, Mg2+, Cu2+, Zn2+, Ca2+, Ni2+, Hg2+ and Co3+, has insignificant influence on quenching phenomenon even at high concentrations (up to 4 mM), implying high resistance of F-CDs to ionic interference. Figure 8b shows the fluorescence quenching of the F-CDs at a gradient concentration of Ag+. With increasing concentration (from 0 to 200 µM), the fluorescence intensity of F-CDs decreases progressively, revealing that the sensing system is sensitive to Ag+ concentration. The linear range of Ag+ detection is 0 - 70 µM with a correlation coefficient (R2) of 0.9928 (Figure 8c). We also investigated the fluorescence spectra of F-CDs (10 µg mL-1) titrated with various concentrations (from 0 to 200 µM) of Ag+ (Figure S11). The linear range of Ag+ detection is 0 - 70 µM and the correlation coefficient (R2) reaches 0.9951. Due to the high biocompatibility, we have investigated the effectiveness of this Ag+ sensing system under biological conditions. Take normal cell (HEK 293) and cancer cell (B16F10) as examples (Figure 8d-g), obvious quenching phenomenon was observed, sufficiently demonstrating high sensitive


Page 12 of 18

Page 13 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Ag+ detection ability of the F-CDs.

Figure 8. (a) Fluorescence intensity of F-CDs in the absence (black column) and presence (red column) of different metal ions; (b) influence of Ag+ on the fluorescence intensity of F-CDs; (c) the linear calibration plot for (F0-F)/F0 versus different concentration of Ag+; F-CDs in HEK 293T cells with (d)/ without (e) Ag+ and B16F10 cells with (f)/without (g) Ag+.

4. CONCLUSIONS In summary, we have developed a F-doping strategy to prolong the emission wavelength of CDs. The F-CDs were synthesized via a solvothermal reaction using aromatic F bearing moiety (4,5-difluorobenzene-1,2-diamine) as the F source and tartaric acid to furnish a large number of carboxy groups. The F atom in aromatic ring can be well retained in the F-CDs. The F-CDs show two maximum emissions at ca. 550 nm (yellow fluorescence) under the excitation at light wavelength from 360 to 500 nm and ca. 600 nm (red fluorescence) under the excitation at light wavelength from 540 to 580 nm, which is quite different from the excitation dependent feature of undoped CDs. Comparing to undoped CDs, a red shift of emission wavelength longer than 50 nm was measured, sufficiently demonstrating the doping effect by F. The F-CDs has remarkable water-solubility, high biocompatibility, as well as excellent stability even under broad pH range, ionic strengths and light illumination. Moreover, the F-CDs emitting strong red fluorescence were used as a novel probe for the highly efficient cell imaging of various normal cells (HEK 293,



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

NIH3T3 and A549 cells) and cancer cells (B16F10, COS-7 and HepG2 cells). Due to the abundant functional groups on the surface of F-CDs, selectively binding the F-CDs to Ag+ has taken place, and thus makes the F-CDs be highly sensitive probe for the detection of Ag+ under both aqueous solution and biological systems. It is indicated that the F-doping method can be used to effectively modify the optical properties of CDs, and the F-CDs are promising probes as an efficient platform for environmental monitoring as well as clinical diagnosis.

ASSOCIATED CONTENT Supporting Information Supporting Information displays Figures S1−S11, including the influence of pH values, ionic strengths, storage and illumination on the fluorescence intensity of F-CDs, the fluorescence intensity of F-CDs and undoped CDs under excitation of 360 nm, 480 nm and 540 nm light, fluorescence images of B16F10 cells incubated with F-CDs, and quenching efficiency of F-CDs by Ag+.

AUTHOR INFROMATION Corresponding Author * E-mail: [email protected] (Aming Xie) * E-mail: weidong@ njust.edu.cn (Wei Dong)

ACKNOWLEDGMENT The financial supported by the National Natural Science Foundation of China (NSFC: 51573078) is gratefully acknowledged.

REFERENCES (1) Liu, Y.; Tian, Y.; Tian, Y.; Wang, Y.; Yang, W. Carbon-Dot-Based Nanosensors for the Detection of Intracellular Redox State. Adv. Mater. 2015, 27, 7156−7160. (2) Shi, W.; Li, X.; Ma, H. A Tunable Ratiometric pH Sensor Based on Carbon Nanodots for the Quantitative Measurement of the Intracellular pH of Whole Cells. Angew. Chem., Int. Ed. 2012, 51, 6432−6435. (3) Ding, C.; Zhu, A.; Tian, Y. Functional Surface Engineering of C-Dots for Fluorescent Biosensing and in Vivo Bioimaging. Acc. Chem. Res. 2014, 47, 20−30. (4) Kwon, W.; Do, S.; Lee, J.; Hwang, S.; Kim, J. K.; Rhee, S.W. Freestanding Luminescent Films of Nitrogen-Rich Carbon Nanodots toward Large-Scale Phosphor-Based White-Light-Emitting Devices. Chem. Mater. 2013, 25, 1893−1899. (5) Strauss, V.; Margraf, J. T.; Dirian, K.; Syrgiannis, Z.; Prato, M.; Wessendorf, C.; Hirsch, A.; Clark, T.; Guldi, D. M. Carbon Nanodots: Supramolecular Electron Donor-Acceptor Hybrids Featuring Peryle-nediimides. Angew. Chem., Int. Ed. 2015, 54, 8292−8297.


Page 14 of 18

Page 15 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(6) Li, X. M.; Rui, M. C.; Song, J. Z.; Shen, Z. H.; Zeng, H. B. Carbon and Graphene Quantum Dots for Optoelectronic and Energy Devices: A Review. Adv. Funct. Mater. 2015, 25, 4929−4947. (7) Zheng, M.; Liu, S.; Li, J.; Qu, D.; Zhao, H.; Guan, X.; Hu, X.; Xie, Z.; Jing, X.; Sun, Z. Integrating Oxaliplatin with Highly Luminescent Carbon Dots: An Unprecedented Theranostic Agent for Personalized Medicine. Adv. Mater. 2014, 26, 3554−3560. (8) Wu, H.; Zeng, F.; Zhang, H.; Xu, J.; Qiu, J.; Wu, S. A nanosystem capable of releasing a photosensitizer bioprecursor under two-photo irradiation for photodynamic therapy. Adv. Sci. 2016, 3, 1500254. (9) Choi, Y.; Kim, S.; Choi, M. H.; Ryoo, S. R.; Park, J.; Min, D.; Kim, B. S. Highly biocompatible carbon nanodots for simultaneous bioimaging and targeted photodynamic therapy in vitro and in vivo. Adv. Funct. Mater. 2014, 24, 5781−5789. (10) Zheng, M.; Ruan, S.; Liu, S.; Sun, T.; Qu, D.; Zhao, H.; Xie, Z.; Gao, H.; Jing, X.; Sun, Z. Self-Targeting Fluorescent Carbon Dots for Diagnosis of Brain Cancer Cells. ACS Nano 2015, 9, 11455−11461. (11) Yang, S. T.; Cao, L.; Luo, P. G.; Lu, F.; Wang, X.; Wang, H.; Meziani, M. J.; Liu, Y.; Qi, G.; Sun, Y. P. Carbon Dots for Optical Imaging in Vivo. J. Am. Chem. Soc. 2009, 131, 11308−11309. (12) Tang, J.; Kong, B.; Wu, H.; Xu, M.; Wang, Y.; Wang, Y.; Zhao, D.; Zheng, G. Carbon nanodots featuring efficient FRET for real-time monitoring of drug delivery and two-photon imaging. Adv. Mater. 2013, 25, 6569−6574. (13) Jiang, K.; Sun, S.; Zhang, L.; Lu, Y.; Wu, A.; Cai, C.; Lin, H. Red, Green, and Blue Luminescence by Carbon Dots: Full-Color Emission Tuning and Multicolor Cellular Imaging. Angew. Chem., Int. Ed. 2015, 54, 5360−5363. (14) Zheng, X. T.; Ananthanarayanan, A.; Luo, K. Q.; Chen, P. Glowing Graphene Quantum Dots and Carbon Dots: Properties, Syntheses, and Biological Applications. Small 2015, 11, 1620– 1636. (15) Baker, S. N.; Baker, G. A. Luminescent carbon nanodots: emergent nanolights. Angew. Chem., Int. Ed. 2010, 49, 6726−6744. (16) Lim, S. Y.; Shen, W.; Gao, Z. Carbon quantum dots and their applications. Chem. Soc. Rev. 2015, 44, 362−381. (17) Ding, H.; Yu, S. B.; Wei, J. S.; Xiong, H. M. Full-Color Light-Emitting Carbon Dots with a Surface-State-Controlled Luminescence Mechanism. ACS Nano 2016, 10, 484−491. (18) Pan, L.; Sun, S.; Zhang, A.; Jiang, Kai.; Zhang, L.; Dong, C.; Huang, Q.; Wu, A.; Lin, H. Truly Fluorescent Excitation-Dependent Carbon Dots and Their Applications in Multicolor Cellular Imaging and Multidimensional Sensing. Adv. Mater. 2015, 27, 7782−7787. (19) Ge, J.; Jia, Q.; Liu, W.; Guo, L.; Liu, Q.; Lan, M.; Zhang, H. Meng, X.; Wang, P. Red-Emissive Carbon Dots for Fluorescent, Photoacoustic, and Thermal Theranostics in Living Mice. Adv. Mater. 2015, 27, 4169–4177. (20) Sun, S.; Zhang, L.; Jiang, K.; Wu, A.; Lin, H. Toward High-Efficient Red Emissive Carbon Dots: Facile Preparation,Unique Properties, and Applications as Multifunctional Theranostic Agents. Chem. Mater. 2016, 28, 8659−8668. (21) Jiang, K.; Sun, S.; Zhang, L.; Wang, Y.; Cai, C.; Lin, H. Bright-Yellow-Emissive N-Doped Carbon Dots: Preparation, Cellular Imaging, and Bifunctional Sensing. ACS Appl. Mater.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(22)

(23)

(24)

(25) (26)

(27)

(28)

(29)

(30) (31) (32) (33)

(34)

(35)

(36) (37)

Interfaces 2015, 7, 23231−23238. Gong, X.; Zhang, Q.; Gao, Y.; Shuang, S.; Choi, M. M.; Dong, C. Phosphorus and Nitrogen Dual-Doped Hollow Carbon Dot as a Nanocarrier for Doxorubicin Delivery and Biological Imaging. ACS Appl. Mater. Interfaces 2016, 8, 11288−11297. Shi, B.; Su, Y.; Zhang, L.; Huang, M.; Liu, R.; Zhao, S. Nitrogen and Phosphorus Co-Doped Carbon Nanodots as a Novel Fluorescent Probe for Highly Sensitive Detection of Fe3+ in Human Serum and Living Cells. ACS Appl. Mater. Interfaces 2016, 8, 10717−10725. Sun, X.; Bruckner, C.; Lei, Y. One-pot and ultrafast synthesis of nitrogen and phosphorus co-doped carbon dots possessing bright dual wavelength fluorescence emission. Nanoscale 2015, 7, 17278−17282. Travlou, N. A.; Secor, Jeff.; Bandosz, T. J. Highly luminescent S-doped carbon dots for the selective detection of ammonia. Carbon 2017, 114, 544−556. Shen, J.; Shang, S.; Chen, X.; Wang, D.; Cai, Y. Highly fluorescent N,S-co-doped carbon dots and their potential applications as antioxidants and sensitive probes for Cr (VI) detection. Sens. Actuators, B 2017, 248, 92–100. Cui, X.; Wang, Y.; Liu, J.; Yang, Q.; Zhang, B.; Gao, Y.; Wang, Y.; Lu, G. Dual functional Nand S-co-doped carbon dots as the sensor for temperature and Fe3+ ions. Sens. Actuators, B 2017, 242, 1272–1280. Borse, V.; Thakur, M.; Sengupta, S.; Srivastava, R. N-doped multi-fluorescent carbon dots for “turn off-on” silver-biothiol dual sensing and mammalian cell imaging application. Sens. Actuators, B 2017, 248, 481–492. Liu, Y.; Duan, W.; Song, W.; Liu, J.; Ren, C.; Wu, J.; Liu, D.; Chen, H. 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. ACS Appl. Mater. Interfaces 2017, 9, 12663– 12672. Purser, S.; Moore, P. R.; Swallow, S.; Gouverneur, V. Fluorine in medicinal chemistry. Chem. Soc. Rev. 2008, 37, 320–330. Buer, B. C.; Meagher, J. L.; Stuckey, J. A.; Marsh, E. N. Structural basis for the enhanced stability of highly fluorinated proteins. Proc. Natl. Acad. Sci. 2012, 109, 4810–4815. Horvath, I. T.; Rabai, J. Facile catalyst separation without water: fluorous biphase hydroformylation of olefins. Science 1994, 266, 72–75. Xiong, S. D.; Li, L.; Jiang, J.; Tong, L. P.; Wu, S.; Xu, Z. S.; Chu, P. K. Cationic fluorine-containing amphiphilic graft copolymers as DNA carriers. Biomaterials 2010, 31, 2673–2685. Percec, V.; Glodde, M.; Bera, T. K.; Miura, Y.; Shiyanovskaya, I.; Singer, K. D.; Balagurusamy, V. S. K.; Heiney, P. A.; Schnell, I.; Rapp, A.; et al. Self-organization of supramolecular helical dendrimers into complex electronic materials. Nature 2002, 419, 384– 387. Kasuya, M. C. Z.; Nakano, S.; Katayama, R.; Hatanaka, K. Evaluation of the hydrophobicity of perfluoroalkyl chains in amphiphilic compounds that are incorporated into cell membrane. J. Fluorine Chem. 2011, 132, 202–206. Neil, E.; Marsh, G. Towards the nonstick egg: designing fluorous proteins. Chem. Biol. 2000, 7, R153–R157. Wang, M.; Liu, H.; Li, L.; Cheng, Y. A fluorinated dendrimer achieves excellent gene


Page 16 of 18

Page 17 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(38) (39) (40) (41) (42)

(43) (44)

(45) (46)

(47)

transfection efficacy at extremely low nitrogen to phosphorus ratios. Nat. Commun. 2014, 5, 3053. Wang, M.; Cheng, Y. The effect of fluorination on the transfection efficacy of surface-engineered dendrimers. Biomaterials 2014, 35, 6603−6613. Liu, H.; Wang, Y.; Wang, M.; Xiao, J.; Cheng Yi. Fluorinated poly(propylenimine) dendrimers as gene vectors. Biomaterials 2014, 35, 5407−5413. Sun W. C.; Gee K. R.; Klaubert, D. H.; Haugland R. P. Synthesis of Fluorinated Fluoresceins. J. Org. Chem. 1997, 62, 6469–6475. Sun W. C.; Gee K. R.; Haugland R. P. Synthesis of novel fluorinated coumarins: Excellent UV-light excitable fluorescent dyes. Bioorg. Med. Chem. Lett. 1998, 8, 3107−3110. Wang, N.; Fan, H.; Sun, J.; Han, Z.; Dong, J.; Ai S. Fluorine-doped carbon nitride quantum dots: Ethylene glycol-assisted synthesis, fluorescent properties, and their application for bacterial imaging. Carbon 2016, 109, 141−148. Eaton, D. F. Reference materials for fluorescence measurement. Pure Appl. Chem. 1988, 60, 1107−1114. Grabolle, M.; Spieles, M.; Lesnyak, V.; Gaponik, N.; Eychmüller, A.; Resch-Genger, U. Determination of the fluorescence quantum yield of quantum dots: suitable procedures and achievable uncertainties. Anal. Chem. 2009, 81, 6285−6294. Karstens, T.; Kobs, K. Rhodamine B and rhodamine 101 as reference substances for fluorescence quantum yield measurements. J. Phys. Chem. 1980, 84, 1871−1872. Kundu, S.; Yadav, R. M.; Narayanan, T. N.; Shelke, M. V.; Vajtai, R.; Ajayan, P. M.; Pillai V. K. Synthesis of N, F and S co-doped graphene quantum dots. Nanoscale 2015, 7, 11515– 11519. Wang, Y.; Di, Y.; Antonietti, M.; Li, H.; Chen, X.; Wang, X. Excellent visible-light photocatalysis of fluorinated polymeric carbon nitride solids, Chem. Mater. 2010, 22, 5119−5121.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

TOC


Page 18 of 18

Large Emission Red-Shift of Carbon Dots by Fluorine Doping and

Recommend Documents