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Rapid synthesis of B-N co-doped yellow emissive carbon quantum dots for cellular imaging
Optical Materials 100 (2020) 109647
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Rapid synthesis of B-N co-doped yellow emissive carbon quantum dots for cellular imaging Yingying Wei a, b, Lin Chen b, Junli Wang b, Xuguang Liu b, c, *, Yongzhen Yang b, **, Shiping Yu b, d a
College of Materials Science and Engineering, Taiyuan University of Technology, Taiyuan, 030024, China Key Laboratory of Interface Science and Engineering in Advanced Materials, Taiyuan University of Technology, Ministry of Education, Taiyuan, 030024, China c Institute of New Carbon Materials, Taiyuan University of Technology, Taiyuan, 030600, China d Interventional Treatment Department, Second Hospital of Shanxi Medical University, Taiyuan, 030001, China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Rapidity B-N co-doped carbon quantum dots Yellow emission Cellular imaging
In order to avoid the interference of autofluorescence and improve tissue penetration capability of carbon quantum dots (CQDs) in biological imaging, the synthesis of long-wavelength emission CQDs was achieved in this work. Boron and nitrogen co-doped CQDs (BN-CQDs) were synthesized by one-step microwave assisted hydrothermal method with o-phenylenediamine as carbon and nitrogen source, boric acid as boron source. The BN-CQDs (QY ¼ 13.79%) synthesized under the optimal conditions exhibit excitation independence, a large Stoke shift, satisfactory fluorescent stability, good biocompatibility and low toxicity, which can meet the basic requirements as cell imaging agent. Cell imaging measurements prove their increased intracellular accumulation with the extension of incubation time with HeLa cells, and clearly observable BN-CQDs imaging for cell morphology. Hence BN-CQDs can be used as a good cell imaging agent.
1. Introduction Cellular imaging, which can observe the internal structure and physiological processes of cells in real time or over a period of time, is conducive to monitoring cellular morphology, structure, dynamic pro cesses and the diagnosis of diseases, and is crucial to the interpretation of many cell biology problems. Traditional cellular imaging probes, such as fluorescent dyes and semiconductor quantum dots, have high prep aration costs and high toxicity, which limit their applications in bio logical field [1,2]. Carbon quantum dots (CQDs), as a new class of carbon nanomaterials, have attracted extensive attention in cellular imaging owing to their low cytotoxicity, superior biocompatibility, good water solubility and high photostability [1,3,4]. However, most of CQDs applied to cell imaging at present are blue-emitting under ultraviolet excitation. They have small Stokes shift, resulting in poor signal-to-noise ratio and self-quenching on current microscope configurations [5,6]. Meanwhile, biological tissues made up of carbohydrates also emit blue light, which interferes imaging of cells by blue emissive CQDs [4,5,7]. To solve this problem, CQDs with excitation dependence were synthe sized whose emission peak is red-shifted by long wavelength excitation
[3,7]. Previous reports have shown that the fluorescence intensity of CQDs decreases significantly when their emission peak is red-shifted, which hinders the further application of CQDs in biological imaging. Therefore, the synthesis of long-wavelength emission CQDs (such as yellow and red etc.) with large Stokes shift becomes the fundamental way to solve the existing problems in cellular imaging by CQDs. The luminescence mechanism of CQDs indicates that the sp2 hy bridization degree of carbon nuclei and surface state ultimately deter mine fluorescence properties of CQDs. The strategy of introducing benzene ring structure into carbon nucleus and increasing sp2 conju gation, and changing the surface functional groups or defects of CQDs are beneficial to the red-shift of emission center [8,9]. Meanwhile, doping heteroatoms with different electronegativity into CQDs can lead to more band structures and introduce new defect energy levels and narrow band gaps, which is also conductive to the red-shift of emission wavelength [10]. For example, when doped into CQDs B atom, with low electronegativity and good ability as electron donor, can conjugation with carbon nucleus, which reduces the LUMO level of CQDs and energy emitted by fluorescence; N, with high electronegativity, can form nitrogen-state functional groups on the surface of CQDs, leading to the
* Corresponding author. Key Laboratory of Interface Science and Engineering in Advanced Materials, Taiyuan University of Technology, Ministry of Education, Taiyuan, 030024, China. ** Corresponding author. E-mail addresses: [email protected] (X. Liu), [email protected] (Y. Yang). https://doi.org/10.1016/j.optmat.2019.109647 Received 28 November 2019; Received in revised form 26 December 2019; Accepted 29 December 2019 Available online 16 January 2020 0925-3467/© 2020 Elsevier B.V. All rights reserved.
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Optical Materials 100 (2020) 109647
generation of new long-wavelength luminescent groups through func tional groups connected to conjugated sp2 region [11,12]. At present, B doped or B-N co-doped CQDs have been preliminarily explored. B-doped CQDs (B-CQDs) were prepared by one-step hydrothermal method from boric acid [11,13] or phenyl boric acid with big sp2 conjugated structure [14,15] as boron source. Some researchers focused on the synthesis of B-N co-doped CQDs (BN-CQDs) from boric acid as boron source and small-molecule urea, diethylenetriamine or ethylenediamine, as nitro gen source by microwave heating, hydrothermal or pyrolysis [12, 16–18]. However, either B-CQDs or BN-CQDs exhibit blue emission. It is obvious that only relying on taking boron-containing materials with big sp2 conjugate as feedstock or the means of B-N co-doping is not enough to realize the red-shift of CQDs’ emission peak. Therefore, integrating the effects of sp2 conjugation and B-N co-doping would be a possible way to achieve the preparation of long-wavelength emission CQDs. Tian et al. [16] reported the synthesis of yellow emission BN-CQDs by one-step hydrothermal method using m-aminobenzene boronic acid with benzene ring as boron source and nitrogen source, the resultant BN-CQDs exhibited a red-shifted emission peak at 540 nm. Li et al. [4] synthesized yellow BN-CQDs with p-phenylenediamine and 3-formyl phenyl boric acid as nitrogen source and boron source by one-step sol vothermal method. They realized the preparation of yellow emission CQDs, but with a synthesis time of 24 h and organic solvent in the process. In summary, large sp2 conjugated size and heteroatom doping are favorable to the red-shift of emission center. Therefore, in this work, B-N co-doped CQDs with excitation independent yellow emission were synthesized by one-step microwave-assisted hydrothermal method at 200 � C within 10 min. o-Phenylenediamine was selected as carbon and nitrogen source, and boron acid as boron source. As a cell imaging agent, high fluorescence intensity, satisfactory fluorescence stability, good biocompatibility and low toxicity are essential. Therefore, the synthesis conditions of BN-CQDs were optimized, and their chemical structure, optical properties and cytotoxicity were investigated towards their application in HeLa cell imaging.
2.3. Apparatus and characterization Transmission electron microscopy (TEM) images and particle size were taken with a JEOL JEM 2100 transmission electron microscope. Raman spectra were obtained using a Japan HORIBA HR800 Raman spectrometer with radiation at 325 nm. X-ray photoelectron spectros copy (XPS) measurements were conducted on a Kratos AXIS ULTRA DLD X-ray photoelectron spectrometer with mono X-ray source Al Kα exci tation (1468.6 eV). Surface functional groups of BN-CQDs were analyzed on a Bruker Tensor 27 Fourier transform infrared spectrometer (FTIR) in the range of 4000 500 cm 1 and recorded on solid samples in a KBr matrix. Ultraviolet–visible (UV–vis) absorption spectra were measured by an ultraviolet spectrophotometer (Jinghua 756 MC, Shanghai, China). Fluorescence spectra, including excitation and emission spectra, were obtained on a Fluoromax-4 Fluorescence spectrometer (Horiba Jobin Yvon, France). Cytotoxicity was detected on a Varioskan Flash Multifunctional microplate reader (Thermo Fisher, USA). Cellular im aging was carried out on a laser confocal apparatus (Leica TCS SP8, Germany). 2.4. Determination of the fluorescence quantum yield Fluorescence quantum yield (QY) of BN-CQDs was determined by a widely accepted relative method, where rhodamine 6G in anhydrous ethanol was chosen as a standard (QY ¼ 95%). To minimize the reab sorption effects, the absorbance of BN-CQDs and reference sample was kept below 0.1. Integrated fluorescence intensity was obtained by calculating the area under the fluorescence curve. A linear fitting line was drawn using the integrated fluorescence intensity against the absorbance with intercept at zero. Finally, QY values were calculated by the following equation: � �� �2 Qx ¼ Qst Gradx= η (1) x=η Gradst st where Q is QY, Grad is the slope of the linear fitting line, η is the refractive index of solvent (1.36 for ethanol and 1.33 for water). The subscripts “st” and “x" refer to the standard solution and BN-CQDs aqueous solution, respectively.
2. Materials and methods 2.1. Chemicals and reagents
2.5. Cell culture
o-Phenylenediamine was produced by Shanghai McLean Biochem ical Technology Co. Ltd. Boric acid was purchased from Tianjin Chem ical Reagent Factory No. 3. Rhodamine 6G was produced by Shanghai Aladdin Biochemical Technology Co. Ltd. Dialysis bags with retained molecular weight of 1000 Da came from Shanghai Yuanye Biological Technology Co. Ltd. Selected standard fetal bovine serum was obtained from Cellmax. 0.25% trypsin, dual-antibody (penicillin and strepto mycin), aseptic phosphate buffered saline (PBS), 1640 medium and Cell Counting Kit-8 (CCK-8) were from Wuhan BOSTER Biological Technol ogy Co. Ltd. HeLa cells were purchased from the cell bank of the Chinese Academy of Sciences, Shanghai.
Cells were cultured in flasks and incubated at 37 � C in a humidified incubator filled with 5% CO2. Each flask contained 5 mL of 1640 me dium supplemented with 10% fetal bovine serum and 1% dual-antibody. 2.6. Cytotoxicity Cytotoxicity of BN-CQDs was analyzed by CCK-8 assay. After reaching 80%–90% confluence, HeLa cells were lifted with trypsin. The trypsinized cells were dispersed and diluted in 1640 medium, followed by centrifugation at 1000 rpm for 5 min. After removing the superna tant, HeLa cells were re-suspended in fresh 1640 medium with a cell density of 2.5 � 104 cells/mL, then they were seeded in 96-well plates, each containing 100 μL of medium. After 24 h cell attachment, HeLa cells were incubated with different concentration of BN-CQDs (0–80 μg/ mL) for 24 h. The cells were then washed three times with PBS to remove uninternalized BN-CQDs. After that 10 μL of CCK-8 dye and 90 μL of 1640 medium were added to each well, followed by incubation for 1 h at 37 � C. The plates were analyzed with a microplate reader. Measure ments of dye optical density (OD) were performed at 450 nm. Cell viability was calculated by the following equation: � ODsample ODblank Cell viabilityð%Þ ¼ *100% (2) ðODcontrol ODblank Þ
2.2. Synthesis of BN-CQDs In a typical procedure of BN-CQDs preparation, 0.27 g of o-phenyl enediamine and 0.15 g of boric acid were dissolved in 10 mL of distilled water, and then the mixture was transferred to a 30 mL microwave re action tube. Subsequently, the reaction tube was put into a Monowave 300 microwave synthesizer (Anton Paar GmbH, Austria) for reaction at predetermined temperature and time period. The resultant solution was cooled to room temperature naturally and filtered through filtration membrane (0.22 μm) to remove large particles. The filtrate was then dialyzed in water through a dialysis bag for two days for further puri fication. Finally, the purified aqueous solution of BN-CQDs was freeze dried to obtain powder BN-CQDs. 2
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Scheme 1. The synthesis process of BN-CQDs and their application in cell imaging.
Fig. 1. The QY fitting lines of BN-CQDs synthesized at 220 � C for different time (a) and at different temperatures for 10 min (b).
2.7. Hemolysis assay
were attached to the wall, they were incubated with BN-CQDs (50 μg/ mL) for 0, 2, 4 and 6 h. Then the cells were washed three times with PBS to remove excess BN-CQDs and fixed for 15 min with 4% para formaldehyde. After fixation, the cells were washed thrice with PBS. Finally, cell images were taken with a laser confocal scanning micro scope at an excitation wavelength of 405 nm, during which the emission signals where collected in range of 540–580 nm. The preparation pro cess and application in cell imaging of BN-CQDs are shown Scheme 1.
Fresh blood was collected from a healthy human and then placed in an anticoagulation tube to avoid coagulation. The blood was cen trifugated at 1000 rpm for 5 min and repeatedly washed with PBS so lution for several times until the supernatant was clear and transparent to obtain clean red blood cells (RBCs). The RBCs were then suspended again in PBS, and resultant suspension was mixed with BN-CQDs PBS solution of equal volume. The final concentration of BN-CQDs was 0, 10, 20, 30, 40, 50 and 80 μg/mL, separately. In contrast, RBCs in PBS (the experimental group without BN-CQDs) and in 1% Triton X-100 were set as the negative and positive controls, respectively. After incubation in a water bath at 37 � C for 3 h, the treated RBC solution was centrifuged at 1000 rpm for 5 min, and the absorbance of supernatant was measured at 575 nm. Each sample had three parallel replicates, the hemolysis was calculated by the following equation: � Isample Inegative control � �100% Hemolysisð%Þ ¼ (3) Ipositive control Inegative control
3. Results and discussion 3.1. Optimization of synthesis conditions QY is one of the criteria to evaluate the fluorescence intensity of fluorescent materials and QY, which is affected by reaction conditions, such as reaction temperature and reaction time. Therefore, BN-CQDs with higher QY were synthesized by optimizing the reaction condi tions. First, the reaction temperature was fixed at 220 � C to investigate the effect of reaction time on QY, as shown in Fig. 1a. It can be clearly seen that with the extension of reaction time, QY first increases and then decreases, reaching the maximum at 10 min, so the optimal reaction time for synthesis of BN-CQDs is 10 min. Second, the reaction time was fixed at 10 min, to study the effect of reaction temperature on QY. The results show that with the increase of reaction temperature, QY also
2.8. Cellular imaging HeLa cells (1 � 105) were placed in a confocal culture dish and cultured overnight in an incubator at 37 � C with 5% CO2. After the cells
Fig. 2. TEM image (a), illustrated by particle size statistics, and Raman spectra (b) of BN-CQDs. 3
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Fig. 3. FTIR spectra (a), full scan XPS spectra (b), high-resolution XPS spectra of C1s, N1s, O1s and B1s (c–f) of BN-CQDs.
3414 cm 1 is attributed to O–H/–NH stretching vibration [17,18], and the distinct absorption bands located at 1620 cm 1 can be attributed to – C and C– – O [19,20]. The BO–H stretching and the vibration of C– B–O–H bending vibration originating from boric acid appear at 3229 and 1196 cm 1, respectively [15,17,20]. The stretching vibration of C–N and C–B is located at 1111 cm 1 [12,15,17,21]. The absorption band at 1449 cm 1 is assigned to vibration of B–N [12,22]. These results show that B and N are doped into BN-CQDs, owing to the dehydration condensation reaction between boric acid and o-phenylenediamine. The relative content of elements and surface structure in BN-CQDs were further investigated by XPS spectral scanning. The binding en ergies of 193.08, 285.08, 400.08 and 532.08 eV in the full spectra of XPS correspond to B1s, C1s, N1s and O1s, respectively, and their relative contents are 24.12%, 28.25%, 5.68% and 41.96%, respectively, con firming the doping of B and N into BN-CQDs. (Fig. 3b). The highresolution spectra of XPS can further clarify the existence form of each element in BN-CQDs. The C1s spectra display three distinct peaks at – C [11,23], 284.44, 285.48 and 288.37 eV, which are assigned to C–C/C– – O, respectively (Fig. 3c). The N1s spectra mainly C–N/C–O and C– include pyridine N, pyrrole N and graphitic N (399.07, 400.56 and 401.40 eV), as shown in Fig. 3d. The O1s spectra (Fig. 3e) show three distinct peaks at 531.64, 532.12 and 533.91 eV, which are ascribed to
shows a trend of first increase and then decrease. The highest QY (13.79%) appears when the reaction temperature is 200 � C. Therefore, 200 � C was set as the optimal reaction temperature for synthesis of BNCQDs (Fig. 1b). In a word, the optimum conditions for synthesis of BNCQDs are: temperature 200 � C, time 10 min. The morphology, structure, optical properties, cytotoxicity and cellular imaging of the BN-CQDs synthesized under optimum conditions were investigated, as will dis cussed in following sections. 3.2. Morphology and structure The morphology and particle size of as-synthesized BN-CQDs were recorded by TEM. As seen in Fig. 2a, BN-CQDs are spherical zerodimensional carbon nanomaterials, with good dispersion and no obvious agglomeration. Additionally, the average particle size of BNCQDs was calculated to be 2.5 nm. Fig. 2b is the Raman spectra of BN-CQDs. The G band at 1591 cm 1 and D band at 1395 cm 1 corre spond to sp2 and sp3 hybrid carbon, respectively, and the relative strength (IG/ID) of crystallized G band and disordered D band is about 1.71, indicating that BN-CQDs mainly consists of sp2 hybrid carbon [9]. FTIR spectra were measured to analyze the surface functional groups of BN-CQDs, as shown in Fig. 3. The broad absorption band at around 4
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Optical Materials 100 (2020) 109647
Fig. 4. (a) UV–vis absorption, excitation, emission spectra of BN-CQDs, (b) emission spectra of BN-CQDs aqueous solutions at different excitation wavelengths. Photographs of as-prepared BN-CQDs solution under sunlight (inset, left) and under 365 nm UV light (inset, right).
Fig. 5. (a) Fluorescence intensity variation of BN-CQDs as a function of illumination time; Emission spectra of BN-CQDs in different concentrations of NaCl solution (b) and pH (c), the illustration of (b) and (c) show the fluorescence intensity variation of BN-CQDs as a function of concentrations of NaCl and pH, respectively; (d) zeta potential variation of BN-CQDs with pH.
– O, B–O and C–O, respectively. The B1s spectra reveal three distinct C– peaks at 186.94 (B-C) [24,25], 192.63 (B–N) and 193.62 eV (B–O) [25] (Fig. 3f). It can be concluded that oxygen-, boron- and nitrogen-containing functional groups are attached on the surface of BN-CQDs, further demonstrating the co-doping of B and N into BN-CQDs.
appears around 420 nm, indicating that BN-CQDs has a large amount of sp2 carbon, which corresponds to the Raman result (Fig. 2b) [27,28]. The optimal excitation and emission wavelengths of BN-CQDs are located at 409 and 564 nm, respectively, demonstrating that BN-CQDs exhibit a large Stokes shift of 155 nm. It can effectively minimize the interference of self-quenching and improve the signal-to-noise ratio in imaging applications. Fig. 4b is the emission spectra of BN-CQDs solution at different excitation wavelengths. A series of emission spectra were obtained by increasing the excitation wavelength of BN-CQDs from 340 nm to 500 nm, their maximum emission wavelength is all located at 564 nm, among which the strongest emission intensity is obtained when the excitation wavelength is fixed at 420 nm. As can be seen from Fig. 1a, the size of BN-CQDs is only relatively uniform, so excitationindependent fluorescence behavior could be explained by identical/
3.3. Optical properties To explore the optical properties of BN-CQDs, UV–vis, excitation and emission spectra of BN-CQDs were measured, as shown in Fig. 4a. There are two obvious absorption peaks in UV–vis spectra, where the first peak at 237 nm is attributed to the π–π* transition of the sp2 in aromatic structures, and the second peak at 288 nm can be ascribed as the n–π* – O [17,26]. In the visible region, a weak absorption band transition of C– 5
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Optical Materials 100 (2020) 109647
Fig. 6. (a) Photographs of RBCs suspension before (a1) and after (a2) treatment by BN-CQDs at different concentrations and Triton X-100 solutions; (b) Hemolysis of RBCs in the presence of different concentrations of BN-CQDs; (c) Cytotoxicity of BN-CQD against HeLa cells at increasing concentrations from 0 to 80 μg/mL.
similar emission sites on each particle of BN-CQDs [29,30]. In addition, there is more overlap between the excitation and UV–vis spectra, indi cating that the yellow emission is mainly related to the surface state [31]. From the inset the photographs in Fig. 4b of BN-CQDs aqueous solution under sunlight and 365 nm UV lamp, it can be clearly seen that the aqueous solution of BN-CQDs emits yellow light when excited by 365 nm UV lamp.
fluorescence intensity of BN-CQDs [17,33,34]. The fluorescence stabil ity experiment of BN-CQDs show that the fluorescence of BN-CQDs was almost unaffected under physiological conditions and continuous ul traviolet radiation for 60 min. 3.5. Cytotoxicity Biocompatibility of BN-CQDs in intracellular environment is one of the most essential requirements of biomedical application. Therefore, it was evaluated by hemolysis rate and cell viability in this work. First, a hemolysis assay with red blood cells (RBCs) was conducted to evaluate the blood compatibility of BN-CQDs. Fig. 6a1 and a2 are macroscopic photographs of RBCs suspension before and after treatment for different concentrations of BN-CQDs and Triton X-100 solutions. RBCs in 0 (PBS, negative control group), 10, 20, 30, 40, 50, 80 μg/mL BN-CQDs solution and Triton X-100 solution (positive control group) are shown from left to right. Following a centrifugation step, the cells are intact in PBS and destroyed completely in Triton X-100 as can be intuitively seen from the photographs that. However, the cells do not rupture significantly in different concentrations of BN-CQDs (10–80 μg/mL), which further confirms the negligible hemolysis caused by BN-CQDs [35] (Fig. 6a2). In order to further determine the hemolysis of BN-CQDs, the amount of hemoglobin released into the supernatant (as an indicator of RBCs lysis) was spectrophotometrically measured, as shown in Fig. 6b. RBCs in PBS show 0% hemolysis, while those in 1% Triton X-100 show 100% he molysis. However, the hemolysis rate of the experimental group (RBCs in BN-CQDs) is less than 0.5%. The above results show that BN-CQDs have good biocompatibility. Second, the cytotoxicity was measured by CCK-8 assay and the test result was obtained after incubation of HeLa cells in BN-CQD solution with the concentration range of 0–80 μg/mL for 24 h, as shown in Fig. 6c. The results indicate that the relative cell viability is more than 90% after 24 h exposure in BN-CQDs at a con centration below 80 μg/mL, revealing almost negligible cytotoxicity of BN-CQDs to cells.
3.4. Fluorescence stability Since the fluorescence stability of BN-CQDs is of great significance for cell imaging, it was investigated under different ultraviolet irradia tion time, NaCl concentration and different pH conditions. The photo bleaching experiment was processed under the excitation wavelength at 420 nm. As shown in Fig. 5a, after 60 min of continuous UV excitation, the fluorescence just slightly decreases, which indicates that BN-CQDs have satisfying photobleaching resistance because of their stable struc ture [20]. To confirm the stability in physiological environment of BN-CQDs under different ionic strength, their fluorescence intensities were measured in different NaCl concentrations ranging in 0–0.4 M. Only slight changes in the fluorescence intensity of BN-CQDs are observed and the emission peak of BN-CQDs is also not affected by NaCl concentration (Fig. 5b). At the same time, the effect of different pH on fluorescence intensity was investigated. It is found that the emission wavelength is insensitive to pH value, and the fluorescence intensity also varies with pH value (Fig. 5c). In the pH range of 3–7, the fluorescence intensity of BN-CQDs gradually increases, achieves the maximum at pH 7, and then slowly decreases, indicating the pH-dependency and consequent possibility to construct a potential pH probe [32]. Zeta potential of BN-CQDs under different pH conditions was measured to study pH-dependent perfor mance, as shown in Fig. 5d. It changes from positive to negative, and gradually becomes more negative as pH value increases from 3 to 11. It indicates that the existence form of fluorescent groups on the surface of BN-CQDs is changed during protonation and deprotonation under different pH conditions, which results in changes in surface charge and 6
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Fig. 7. Confocal microscopy images of BN-CQDs (50 μg/mL) incubated with HeLa cells for different times (0, 2, 4 and 6 h).
3.6. Cellular imaging
photobleaching resistance and particular pH sensitivity. They also have low cytotoxicity and good biocompatibility. Given these excellent per formances, as-synthesized BN-CQDs were used as cellular imaging agent in imaging of HeLa cells. The confocal microscopy images of HeLa cells show that BN-CQDs can be effectively absorbed by HeLa cells, mainly located at cytoplasm, and there is no significant change in cell morphology and survival rate. Therefore, BN-CQDs are a promising bioimaging agent.
Given the good biocompatibility and low cytotoxicity of BN-CQDs, the safe dose of BN-CQDs (50 μg/mL) was incubated with HeLa cells for different time (0, 2, 4 and 6 h), and the cellular imaging results were recorded with a confocal microscope at 405 nm as the excitation wavelength. The images of cells treated by BN-CQDs clearly display that BN-CQDs can mark cells, as shown in Fig. 7. The low cytotoxicity and biocom patibility of BN-CQDs are further confirmed because no significant morphological damage of the cells is observed. When HeLa cells are incubated with BN-CQDs for 2 h, they show weak yellow fluorescence. With the extension of incubation time, the fluorescence of the cells gradually becomes bright, because the amount of BN-CQDs entering the cells gradually increases. It can be seen that BN-CQDs are mainly located in cytoplasm, indicating that BN-CQDs could be absorbed and accu mulated by the cells. In addition, the morphology of the cells incubated with BN-CQDs is clearer than that of the blank group. These results of cell imaging prove the feasibility of applying BN-CQDs to cell imaging.
Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. CRediT authorship contribution statement Yingying Wei: Data curation, Formal analysis, Writing - original draft, Writing - review & editing. Lin Chen: Writing - review & editing. Junli Wang: Data curation. Xuguang Liu: Writing - review & editing. Yongzhen Yang: Writing - review & editing. Shiping Yu: Writing review & editing.
4. Conclusion To sum up, this work reports yellow light BN-CQDs applied to cellular imaging, which were synthesized by using the strategy of combining the large sp2 conjugate size with B-N co-doping to realize the red-shift of emission center. Specifically, o-phenylenediamine and boric acid were taken as carbon and nitrogen source and boron dopant, respectively, in a simple and fast microwave-assisted hydrothermal method. The reaction conditions were optimized to achieve a QY of 13.79% at 200 � C for 10 min. The BN-CQDs synthesized under optimal conditions have an average size of 2.5 nm. With oxygen-, boron- and nitrogen-containing functional groups attached their surface. They exhibit remarkable fluorescent properties, including strong absorption in UV region, bright yellow emission under 420 nm excitation, a large Stoke shift, obvious excitation-independent fluorescence behavior,
Acknowledgements This work was supported by the National Natural Science Foundation of China (51803148, U1710117, U1610255, 51972221), the Shanxi Provincial Key Innovative Research Team in Science and Technology (201605D131045-10) and Shanxi Provincial Excellent Talents Science and Technology Innovation Project (201805D211001). References [1] A.M. Alam, B.Y. Park, Z.K. Ghouri, M. Park, H.Y. Kim, Synthesis of carbon quantum dots from cabbage with down- and up-conversion photoluminescence properties:
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Optical Materials journal homepage: http://www.elsevier.com/locate/optmat
Rapid synthesis of B-N co-doped yellow emissive carbon quantum dots for cellular imaging Yingying Wei a, b, Lin Chen b, Junli Wang b, Xuguang Liu b, c, *, Yongzhen Yang b, **, Shiping Yu b, d a
College of Materials Science and Engineering, Taiyuan University of Technology, Taiyuan, 030024, China Key Laboratory of Interface Science and Engineering in Advanced Materials, Taiyuan University of Technology, Ministry of Education, Taiyuan, 030024, China c Institute of New Carbon Materials, Taiyuan University of Technology, Taiyuan, 030600, China d Interventional Treatment Department, Second Hospital of Shanxi Medical University, Taiyuan, 030001, China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Rapidity B-N co-doped carbon quantum dots Yellow emission Cellular imaging
In order to avoid the interference of autofluorescence and improve tissue penetration capability of carbon quantum dots (CQDs) in biological imaging, the synthesis of long-wavelength emission CQDs was achieved in this work. Boron and nitrogen co-doped CQDs (BN-CQDs) were synthesized by one-step microwave assisted hydrothermal method with o-phenylenediamine as carbon and nitrogen source, boric acid as boron source. The BN-CQDs (QY ¼ 13.79%) synthesized under the optimal conditions exhibit excitation independence, a large Stoke shift, satisfactory fluorescent stability, good biocompatibility and low toxicity, which can meet the basic requirements as cell imaging agent. Cell imaging measurements prove their increased intracellular accumulation with the extension of incubation time with HeLa cells, and clearly observable BN-CQDs imaging for cell morphology. Hence BN-CQDs can be used as a good cell imaging agent.
1. Introduction Cellular imaging, which can observe the internal structure and physiological processes of cells in real time or over a period of time, is conducive to monitoring cellular morphology, structure, dynamic pro cesses and the diagnosis of diseases, and is crucial to the interpretation of many cell biology problems. Traditional cellular imaging probes, such as fluorescent dyes and semiconductor quantum dots, have high prep aration costs and high toxicity, which limit their applications in bio logical field [1,2]. Carbon quantum dots (CQDs), as a new class of carbon nanomaterials, have attracted extensive attention in cellular imaging owing to their low cytotoxicity, superior biocompatibility, good water solubility and high photostability [1,3,4]. However, most of CQDs applied to cell imaging at present are blue-emitting under ultraviolet excitation. They have small Stokes shift, resulting in poor signal-to-noise ratio and self-quenching on current microscope configurations [5,6]. Meanwhile, biological tissues made up of carbohydrates also emit blue light, which interferes imaging of cells by blue emissive CQDs [4,5,7]. To solve this problem, CQDs with excitation dependence were synthe sized whose emission peak is red-shifted by long wavelength excitation
[3,7]. Previous reports have shown that the fluorescence intensity of CQDs decreases significantly when their emission peak is red-shifted, which hinders the further application of CQDs in biological imaging. Therefore, the synthesis of long-wavelength emission CQDs (such as yellow and red etc.) with large Stokes shift becomes the fundamental way to solve the existing problems in cellular imaging by CQDs. The luminescence mechanism of CQDs indicates that the sp2 hy bridization degree of carbon nuclei and surface state ultimately deter mine fluorescence properties of CQDs. The strategy of introducing benzene ring structure into carbon nucleus and increasing sp2 conju gation, and changing the surface functional groups or defects of CQDs are beneficial to the red-shift of emission center [8,9]. Meanwhile, doping heteroatoms with different electronegativity into CQDs can lead to more band structures and introduce new defect energy levels and narrow band gaps, which is also conductive to the red-shift of emission wavelength [10]. For example, when doped into CQDs B atom, with low electronegativity and good ability as electron donor, can conjugation with carbon nucleus, which reduces the LUMO level of CQDs and energy emitted by fluorescence; N, with high electronegativity, can form nitrogen-state functional groups on the surface of CQDs, leading to the
* Corresponding author. Key Laboratory of Interface Science and Engineering in Advanced Materials, Taiyuan University of Technology, Ministry of Education, Taiyuan, 030024, China. ** Corresponding author. E-mail addresses: [email protected] (X. Liu), [email protected] (Y. Yang). https://doi.org/10.1016/j.optmat.2019.109647 Received 28 November 2019; Received in revised form 26 December 2019; Accepted 29 December 2019 Available online 16 January 2020 0925-3467/© 2020 Elsevier B.V. All rights reserved.
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generation of new long-wavelength luminescent groups through func tional groups connected to conjugated sp2 region [11,12]. At present, B doped or B-N co-doped CQDs have been preliminarily explored. B-doped CQDs (B-CQDs) were prepared by one-step hydrothermal method from boric acid [11,13] or phenyl boric acid with big sp2 conjugated structure [14,15] as boron source. Some researchers focused on the synthesis of B-N co-doped CQDs (BN-CQDs) from boric acid as boron source and small-molecule urea, diethylenetriamine or ethylenediamine, as nitro gen source by microwave heating, hydrothermal or pyrolysis [12, 16–18]. However, either B-CQDs or BN-CQDs exhibit blue emission. It is obvious that only relying on taking boron-containing materials with big sp2 conjugate as feedstock or the means of B-N co-doping is not enough to realize the red-shift of CQDs’ emission peak. Therefore, integrating the effects of sp2 conjugation and B-N co-doping would be a possible way to achieve the preparation of long-wavelength emission CQDs. Tian et al. [16] reported the synthesis of yellow emission BN-CQDs by one-step hydrothermal method using m-aminobenzene boronic acid with benzene ring as boron source and nitrogen source, the resultant BN-CQDs exhibited a red-shifted emission peak at 540 nm. Li et al. [4] synthesized yellow BN-CQDs with p-phenylenediamine and 3-formyl phenyl boric acid as nitrogen source and boron source by one-step sol vothermal method. They realized the preparation of yellow emission CQDs, but with a synthesis time of 24 h and organic solvent in the process. In summary, large sp2 conjugated size and heteroatom doping are favorable to the red-shift of emission center. Therefore, in this work, B-N co-doped CQDs with excitation independent yellow emission were synthesized by one-step microwave-assisted hydrothermal method at 200 � C within 10 min. o-Phenylenediamine was selected as carbon and nitrogen source, and boron acid as boron source. As a cell imaging agent, high fluorescence intensity, satisfactory fluorescence stability, good biocompatibility and low toxicity are essential. Therefore, the synthesis conditions of BN-CQDs were optimized, and their chemical structure, optical properties and cytotoxicity were investigated towards their application in HeLa cell imaging.
2.3. Apparatus and characterization Transmission electron microscopy (TEM) images and particle size were taken with a JEOL JEM 2100 transmission electron microscope. Raman spectra were obtained using a Japan HORIBA HR800 Raman spectrometer with radiation at 325 nm. X-ray photoelectron spectros copy (XPS) measurements were conducted on a Kratos AXIS ULTRA DLD X-ray photoelectron spectrometer with mono X-ray source Al Kα exci tation (1468.6 eV). Surface functional groups of BN-CQDs were analyzed on a Bruker Tensor 27 Fourier transform infrared spectrometer (FTIR) in the range of 4000 500 cm 1 and recorded on solid samples in a KBr matrix. Ultraviolet–visible (UV–vis) absorption spectra were measured by an ultraviolet spectrophotometer (Jinghua 756 MC, Shanghai, China). Fluorescence spectra, including excitation and emission spectra, were obtained on a Fluoromax-4 Fluorescence spectrometer (Horiba Jobin Yvon, France). Cytotoxicity was detected on a Varioskan Flash Multifunctional microplate reader (Thermo Fisher, USA). Cellular im aging was carried out on a laser confocal apparatus (Leica TCS SP8, Germany). 2.4. Determination of the fluorescence quantum yield Fluorescence quantum yield (QY) of BN-CQDs was determined by a widely accepted relative method, where rhodamine 6G in anhydrous ethanol was chosen as a standard (QY ¼ 95%). To minimize the reab sorption effects, the absorbance of BN-CQDs and reference sample was kept below 0.1. Integrated fluorescence intensity was obtained by calculating the area under the fluorescence curve. A linear fitting line was drawn using the integrated fluorescence intensity against the absorbance with intercept at zero. Finally, QY values were calculated by the following equation: � �� �2 Qx ¼ Qst Gradx= η (1) x=η Gradst st where Q is QY, Grad is the slope of the linear fitting line, η is the refractive index of solvent (1.36 for ethanol and 1.33 for water). The subscripts “st” and “x" refer to the standard solution and BN-CQDs aqueous solution, respectively.
2. Materials and methods 2.1. Chemicals and reagents
2.5. Cell culture
o-Phenylenediamine was produced by Shanghai McLean Biochem ical Technology Co. Ltd. Boric acid was purchased from Tianjin Chem ical Reagent Factory No. 3. Rhodamine 6G was produced by Shanghai Aladdin Biochemical Technology Co. Ltd. Dialysis bags with retained molecular weight of 1000 Da came from Shanghai Yuanye Biological Technology Co. Ltd. Selected standard fetal bovine serum was obtained from Cellmax. 0.25% trypsin, dual-antibody (penicillin and strepto mycin), aseptic phosphate buffered saline (PBS), 1640 medium and Cell Counting Kit-8 (CCK-8) were from Wuhan BOSTER Biological Technol ogy Co. Ltd. HeLa cells were purchased from the cell bank of the Chinese Academy of Sciences, Shanghai.
Cells were cultured in flasks and incubated at 37 � C in a humidified incubator filled with 5% CO2. Each flask contained 5 mL of 1640 me dium supplemented with 10% fetal bovine serum and 1% dual-antibody. 2.6. Cytotoxicity Cytotoxicity of BN-CQDs was analyzed by CCK-8 assay. After reaching 80%–90% confluence, HeLa cells were lifted with trypsin. The trypsinized cells were dispersed and diluted in 1640 medium, followed by centrifugation at 1000 rpm for 5 min. After removing the superna tant, HeLa cells were re-suspended in fresh 1640 medium with a cell density of 2.5 � 104 cells/mL, then they were seeded in 96-well plates, each containing 100 μL of medium. After 24 h cell attachment, HeLa cells were incubated with different concentration of BN-CQDs (0–80 μg/ mL) for 24 h. The cells were then washed three times with PBS to remove uninternalized BN-CQDs. After that 10 μL of CCK-8 dye and 90 μL of 1640 medium were added to each well, followed by incubation for 1 h at 37 � C. The plates were analyzed with a microplate reader. Measure ments of dye optical density (OD) were performed at 450 nm. Cell viability was calculated by the following equation: � ODsample ODblank Cell viabilityð%Þ ¼ *100% (2) ðODcontrol ODblank Þ
2.2. Synthesis of BN-CQDs In a typical procedure of BN-CQDs preparation, 0.27 g of o-phenyl enediamine and 0.15 g of boric acid were dissolved in 10 mL of distilled water, and then the mixture was transferred to a 30 mL microwave re action tube. Subsequently, the reaction tube was put into a Monowave 300 microwave synthesizer (Anton Paar GmbH, Austria) for reaction at predetermined temperature and time period. The resultant solution was cooled to room temperature naturally and filtered through filtration membrane (0.22 μm) to remove large particles. The filtrate was then dialyzed in water through a dialysis bag for two days for further puri fication. Finally, the purified aqueous solution of BN-CQDs was freeze dried to obtain powder BN-CQDs. 2
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Scheme 1. The synthesis process of BN-CQDs and their application in cell imaging.
Fig. 1. The QY fitting lines of BN-CQDs synthesized at 220 � C for different time (a) and at different temperatures for 10 min (b).
2.7. Hemolysis assay
were attached to the wall, they were incubated with BN-CQDs (50 μg/ mL) for 0, 2, 4 and 6 h. Then the cells were washed three times with PBS to remove excess BN-CQDs and fixed for 15 min with 4% para formaldehyde. After fixation, the cells were washed thrice with PBS. Finally, cell images were taken with a laser confocal scanning micro scope at an excitation wavelength of 405 nm, during which the emission signals where collected in range of 540–580 nm. The preparation pro cess and application in cell imaging of BN-CQDs are shown Scheme 1.
Fresh blood was collected from a healthy human and then placed in an anticoagulation tube to avoid coagulation. The blood was cen trifugated at 1000 rpm for 5 min and repeatedly washed with PBS so lution for several times until the supernatant was clear and transparent to obtain clean red blood cells (RBCs). The RBCs were then suspended again in PBS, and resultant suspension was mixed with BN-CQDs PBS solution of equal volume. The final concentration of BN-CQDs was 0, 10, 20, 30, 40, 50 and 80 μg/mL, separately. In contrast, RBCs in PBS (the experimental group without BN-CQDs) and in 1% Triton X-100 were set as the negative and positive controls, respectively. After incubation in a water bath at 37 � C for 3 h, the treated RBC solution was centrifuged at 1000 rpm for 5 min, and the absorbance of supernatant was measured at 575 nm. Each sample had three parallel replicates, the hemolysis was calculated by the following equation: � Isample Inegative control � �100% Hemolysisð%Þ ¼ (3) Ipositive control Inegative control
3. Results and discussion 3.1. Optimization of synthesis conditions QY is one of the criteria to evaluate the fluorescence intensity of fluorescent materials and QY, which is affected by reaction conditions, such as reaction temperature and reaction time. Therefore, BN-CQDs with higher QY were synthesized by optimizing the reaction condi tions. First, the reaction temperature was fixed at 220 � C to investigate the effect of reaction time on QY, as shown in Fig. 1a. It can be clearly seen that with the extension of reaction time, QY first increases and then decreases, reaching the maximum at 10 min, so the optimal reaction time for synthesis of BN-CQDs is 10 min. Second, the reaction time was fixed at 10 min, to study the effect of reaction temperature on QY. The results show that with the increase of reaction temperature, QY also
2.8. Cellular imaging HeLa cells (1 � 105) were placed in a confocal culture dish and cultured overnight in an incubator at 37 � C with 5% CO2. After the cells
Fig. 2. TEM image (a), illustrated by particle size statistics, and Raman spectra (b) of BN-CQDs. 3
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Fig. 3. FTIR spectra (a), full scan XPS spectra (b), high-resolution XPS spectra of C1s, N1s, O1s and B1s (c–f) of BN-CQDs.
3414 cm 1 is attributed to O–H/–NH stretching vibration [17,18], and the distinct absorption bands located at 1620 cm 1 can be attributed to – C and C– – O [19,20]. The BO–H stretching and the vibration of C– B–O–H bending vibration originating from boric acid appear at 3229 and 1196 cm 1, respectively [15,17,20]. The stretching vibration of C–N and C–B is located at 1111 cm 1 [12,15,17,21]. The absorption band at 1449 cm 1 is assigned to vibration of B–N [12,22]. These results show that B and N are doped into BN-CQDs, owing to the dehydration condensation reaction between boric acid and o-phenylenediamine. The relative content of elements and surface structure in BN-CQDs were further investigated by XPS spectral scanning. The binding en ergies of 193.08, 285.08, 400.08 and 532.08 eV in the full spectra of XPS correspond to B1s, C1s, N1s and O1s, respectively, and their relative contents are 24.12%, 28.25%, 5.68% and 41.96%, respectively, con firming the doping of B and N into BN-CQDs. (Fig. 3b). The highresolution spectra of XPS can further clarify the existence form of each element in BN-CQDs. The C1s spectra display three distinct peaks at – C [11,23], 284.44, 285.48 and 288.37 eV, which are assigned to C–C/C– – O, respectively (Fig. 3c). The N1s spectra mainly C–N/C–O and C– include pyridine N, pyrrole N and graphitic N (399.07, 400.56 and 401.40 eV), as shown in Fig. 3d. The O1s spectra (Fig. 3e) show three distinct peaks at 531.64, 532.12 and 533.91 eV, which are ascribed to
shows a trend of first increase and then decrease. The highest QY (13.79%) appears when the reaction temperature is 200 � C. Therefore, 200 � C was set as the optimal reaction temperature for synthesis of BNCQDs (Fig. 1b). In a word, the optimum conditions for synthesis of BNCQDs are: temperature 200 � C, time 10 min. The morphology, structure, optical properties, cytotoxicity and cellular imaging of the BN-CQDs synthesized under optimum conditions were investigated, as will dis cussed in following sections. 3.2. Morphology and structure The morphology and particle size of as-synthesized BN-CQDs were recorded by TEM. As seen in Fig. 2a, BN-CQDs are spherical zerodimensional carbon nanomaterials, with good dispersion and no obvious agglomeration. Additionally, the average particle size of BNCQDs was calculated to be 2.5 nm. Fig. 2b is the Raman spectra of BN-CQDs. The G band at 1591 cm 1 and D band at 1395 cm 1 corre spond to sp2 and sp3 hybrid carbon, respectively, and the relative strength (IG/ID) of crystallized G band and disordered D band is about 1.71, indicating that BN-CQDs mainly consists of sp2 hybrid carbon [9]. FTIR spectra were measured to analyze the surface functional groups of BN-CQDs, as shown in Fig. 3. The broad absorption band at around 4
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Fig. 4. (a) UV–vis absorption, excitation, emission spectra of BN-CQDs, (b) emission spectra of BN-CQDs aqueous solutions at different excitation wavelengths. Photographs of as-prepared BN-CQDs solution under sunlight (inset, left) and under 365 nm UV light (inset, right).
Fig. 5. (a) Fluorescence intensity variation of BN-CQDs as a function of illumination time; Emission spectra of BN-CQDs in different concentrations of NaCl solution (b) and pH (c), the illustration of (b) and (c) show the fluorescence intensity variation of BN-CQDs as a function of concentrations of NaCl and pH, respectively; (d) zeta potential variation of BN-CQDs with pH.
– O, B–O and C–O, respectively. The B1s spectra reveal three distinct C– peaks at 186.94 (B-C) [24,25], 192.63 (B–N) and 193.62 eV (B–O) [25] (Fig. 3f). It can be concluded that oxygen-, boron- and nitrogen-containing functional groups are attached on the surface of BN-CQDs, further demonstrating the co-doping of B and N into BN-CQDs.
appears around 420 nm, indicating that BN-CQDs has a large amount of sp2 carbon, which corresponds to the Raman result (Fig. 2b) [27,28]. The optimal excitation and emission wavelengths of BN-CQDs are located at 409 and 564 nm, respectively, demonstrating that BN-CQDs exhibit a large Stokes shift of 155 nm. It can effectively minimize the interference of self-quenching and improve the signal-to-noise ratio in imaging applications. Fig. 4b is the emission spectra of BN-CQDs solution at different excitation wavelengths. A series of emission spectra were obtained by increasing the excitation wavelength of BN-CQDs from 340 nm to 500 nm, their maximum emission wavelength is all located at 564 nm, among which the strongest emission intensity is obtained when the excitation wavelength is fixed at 420 nm. As can be seen from Fig. 1a, the size of BN-CQDs is only relatively uniform, so excitationindependent fluorescence behavior could be explained by identical/
3.3. Optical properties To explore the optical properties of BN-CQDs, UV–vis, excitation and emission spectra of BN-CQDs were measured, as shown in Fig. 4a. There are two obvious absorption peaks in UV–vis spectra, where the first peak at 237 nm is attributed to the π–π* transition of the sp2 in aromatic structures, and the second peak at 288 nm can be ascribed as the n–π* – O [17,26]. In the visible region, a weak absorption band transition of C– 5
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Fig. 6. (a) Photographs of RBCs suspension before (a1) and after (a2) treatment by BN-CQDs at different concentrations and Triton X-100 solutions; (b) Hemolysis of RBCs in the presence of different concentrations of BN-CQDs; (c) Cytotoxicity of BN-CQD against HeLa cells at increasing concentrations from 0 to 80 μg/mL.
similar emission sites on each particle of BN-CQDs [29,30]. In addition, there is more overlap between the excitation and UV–vis spectra, indi cating that the yellow emission is mainly related to the surface state [31]. From the inset the photographs in Fig. 4b of BN-CQDs aqueous solution under sunlight and 365 nm UV lamp, it can be clearly seen that the aqueous solution of BN-CQDs emits yellow light when excited by 365 nm UV lamp.
fluorescence intensity of BN-CQDs [17,33,34]. The fluorescence stabil ity experiment of BN-CQDs show that the fluorescence of BN-CQDs was almost unaffected under physiological conditions and continuous ul traviolet radiation for 60 min. 3.5. Cytotoxicity Biocompatibility of BN-CQDs in intracellular environment is one of the most essential requirements of biomedical application. Therefore, it was evaluated by hemolysis rate and cell viability in this work. First, a hemolysis assay with red blood cells (RBCs) was conducted to evaluate the blood compatibility of BN-CQDs. Fig. 6a1 and a2 are macroscopic photographs of RBCs suspension before and after treatment for different concentrations of BN-CQDs and Triton X-100 solutions. RBCs in 0 (PBS, negative control group), 10, 20, 30, 40, 50, 80 μg/mL BN-CQDs solution and Triton X-100 solution (positive control group) are shown from left to right. Following a centrifugation step, the cells are intact in PBS and destroyed completely in Triton X-100 as can be intuitively seen from the photographs that. However, the cells do not rupture significantly in different concentrations of BN-CQDs (10–80 μg/mL), which further confirms the negligible hemolysis caused by BN-CQDs [35] (Fig. 6a2). In order to further determine the hemolysis of BN-CQDs, the amount of hemoglobin released into the supernatant (as an indicator of RBCs lysis) was spectrophotometrically measured, as shown in Fig. 6b. RBCs in PBS show 0% hemolysis, while those in 1% Triton X-100 show 100% he molysis. However, the hemolysis rate of the experimental group (RBCs in BN-CQDs) is less than 0.5%. The above results show that BN-CQDs have good biocompatibility. Second, the cytotoxicity was measured by CCK-8 assay and the test result was obtained after incubation of HeLa cells in BN-CQD solution with the concentration range of 0–80 μg/mL for 24 h, as shown in Fig. 6c. The results indicate that the relative cell viability is more than 90% after 24 h exposure in BN-CQDs at a con centration below 80 μg/mL, revealing almost negligible cytotoxicity of BN-CQDs to cells.
3.4. Fluorescence stability Since the fluorescence stability of BN-CQDs is of great significance for cell imaging, it was investigated under different ultraviolet irradia tion time, NaCl concentration and different pH conditions. The photo bleaching experiment was processed under the excitation wavelength at 420 nm. As shown in Fig. 5a, after 60 min of continuous UV excitation, the fluorescence just slightly decreases, which indicates that BN-CQDs have satisfying photobleaching resistance because of their stable struc ture [20]. To confirm the stability in physiological environment of BN-CQDs under different ionic strength, their fluorescence intensities were measured in different NaCl concentrations ranging in 0–0.4 M. Only slight changes in the fluorescence intensity of BN-CQDs are observed and the emission peak of BN-CQDs is also not affected by NaCl concentration (Fig. 5b). At the same time, the effect of different pH on fluorescence intensity was investigated. It is found that the emission wavelength is insensitive to pH value, and the fluorescence intensity also varies with pH value (Fig. 5c). In the pH range of 3–7, the fluorescence intensity of BN-CQDs gradually increases, achieves the maximum at pH 7, and then slowly decreases, indicating the pH-dependency and consequent possibility to construct a potential pH probe [32]. Zeta potential of BN-CQDs under different pH conditions was measured to study pH-dependent perfor mance, as shown in Fig. 5d. It changes from positive to negative, and gradually becomes more negative as pH value increases from 3 to 11. It indicates that the existence form of fluorescent groups on the surface of BN-CQDs is changed during protonation and deprotonation under different pH conditions, which results in changes in surface charge and 6
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Optical Materials 100 (2020) 109647
Fig. 7. Confocal microscopy images of BN-CQDs (50 μg/mL) incubated with HeLa cells for different times (0, 2, 4 and 6 h).
3.6. Cellular imaging
photobleaching resistance and particular pH sensitivity. They also have low cytotoxicity and good biocompatibility. Given these excellent per formances, as-synthesized BN-CQDs were used as cellular imaging agent in imaging of HeLa cells. The confocal microscopy images of HeLa cells show that BN-CQDs can be effectively absorbed by HeLa cells, mainly located at cytoplasm, and there is no significant change in cell morphology and survival rate. Therefore, BN-CQDs are a promising bioimaging agent.
Given the good biocompatibility and low cytotoxicity of BN-CQDs, the safe dose of BN-CQDs (50 μg/mL) was incubated with HeLa cells for different time (0, 2, 4 and 6 h), and the cellular imaging results were recorded with a confocal microscope at 405 nm as the excitation wavelength. The images of cells treated by BN-CQDs clearly display that BN-CQDs can mark cells, as shown in Fig. 7. The low cytotoxicity and biocom patibility of BN-CQDs are further confirmed because no significant morphological damage of the cells is observed. When HeLa cells are incubated with BN-CQDs for 2 h, they show weak yellow fluorescence. With the extension of incubation time, the fluorescence of the cells gradually becomes bright, because the amount of BN-CQDs entering the cells gradually increases. It can be seen that BN-CQDs are mainly located in cytoplasm, indicating that BN-CQDs could be absorbed and accu mulated by the cells. In addition, the morphology of the cells incubated with BN-CQDs is clearer than that of the blank group. These results of cell imaging prove the feasibility of applying BN-CQDs to cell imaging.
Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. CRediT authorship contribution statement Yingying Wei: Data curation, Formal analysis, Writing - original draft, Writing - review & editing. Lin Chen: Writing - review & editing. Junli Wang: Data curation. Xuguang Liu: Writing - review & editing. Yongzhen Yang: Writing - review & editing. Shiping Yu: Writing review & editing.
4. Conclusion To sum up, this work reports yellow light BN-CQDs applied to cellular imaging, which were synthesized by using the strategy of combining the large sp2 conjugate size with B-N co-doping to realize the red-shift of emission center. Specifically, o-phenylenediamine and boric acid were taken as carbon and nitrogen source and boron dopant, respectively, in a simple and fast microwave-assisted hydrothermal method. The reaction conditions were optimized to achieve a QY of 13.79% at 200 � C for 10 min. The BN-CQDs synthesized under optimal conditions have an average size of 2.5 nm. With oxygen-, boron- and nitrogen-containing functional groups attached their surface. They exhibit remarkable fluorescent properties, including strong absorption in UV region, bright yellow emission under 420 nm excitation, a large Stoke shift, obvious excitation-independent fluorescence behavior,
Acknowledgements This work was supported by the National Natural Science Foundation of China (51803148, U1710117, U1610255, 51972221), the Shanxi Provincial Key Innovative Research Team in Science and Technology (201605D131045-10) and Shanxi Provincial Excellent Talents Science and Technology Innovation Project (201805D211001). References [1] A.M. Alam, B.Y. Park, Z.K. Ghouri, M. Park, H.Y. Kim, Synthesis of carbon quantum dots from cabbage with down- and up-conversion photoluminescence properties:
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