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Click multiwalled carbon nanotubes: A novel method for preparation of carboxyl groups functionalized carbon quantum dots
Materials Science & Engineering C 108 (2020) 110376
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Materials Science & Engineering C journal homepage: www.elsevier.com/locate/msec
Click multiwalled carbon nanotubes: A novel method for preparation of carboxyl groups functionalized carbon quantum dots
T
Ziyang Hea,b, Hongye Huanga,b, Ruming Jiangb, Liucheng Maob, Meiying Liub, Junyu Chenb, Fengjie Dengb, Naigen Zhoua,∗∗, Xiaoyong Zhangb,∗, Yen Weic,d,∗∗∗ a
School of Materials Science and Engineering, Nanchang University, Nanchang, Jiangxi, 330031, China Department of Chemistry, Nanchang University, 999 Xuefu Avenue, Nanchang, 330031, China c Department of Chemistry and the Tsinghua Center for Frontier Polymer Research, Tsinghua University, Beijing, 100084, PR China d Department of Chemistry and Center for Nanotechnology and Institute of Biomedical Technology, Chung-Yuan Christian University, Chung-Li, 32023, Taiwan b
A R T I C LE I N FO
A B S T R A C T
Keywords: Multiwalled carbon nanotubes Click reaction Functionalized carbon quantum dots Biological imaging
As potential alternatives to conventional semiconductor quantum dots, fluorescent carbon quantum dots (CQDs) have received increasing research attention in biomedical fields owing to their splendid advantages of low cytotoxicity, strong fluorescence and excellent water dispersion. However, the preparation procedures of CQDs with designable chemical properties and functions are complicated and low efficient. In this work, we developed a facile, economical and straightforward strategy to prepare CQDs by a one-step thiol-ene click reaction between multiwalled carbon nanotubes (CNTs) and thiomalic acid (TA). The successful synthesis of CQDs was confirmed by a series of characterization data. The results manifested that CQDs were well combined with TA through surface thiol-ene click chemistry. In addition, the optical property is also desirable, the maximum emission wavelength was located in 500 nm and CQDs still could emit strong blue fluorescent light after irradiation with UV irradiation for 3 h. Besides, the pH value makes no significant changes for fluorescence emission wavelength of CQDs and CQDs can emit strongest fluorescence in weak acid solution. Furthermore, CQDs could be internalized by cells and show great cell dyeing performance and low cytotoxicity. All these features imply that TA functionalized CQDs possess great potential for biological imaging. The one-step thiol-ene click strategy provided a novel tool to prepare functionalized CQDs with great potential for biomedical applications.
1. Introduction Semiconductor quantum dots have attracted great research attention for various applications since their discovered many years ago [1–4]. Compared with organic fluorescent dyes, semiconductor quantum dots possess admirable properties such as tunable emission wavelengths, great optical stability, long fluorescence lifetime and high fluorescence intensity [5,6]. These unique properties make semiconductor quantum dots great potential in cell imaging, sensors, catalysis and other fields. However, semiconductor quantum dots still own some defects, such as high cytotoxicity, poor water dispersion and complex preparation procedure [7], which impeded the applications of semiconductor quantum dots in biomedical fields. Some fluorescent polymeric nanoparticles based on the self-polymerization of organic dyes containing amphiphilic copolymers have also be reported for
preparation of fluorescent probes. However, the synthesis of these organic dyes and fabrication of these fluorescent copolymers are still rather complex and require specific technique and equipment [8–12]. Carbon quantum dots (CQDs) are expected to replace semiconductor quantum dots, because CQDs not only inherit the superiorities of semiconductor quantum dots, but also have the advantages of good biocompatibility, low cytotoxicity and excellent water dispersion. In recent years, numerous reports have demonstrated that CQDs possess excellent biocompatibility and optical properties [13–21]. For example, Li et al. have synthesized N, P-doped carbon dots and proved their fine biocompatibility. They demonstrated that HeLa cells can maintain high survival rate after incubation with 300 μg mL−1 CQDs for 24 h [22]. More recently, Huang et al. reported the synthesis of S, N doped CQDs through hydrothermal treatment of fungus fibers and these CQDs could be utilized for cell imaging and sensing tetracyclines [23]. Moreover,
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Corresponding author. Corresponding author. ∗∗∗ Corresponding author. Department of Chemistry and the Tsinghua Center for Frontier Polymer Research, Tsinghua University, Beijing, 100084, PR China. E-mail addresses: [email protected] (N. Zhou), [email protected] (X. Zhang), [email protected] (Y. Wei). ∗∗
https://doi.org/10.1016/j.msec.2019.110376 Received 16 September 2019; Received in revised form 29 October 2019; Accepted 29 October 2019 Available online 03 November 2019 0928-4931/ © 2019 Elsevier B.V. All rights reserved.
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the excellent fluorescent property of CQDs makes them well applied in cells imaging [24,25]. CQDs can keep strong fluorescence even exposed to high-energy laser for hours, which demonstrates CQDs possess excellent photostability. The excellent water dispersibility is also important for CQDs in biological imaging application. The introduction of hydrophilic groups on CQDs can not only greatly improve their water dispersion, but also provide the ability for further surface modification and drug loading. To date, many synthetic methods for preparation of CQDs have been developed, that mainly divided into top-down and bottom-up routes. The top-down approaches are mainly relied on smashing carbon skeleton into CQDs, and the bottom-up routes are synthesized CQDs through dehydration of some organic small molecules. Some effective synthesis methods such as electrochemical synthesis [26], arc discharge method [27], laser ablation methods [28] et al. are widely applied in fabricating CQDs. However, these physical preparation methods for CQDs often require expensive laboratory equipment and complex preparation procedures. In addition, CQDs obtained by these physical methods could not control or adjust the surface chemical properties of CQDs [29,30]. These defects are seriously impeded the application of CQDs in biological imaging. In terms of chemical methods, it usually takes several steps to synthesize CQDs complicatedly and tediously [15,25,31]. On the other hand, the preparation of CQDs with specific surface functional groups and designable optical properties is still rarely reported and of great challenge. Shen et al. reported a hydrothermal method to prepare CQDs from sweet potato and the obtained CQDs is selective and sensitive for Fe3+ detection and applied in cell imaging [32], Ma et al. developed a method to prepare green CQDs via combining polyethyleneimine and sucrose at mild conditions [33]. Although their approaches are simple and effective, it is difficult to design specific groups functionalized CQDs. Therefore, the development of novel and facile procedure to meet the requirements for preparation of CQDs through specific reactions is of great research interest. In this contribution, we developed an efficient, simple and one-step strategy for synthesis of functionalized CQDs with carboxyl groups based on the thiol-ene click reaction. Because sp2 carbon abundantly exists in the surface of CNTs and the sp2 carbon can react with compounds containing thiol groups through thiol-ene click reaction and the intact CNTs would be transformed to 0D CQDs. CNTs can be regarded as the coiled sheets of graphene and lack of a bandgap. When CNTs were smashed to 0D CQDs through chemical or physical methods, the CQDs would possess a non-zero bandgap and fluorescence, which can be ascribed to quantum confinement and edge effect [34,35]. We prepared luminescent CQDs via the thiol-ene click reaction between CNTs and thiomalic acid (TA). Compared with general approaches, this synthetic method is simple, inexpensive and efficient. Most importantly, the thiol-ene click strategy can prepare devisable functionalized CQDs through employing diversified sulfhydryl compounds. Besides, CQDs prepared by the simple one-step chemical strategy display excellent fluorescent properties, low cytotoxicity and commendable water dispersion. Furthermore, CQDs could possess accordant small particle size, which enhanced cell permeability of CQDs. Moreover, CQDs are covered with abundant carboxyl groups, which improved water dispersibility and biocompatibility. All of these merits endow CQDs tremendous potential in cell imaging and drug delivery.
(Mw: 46.07 Da, 64-17-5, AR) were obtained from Tianjin Damao Chemical Reagent Co. Ltd., All chemical agents were used as received without further purification. 2.2. Characterization X-ray photoelectron spectroscopy (XPS) measurements were performed using a XPS instrument (ESCALAB250Xi) (Thermo Fisher Scientific Inc. USA) with Al Kα X-ray source. Transmission electronic microscope (TEM) were conducted on a JEM-2100 TEM (Japan Electron Optics Laboratory Co., Ltd.) was operated at an accelerating voltage of 200 kV. Fourier Transform Infrared (FT-IR) spectra were recorded on a Nicolet 5700 FT-IR instrument (Nicolet Instrument Company, USA) over a range from 400 to 4000 cm−1. 1H nuclear magnetic resonance (NMR) spectra were recorded on Bruker Avance400 spectrometer with D2O as the solvent. The fluorescent data were obtained from the Fluorescence spectrophotometer (FSP, model: C11367-11), which purchased from Hamamatsu (Japan). The different solution pH for the study was deployed by 0.1 mol/L HCl aqueous solution and 0.1 mol/L NaOH aqueous solution. 2.3. Procedure for preparation of CQDs The procedure for preparation of CQDs from multiwalled carbon nanotubes was shown in Scheme 1. First, carbon nanotubes (200 mg) was dispersed in 25 mL anhydrous DMF and executed with ultrasonicprocessing for 12 min. After that, TA (1.0 g) and dicumyl peroxide (1.0 g) were added into DMF. Then the solution was transferred to reaction flask and heated at 180 °C for 48 h with reflux condition and nitrogen protection. After the reaction, the reaction flask was cooled to room temperature naturally. Reaction product was centrifuged at a high speed (8000 rpm) for 10 min and the yellow liquid can be separated. In order to obtain the pure fluorescent CQDs, the yellow liquid was dialyzed against distilled water with dialysis bag with 1000 Da molecular weight cut-off for 24 h to eliminate the unreacted impurities and we remove the water by reduced pressure distillation and vacuum drying. Finally, we acquired yellow-brown viscous liquid. 2.4. Cytotoxicity evaluation of CQDs The cell viability is an important parameter for application in cell imaging. L929 cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). According to our previous reports, the cytotoxicity of CQDs was evaluated by cell counting kit-8 (CCK-8) assay [36,37]. First, in 160 μL of media (containing 10% fetal bovine serum (FBS)), L929 cells were seeded in 96-well microplates at a density of 5 × 104 cells per mL. After 24 h, L929 cells were incubated with 0–160 μg mL−1 CQDs for 24 h and the cells were washed with phosphate buffered saline (PBS) three times to remove uninternalized nanoparticles. Then, 10 μL of CCK-8 dye and 100 μL of Dulbecco's Modified Eagle's Medium (DMEM) cell culture media were added to each well, followed by incubation for 2 h at 37 °C. The plates were then analyzed with a microplate reader (Victor III, Perkin-Elmer). The values were proportional to the number of live cells. The percent reduction of
2. Experiment sections 2.1. Materials The following materials were used: multiwalled carbon nanotubes were purchased from Chengdu Organic Chemicals Co., Ltd. (Chinese Academy of Sciences). Dicumyl peroxyide (Mw: 270.37 Da, 80-43-3, 98%) and mercaptosuccinic acid (Mw: 150.15 Da, 70-49-5, 98%) were purchased from the Aladdin Industrial Co., Ltd. (Shanghai, China). N, N-Dimethylformamide (DMF, Mw: 73.09 Da, 68-12-2, AR) and ethanol
Scheme 1. Schematic showing the synthesis of CQDs through the thiol-ene click reaction. 2
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TA. Moreover, we also found that the signals about symmetrical stretching vibration and a symmetrical stretching vibration of methylene groups with weak intensities were also emerged at peaks of 2977.6 and 2929.4 cm−1, respectively. The adsorption peak at 675 cm−1 could be assigned to the wagging vibration of –CH2-. All of the above results indicated that carboxyl groups and TA have introduced on surface of CQDs. Therefore, we could confirm that thiol-ene click reaction was successful. XPS techniques were also used for characterization analysis. As shown in Fig. 2A, in the full survey XPS spectrum of the CQDs, three predominant peaks at 163.6 eV (showed in Fig. 2D), 284.3 eV (showed in Fig. 2B) and 532.3 eV (showed in Fig. 2C) are corresponding to the binding energies of S 2p, C 1s and O 1s, respectively. These signals clearly indicated that the CQDs mainly consist of three elements of carbon, oxygen and sulfur. Furthermore, the atomic concentrations of C, O, and S were calculated based on XPS measurement. As shown in Table S1, the contents of three elements in CQDs were 95.97% (C), 3.23% (O) and 0.79% (S), respectively. As is well-known, CNTs are thoroughly composed of carbon, the emergence of sulfur and oxygen in XPS spectra could clearly evidence the successful immobilization of TA on the surface of CNTs and obtained carboxyl groups functionalized CQDs. More importantly, we also found that the ratio of S and O is about 1:4, which is well consistent with ratio of S/O in TA. It is therefore, we could also clearly evidence successful preparation of carboxyl groups functionalized CQDs through the one-step thiol-ene click reaction between CNTs and TA. Based on the above characterization, we could confirm that the thiol-ene click reaction has occurred successfully. The TEM was employed to characterize the size and morphology of samples. Fig. 3A and B clearly show representative TEM images of CQDs. It could be seen that formed CQDs maintain a spherical shape and dispersed uniformly without apparent aggregation on TEM grid surface. Besides, as displayed in Fig. 3C and D, the diameter of individual pristine CQDs was less than 10 nm. The dynamic light scattering (DLS) technique was employed to characterize the hydrodynamic size distribution of CQDs. As shown in Fig. S2, the results indicated the hydrodynamic size of CQDs is 342.0 ± 96.5 nm with polydispersity index (PDI = 0.403). The high surface energy can lead to the aggregation of CQDs and carboxyl groups can form hydrogen bonding between CQDs. Besides, the hydrated particle size includes the nuclei of nanoparticles and expanded micelles while the TEM show the dry particle, these factors above can make the hydrodynamic size of CQDs is much bigger than that of TEM characterization. According to all the above characterization data, we could demonstrate CQDs have been successfully synthesized by the thiol-ene click reaction. Hence, in the following section, the optical properties and biocompatibility of CQDs will be evaluated. The small particle size and abundant surface functional groups of CQDs not only render them good water dispersibility but also endow their
CCK-8 was compared to the control (cells not exposed to nanoparticles), which represented a 100% CCK-8 reduction. Three replicate wells were used for each control and test concentration per microplate, and the experiment was repeated three times. Cell survival is expressed as the absorbance relative to that of untreated controls. The results are presented as the mean ± standard deviation (SD). 2.5. Confocal microscopic imaging L929 cells were cultured on a culture dish maintained at 37 °C under a humidified condition of 5% CO2 in culture medium and until cells reached 60–70% confluence. L929 cells were treated with a final concentration of 20 μg mL−1 of CQDs for 3 h. Cells were then washed three times with PBS to remove uninternalized nanoparticles and fixed with 4% paraformaldehyde for 10 min at room temperature. Cell images were taken with a confocal laser scanning microscope (CLSM) Zeiss 710 3-channel (Zeiss, Germany) at an excitation wavelength of 405 nm. 3. Results and discussion Owing to the admirable properties such as low cell toxicity, high water dispersibility and excellent fluorescent properties, CQDs are considered as enormous potential materials in biological applications. However, the existing synthesis methods are often unsatisfactory, which requires tedious steps or expensive instruments and poor designability. Besides, proper particle size and water dispersion are also important. Therefore, a simple, cheap and efficient synthetic method is necessary. In this work, we developed a one-step strategy for synthesis of CQDs. The evidence of success consists of a series of characterization methods. 1H NMR spectrum was first introduced to prove the successful introduction of TA on the surface of CQDs. As shown in Fig. 1A, a number of chemical shift signals at 2–3 ppm could be observed and two chemical shifts appeared at about 3.3 ppm, compared with the spectrum of unadorned TA (shown in Fig. S1), we can find the similar chemical shifts also existed in the 1H NMR spectrum of TA. This illustrated that TA was introduced on the surface of CNTs successfully via the thiol-ene click chemistry and formation of CQDs with carboxyl groups. In order to gain further structural characterization, the FT-IR spectroscopy was performed to evidence the surface functional groups on CQDs. The FT-IR spectra of CNTs and CQDs are shown in Fig. 1B, it is clear that almost no obvious peaks were appeared in the FT-IR spectrum of CNTs, which indicated less functional groups existence on the surface of CNTs. Whereas several obvious peaks could be observed on the FT-IR spectrum of CQDs. Apparently, the peak at 3450 cm−1 could be ascribed to –OH and peak at 1700 cm−1 could be assigned to –C]O. Correspondingly, the COO− symmetric stretching vibration (vs COO−) at 1421 cm−1 was also observed in FT-IR spectrum. Obviously, the –OH and the –C]O groups of CQDs were originated from introduction of
Fig. 1. The 1H NMR spectrum of the CQDs in D2O (A). The FT-IR spectra of pristine CNTs and CQDs samples (B). Many characteristic signals have indicated that carboxyl groups have been immobilized on the surface of CQDs. 3
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Fig. 2. XPS spectra of the CQDs. (A) Survey scan ranging from 0 to 1200 eV, (B) C 1s, (C) O 1s and (D) S 2p spectra of the CQDs.
wavelength at 410 nm. On the other hand, when the emission wavelength was set at 500 nm, the excitation peak was found at 410 nm. The Stoke shift is 90 nm, which makes CQDs great potential for biological imaging owing to the few overlap between the excitation and emission wavelengths. Besides, the uniform CQDs aqueous solution was exhibited in insert of Fig. 4B (left cuvette) and exhibits light yellow in natural light. Especially, the mark “Q” as background could be observed clearly through the cuvette containing the samples, which displayed the great water dispersibility of CQDs. Moreover, in insert of Fig. 4B (right cuvette), the water suspension of CQDs emit strong blue fluorescence under irradiated by UV lamp (365 nm), which emerged the good fluorescence properties of CQDs. In order to further evaluate the
capability be internalized by cells. More importantly, the carboxyl groups introduced on the surface CQDs could be used for loading drugs and further conjugation reactions. In order to further evaluate optical properties, UV–Vis spectrum was employed. As displayed in Fig. 4A, it is clearly seen that two adsorption peaks are located at 208 and 266 nm, respectively. The adsorption peak at 208 nm could be ascribed to π→π* transition of aromatic rings and the adsorption peak at 208 nm could be attributed to n→π* transition of C]O resulting from TA. The UV–Vis spectrum further proved the TA was conjugated on the surface of CNTs and formation of CQDs. Fig. 4B shows typical fluorescent spectra of CQDs, the maximum emission wavelength was settled at 500 nm when the CQDs were excited with the
Fig. 3. (A) TEM image of CQDs, scale bar = 500 nm; (B) enlarged TEM image of CQDs, scale bar = 100 nm; (C) and (D) enlarged TEM images of CQDs, scale bar = 10 nm. 4
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Fig. 4. (A) UV–Vis spectrum of the CQDs in ethanol solution. (B) Photoluminescence excitation and emission spectra of CQDs in aqueous solutions. The excitation spectrum was obtained by set the emission wavelength at 500 nm while the emission spectrum was obtained via a 410 nm excitation wavelength. Insert shows the photographs of the CQDs in aqueous solution under natural light (left cuvette) and UV light (λ = 365 nm, right cuvette). (C) Fluorescence spectra of the CQDs in aqueous solution excited by different wavelengths of light. (D) The effects of excitation wavelengths on the fluorescence emission intensity.
fluorescence property of CQDs, the influence of excitation wavelengths on the emission of CQDs was also investigated (Fig. 4C). For example, when the excitation wavelengths were increased from 320 to 520 nm with 20 nm escalation, the emission peaks of CQDs were gradually red shift correspondingly. On the other hand, we also found that the fluorescence intensities of CQDs were also changed with the increasing of excitation wavelengths. As shown in Fig. 4D, the fluorescence intensities were increased when the excitation wavelengths were increased from 320 to 410 nm and reached the maximum value at 410 nm. After that, the intensities were significantly decreased after when the excitation wavelengths were further increased from 410 to 530 nm. This fluorescence phenomenon is well similar with other CQDs. This is possibly ascribed to the heterogeneity of CQDs. The above results demonstrated that the CQDs could be existed with various excitation wavelength and displayed different emission color. When the excitation wavelength was set at 405 nm, green fluorescence will emit. However, when the excitation wavelength was changed to 488 nm, the yellow fluorescence will emit. This provide multi-color imaging using CQDs when the excitation lasers were changed. Great photostability is also important for potential bioimaging applications. To confirm the stability of the CQDs under high-energy ray, we measured the changes of fluorescence intensity of CQDs under ultraviolet light (λ = 365 nm) at different times. As shown in Fig. S3, the fluorescence intensity of CQDs aqueous solution is 806.87 a.u. After being exposed in UV light for 1 h, the fluorescence intensity of CQDs aqueous solution is decreased to 735.69 a.u. from 806.87 a.u. The fluorescence intensity decreases only by 8.83%. Even after irradiating for 3 h, the fluorescence intensity can still keep at 80.8% of initial intensity (651.98 a.u.), which could demonstrate CQDs possess decent fluorescence stability. This guarantees the applications of CQDs in cell imaging and other fields. Besides fluorescence stability, the pH effects on fluorescence are also worth to exploring. A series of different pH values of CQDs aqueous solution were prepared. The changes of fluorescence in different pH conditions are displayed in Fig. 5, it should be easy to discover that the emission wavelength is almost impervious to pH values. However, fluorescence intensity of CQDs in aqueous
Fig. 5. The fluorescence intensity of CQDs in the aqueous solution with different pH values.
solution would be influenced by different pH values. As shown in Fig. S4, the fluorescence intensity heightens gradually with the changes of pH values from pH 1.0 to pH 5.0, the fluorescence intensity reaches the strongest at the pH 5.0 and slowly decreases with the pH values improved. This character endows CQDs great potential application in pH measurement as highly effective fluorescent probes. Excellent water dispersion is also necessary property for biological imaging. However, the pristine CNTs possess bad water dispersion, which was a serious obstacle to biological application. In this work, we employed hydrophilic TA to ameliorate its dispersibility. Therefore, we assessed the dispersibility in water and alcohol by comparing the dispersion effect under different time conditions. As shown in Fig. 6, it is obvious that the most pristine CNTs sink to the bottom quickly within 10 min whether in an aqueous solution or an ethanol solution. On the contrary, the CQDs could maintain uniform distribution in water 5
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CQDs at concentration of 160 μg mL−1 for 24 h, the cell viability values still kept above 90%. The results above could deduce that the CQDs possess great biocompatibility and extensive application prospect in biomedicine fields. Previously, fluorescent polymeric nanoparticles from the self-assembly of amphiphilic copolymers that contain hydrophobic fluorescent organic dyes and hydrophilic components have also be reported by some groups [38–40]. Especially, the preparation of fluorescent polymeric nanoparticles with aggregation-induced emission (AIE) feature has attracted the most research interest because the unique AIE characteristic of these fluorescent nanoparticles could effective overcome the fluorescence quenching of conventional organic dyes [41–44]. Some groups have demonstrated that these AIE-active fluorescent polymeric nanoparticles could be synthesized by a number of methods and could be utilized for different biomedical applications such as biological imaging, cancer treatment, bio/chem sensors [45–52]. Although these fluorescent polymeric nanoparticles would be well designed, the synthesis of AIE-active organic dyes is complex, expensive and time consuming. On the other hand, the size and morphology of final self-assemblies is not controllable and final particle size is relatively large. In this work, we demonstrated the preparation of CQDs through the one-pot thiol-ene click reaction between CNTs with TA. This procedure is rather simple and effective. The particle size is less than 10 nm and the surface functional groups could be well adjusted through choosing thiol-containing molecules. Therefore, we believe that the method described in this work should be a general and useful route to prepare a functionalized CQDs [35]. We have confirmed that CQDs has good biocompatibility above. In the following section, the cell uptake behavior of CQDs would be evaluated via CLSM to explore their potential in cell imaging. As shown in Fig. 7B, in the bright field image, L929 cells still could keep their natural size and morphology after they were incubated with 20 μg mL−1 of CQDs for 3 h, which further evaluated the splendid hypotoxicity and biocompatibility. The Fig. 7C exhibited the fluorescence image of L929 cells under 405 nm laser. There are no fluorescence
Fig. 6. Photographs of CNTs in water (A), CNTs in alcohol (B), CQDs in water (C) and CQDs in alcohol (D) for different resting time points.
solution or alcohol solution more than 12 h. Therefore, we could assert the CQDs possess excellent dispersion in water or organic solvent far beyond CNTs, which grant CQDs possess great potential in further biomedical applications. The results above indicated that the CQDs possess suitable size and superb dispersibility. Besides, the strong green fluorescence property in aqueous solution makes CQDs desirable probes for biological imaging. However, assessment of biocompatibility is also important for biological applications. Hence, in this work, we adopted a typical CCK-8 assay to evaluate the cytotoxicity of CQDs. As shown in Fig. 7A, the cells were incubated with different concentrations of CQDs (from 0160 μg mL−1) for 24 h. It could be clearly observed that no significant decrease was found based on cell viability values. Even cultured with
Fig. 7. Cytotoxicity testing results of CQDs via CCK-8 assay in different concentrations of CQDs for 24 h (A). Cell imaging of CQDs, bright field (B), fluorescent image (C), and the overlap image of A and B (D), where the scale bar = 20 μm. The cells were excited with a single laser (λ = 405 nm). 6
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signals in cell nucleus could be observed and some strong fluorescence signal surrounded, which illustrated the CQDs mainly accumulated in cytoplasm through non-specific endocytosis. The Fig. 7D shows the overlapped image established on bright field and fluorescence image. It can be viewed that the fluorescence signals covered on the cell locations and the fluorescence signals mainly distributed in cytoplasm rather than cell nucleus. The results further evaluate the great biocompatibility and demonstrated the CQDs were internalized by L929 cells. Therefore, CQDs could turn into promising biomaterials as fluorescent probes applied in biological imaging.
X. Zhang, Y. Wei, Dyes Pigments 149 (2018) 581–587. [11] R. Jiang, M. Liu, H. Huang, L. Mao, Q. Huang, Y. Wen, Q.Y. Cao, J. Tian, X. Zhang, Y. Wei, Dyes Pigments 153 (2018) 99–105. [12] R. Jiang, M. Liu, H. Huang, L. Mao, Q. Huang, Y. Wen, Q.-y. Cao, J. Tian, X. Zhang, Y. Wei, Dyes Pigments 153 (2018) 99–105. [13] K. B, Z. A, D. C, Z. X, L. B, T. Y, Adv. Mater. 24 (2012) 5844–5848. [14] V. Vaijayanthimala, H.C. Chang, Nanomedicine 4 (2009) 47–55. [15] A. Zhu, Q. Qu, X. Shao, B. Kong, Y. Tian, Angew. Chem. Int. Ed. 124 (2012) 7297–7301. [16] P.G. Luo, S. Sahu, S.-T. Yang, S.K. Sonkar, J. Wang, H. Wang, G.E. LeCroy, L. Cao, Y.-P. Sun, J. Mater. Chem. B 1 (2013) 2116–2127. [17] P.G. Luo, F. Yang, S.-T. Yang, S.K. Sonkar, L. Yang, J.J. Broglie, Y. Liu, Y.-P. Sun, RSC Adv. 4 (2014) 10791–10807. [18] X. Wang, L. Cao, S.-T. Yang, F. Lu, M.J. Meziani, L. Tian, K.W. Sun, M.A. Bloodgood, Y.-P. Sun, Angew. Chem., Int. Ed. Engl. 49 (2010) 5310–5314. [19] H. Ouyang, M. Zhou, Y. Guo, M. He, H. Huang, X. Ye, Y. Feng, X. Zhou, S. Yang, Fitoterapia 96 (2014) 152–158. [20] X. Zhang, J. Li, B. Xie, B. Wu, S. Lei, Y. Yao, M. He, H. Ouyang, Y. Feng, W. Xu, S. Yang, Front. Pharmacol. 9 (2018). [21] X. Zhang, M. He, S. Lei, B. Wu, T. Tan, H. Ouyang, W. Xu, Y. Feng, J. Pharmaceut. Biomed. 156 (2018) 221–231. [22] H. Li, F.Q. Shao, S.Y. Zou, Q.J. Yang, H. Huang, J.J. Feng, A.J. Wang, Microchim. Acta 183 (2016) 1–6. [23] C. Shi, H. Qi, R. Ma, Z. Sun, L. Xiao, G. Wei, Z. Huang, S. Liu, J. Li, M. Dong, J. Fan, Z. Guo, Mater. Sci. Eng. C 105 (2019) 110132. [24] Q. Qu, A. Zhu, X. Shao, G. Shi, Y. Tian, Chem. Commun. 48 (2012) 5473–5475. [25] Z. Yan, J. Shu, Y. Yu, Z. Zhang, Z. Liu, J. Chen, Luminescence 30 (2015) 388–392. [26] H. Li, H. Ming, Y. Liu, H. Yu, X. He, H. Huang, K. Pan, Z. Kang, S.T. Lee, New J. Chem. 35 (2011) 2666–2670. [27] S. Dey, A. Govindaraj, K. Biswas, C.N.R. Rao, Chem. Phys. Lett. 595–596 (2014) 203–208. [28] X. Li, H. Wang, Y. Shimizu, A. Pyatenko, K. Kawaguchi, N. Koshizaki, Chem. Commun. 47 (2010) 932–934. [29] M. Lu, L. Zhou, Mater. Sci. Eng. C 101 (2019) 352–359. [30] C. Cheng, M. Xing, Q. Wu, Mater. Sci. Eng. C 99 (2019) 611–619. [31] X. Su, Y. Xu, Y. Che, X. Liao, Y. Jiang, J. Biomed. Mater. Res. A 103 (2015) 3956–3964. [32] J. Shen, S. Shang, X. Chen, D. Wang, Y. Cai, Mater. Sci. Eng. C 76 (2017) 856–864. [33] C. Ma, X. Zhang, L. Yang, Y. Wu, H. Liu, X. Zhang, Y. Wei, Mater. Sci. Eng. C 68 (2016) 37–42. [34] S. Zhu, J. Zhang, C. Qiao, S. Tang, Y. Li, W. Yuan, B. Li, L. Tian, F. Liu, R. Hu, Chem. Commun. 47 (2011) 6858–6860. [35] S. Zhu, Y. Song, X. Zhao, J. Shao, J. Zhang, B. Yang, Nano Res. 8 (2015) 355–381. [36] X. Zhang, M. Liu, X. Zhang, F. Deng, C. Zhou, J. Hui, W. Liu, Y. Wei, Toxicol. Res. 4 (2015) 160–168. [37] X. Zhang, H. Qi, S. Wang, L. Feng, Y. Ji, L. Tao, S. Li, Y. Wei, Toxicol. Res. 1 (2012) 201–205. [38] X. Zhang, K. Wang, M. Liu, X. Zhang, L. Tao, Y. Chen, Y. Wei, Nanoscale 7 (2015) 11486–11508. [39] Y. Shi, R. Jiang, M. Liu, L. Fu, G. Zeng, Q. Wan, L. Mao, F. Deng, X. Zhang, Y. Wei, Mater. Sci. Eng. C 77 (2017) 972–977. [40] J. Mei, N.L. Leung, R.T. Kwok, J.W. Lam, B.Z. Tang, Chem. Rev. 115 (2015) 11718–11940. [41] X. Zhang, X. Zhang, B. Yang, M. Liu, W. Liu, Y. Chen, Y. Wei, Polym. Chem. 5 (2014) 356–360. [42] X. Zhang, X. Zhang, B. Yang, M. Liu, W. Liu, Y. Chen, Y. Wei, Polym. Chem. 5 (2014) 399–404. [43] Q. Wan, Q. Huang, M. Liu, D. Xu, H. Huang, X. Zhang, Y. Wei, Appl. Mater. Today 9 (2017) 145–160. [44] J. Mei, Y. Hong, J.W. Lam, A. Qin, Y. Tang, B.Z. Tang, Adv. Mater. 26 (2014) 5429–5479. [45] Q.-y. Cao, R. Jiang, M. Liu, Q. Wan, D. Xu, J. Tian, H. Huang, Y. Wen, X. Zhang, Y. Wei, Mater. Sci. Eng. C 80 (2017) 578–583. [46] Q.-y. Cao, R. Jiang, M. Liu, Q. Wan, D. Xu, J. Tian, H. Huang, Y. Wen, X. Zhang, Y. Wei, Mater. Sci. Eng. C 80 (2017) 411–416. [47] H. Huang, D. Xu, M. Liu, R. Jiang, L. Mao, Q. Huang, Q. Wan, Y. Wen, X. Zhang, Y. Wei, Mater. Sci. Eng. C 78 (2017) 862–867. [48] L. Huang, S. Yang, J. Chen, J. Tian, Q. Huang, H. Huang, Y. Wen, F. Deng, X. Zhang, Y. Wei, Mater. Sci. Eng. C 94 (2019) 270–278. [49] R. Jiang, H. Liu, M. Liu, J. Tian, Q. Huang, H. Huang, Y. Wen, Q.-y. Cao, X. Zhang, Y. Wei, Mater. Sci. Eng. C 81 (2017) 416–421. [50] R. Jiang, M. Liu, C. Li, Q. Huang, H. Huang, Q. Wan, Y. Wen, Q.-y. Cao, X. Zhang, Y. Wei, Mater. Sci. Eng. C 80 (2017) 708–714. [51] L. Mao, Y. Liu, S. Yang, Y. Li, X. Zhang, Y. Wei, Dyes Pigments 162 (2019) 611–623. [52] Y. Hong, J.W. Lam, B.Z. Tang, Chem. Soc. Rev. 40 (2011) 5361–5388.
4. Conclusions In conclusion, we have successfully synthesized carboxyl groups functionalized CQDs by a simple, efficient one-pot thiol-ene click approach, which reacted between CNTs and TA. These functionalized CQDs were well isolated from CNTs and emitted strong, stable and excitation independent fluorescence. In addition, the CQDs exhibited wonderful fluorescence properties and great biocompatibility, which permit CQDs to be adopted as efficient fluorescent agents for in vitro/in vivo imaging. Besides, superb pH adaptability and good photostability guarantee extensive application prospects of fluorescent CQDs. Taken together, this work provides a facile, inexpensive approach for synthesis of carboxyl groups functionalized CQDs and they possess great potential in numerous biomedical applications. More importantly, this method could also open a novel avenue for preparation of other functionalized CQDs with good designability and these CQDs could be of great interest for different biomedical applications. Declaration of competing interest The authors declared that there is no conflict of interest. Acknowledgments This research was supported by the National Natural Science Foundation of China (Nos. 21788102, 21865016, 51363016, 21474057, 21564006, 21561022, 21644014). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.msec.2019.110376. References [1] X. Gao, Y. Cui, R.M. Levenson, L.W. Chung, S. Nie, Nat. Biotechnol. 22 (2004) 969–976. [2] L.C. Mattheakis, J.M. Dias, Y.J. Choi, J. Gong, M.P. Bruchez, J. Liu, E. Wang, Anal. Biochem. 327 (2004) 200–208. [3] G. R, Z. M, W. I, Angew. Chem. Int. Ed. 47 (2010) 7602–7625. [4] M. X, P. FF, B. LA, T. JM, D. S, L. JJ, S. G, W. AM, G. SS, W. S, Science 307 (2005) 538–544. [5] R. E.B, S. Nie, J. Am. Chem. Soc. 125 (2003) 7100–7106. [6] A.D. Yoffe, Adv. Phys. 50 (2001) 1–208. [7] J. Chen, M. Liu, Q. Huang, L. Huang, H. Huang, F. Deng, Y. Wen, J. Tian, X. Zhang, Y. Wei, Chem. Eng. J. 337 (2018) 82–90. [8] H. Huang, M. Liu, S. Luo, K. Wang, Q. Wan, F. Deng, D. Xu, X. Zhang, Y. Wei, Chem. Eng. J. 304 (2016) 149–155. [9] R. Jiang, M. Liu, T. Chen, H. Huang, Q. Huang, J. Tian, Y. Wen, Q.-y. Cao, X. Zhang, Y. Wei, Dyes Pigments 148 (2018) 52–60. [10] R. Jiang, M. Liu, H. Huang, L. Huang, Q. Huang, Y. Wen, Q.-y. Cao, J. Tian,
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Materials Science & Engineering C journal homepage: www.elsevier.com/locate/msec
Click multiwalled carbon nanotubes: A novel method for preparation of carboxyl groups functionalized carbon quantum dots
T
Ziyang Hea,b, Hongye Huanga,b, Ruming Jiangb, Liucheng Maob, Meiying Liub, Junyu Chenb, Fengjie Dengb, Naigen Zhoua,∗∗, Xiaoyong Zhangb,∗, Yen Weic,d,∗∗∗ a
School of Materials Science and Engineering, Nanchang University, Nanchang, Jiangxi, 330031, China Department of Chemistry, Nanchang University, 999 Xuefu Avenue, Nanchang, 330031, China c Department of Chemistry and the Tsinghua Center for Frontier Polymer Research, Tsinghua University, Beijing, 100084, PR China d Department of Chemistry and Center for Nanotechnology and Institute of Biomedical Technology, Chung-Yuan Christian University, Chung-Li, 32023, Taiwan b
A R T I C LE I N FO
A B S T R A C T
Keywords: Multiwalled carbon nanotubes Click reaction Functionalized carbon quantum dots Biological imaging
As potential alternatives to conventional semiconductor quantum dots, fluorescent carbon quantum dots (CQDs) have received increasing research attention in biomedical fields owing to their splendid advantages of low cytotoxicity, strong fluorescence and excellent water dispersion. However, the preparation procedures of CQDs with designable chemical properties and functions are complicated and low efficient. In this work, we developed a facile, economical and straightforward strategy to prepare CQDs by a one-step thiol-ene click reaction between multiwalled carbon nanotubes (CNTs) and thiomalic acid (TA). The successful synthesis of CQDs was confirmed by a series of characterization data. The results manifested that CQDs were well combined with TA through surface thiol-ene click chemistry. In addition, the optical property is also desirable, the maximum emission wavelength was located in 500 nm and CQDs still could emit strong blue fluorescent light after irradiation with UV irradiation for 3 h. Besides, the pH value makes no significant changes for fluorescence emission wavelength of CQDs and CQDs can emit strongest fluorescence in weak acid solution. Furthermore, CQDs could be internalized by cells and show great cell dyeing performance and low cytotoxicity. All these features imply that TA functionalized CQDs possess great potential for biological imaging. The one-step thiol-ene click strategy provided a novel tool to prepare functionalized CQDs with great potential for biomedical applications.
1. Introduction Semiconductor quantum dots have attracted great research attention for various applications since their discovered many years ago [1–4]. Compared with organic fluorescent dyes, semiconductor quantum dots possess admirable properties such as tunable emission wavelengths, great optical stability, long fluorescence lifetime and high fluorescence intensity [5,6]. These unique properties make semiconductor quantum dots great potential in cell imaging, sensors, catalysis and other fields. However, semiconductor quantum dots still own some defects, such as high cytotoxicity, poor water dispersion and complex preparation procedure [7], which impeded the applications of semiconductor quantum dots in biomedical fields. Some fluorescent polymeric nanoparticles based on the self-polymerization of organic dyes containing amphiphilic copolymers have also be reported for
preparation of fluorescent probes. However, the synthesis of these organic dyes and fabrication of these fluorescent copolymers are still rather complex and require specific technique and equipment [8–12]. Carbon quantum dots (CQDs) are expected to replace semiconductor quantum dots, because CQDs not only inherit the superiorities of semiconductor quantum dots, but also have the advantages of good biocompatibility, low cytotoxicity and excellent water dispersion. In recent years, numerous reports have demonstrated that CQDs possess excellent biocompatibility and optical properties [13–21]. For example, Li et al. have synthesized N, P-doped carbon dots and proved their fine biocompatibility. They demonstrated that HeLa cells can maintain high survival rate after incubation with 300 μg mL−1 CQDs for 24 h [22]. More recently, Huang et al. reported the synthesis of S, N doped CQDs through hydrothermal treatment of fungus fibers and these CQDs could be utilized for cell imaging and sensing tetracyclines [23]. Moreover,
∗
Corresponding author. Corresponding author. ∗∗∗ Corresponding author. Department of Chemistry and the Tsinghua Center for Frontier Polymer Research, Tsinghua University, Beijing, 100084, PR China. E-mail addresses: [email protected] (N. Zhou), [email protected] (X. Zhang), [email protected] (Y. Wei). ∗∗
https://doi.org/10.1016/j.msec.2019.110376 Received 16 September 2019; Received in revised form 29 October 2019; Accepted 29 October 2019 Available online 03 November 2019 0928-4931/ © 2019 Elsevier B.V. All rights reserved.
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the excellent fluorescent property of CQDs makes them well applied in cells imaging [24,25]. CQDs can keep strong fluorescence even exposed to high-energy laser for hours, which demonstrates CQDs possess excellent photostability. The excellent water dispersibility is also important for CQDs in biological imaging application. The introduction of hydrophilic groups on CQDs can not only greatly improve their water dispersion, but also provide the ability for further surface modification and drug loading. To date, many synthetic methods for preparation of CQDs have been developed, that mainly divided into top-down and bottom-up routes. The top-down approaches are mainly relied on smashing carbon skeleton into CQDs, and the bottom-up routes are synthesized CQDs through dehydration of some organic small molecules. Some effective synthesis methods such as electrochemical synthesis [26], arc discharge method [27], laser ablation methods [28] et al. are widely applied in fabricating CQDs. However, these physical preparation methods for CQDs often require expensive laboratory equipment and complex preparation procedures. In addition, CQDs obtained by these physical methods could not control or adjust the surface chemical properties of CQDs [29,30]. These defects are seriously impeded the application of CQDs in biological imaging. In terms of chemical methods, it usually takes several steps to synthesize CQDs complicatedly and tediously [15,25,31]. On the other hand, the preparation of CQDs with specific surface functional groups and designable optical properties is still rarely reported and of great challenge. Shen et al. reported a hydrothermal method to prepare CQDs from sweet potato and the obtained CQDs is selective and sensitive for Fe3+ detection and applied in cell imaging [32], Ma et al. developed a method to prepare green CQDs via combining polyethyleneimine and sucrose at mild conditions [33]. Although their approaches are simple and effective, it is difficult to design specific groups functionalized CQDs. Therefore, the development of novel and facile procedure to meet the requirements for preparation of CQDs through specific reactions is of great research interest. In this contribution, we developed an efficient, simple and one-step strategy for synthesis of functionalized CQDs with carboxyl groups based on the thiol-ene click reaction. Because sp2 carbon abundantly exists in the surface of CNTs and the sp2 carbon can react with compounds containing thiol groups through thiol-ene click reaction and the intact CNTs would be transformed to 0D CQDs. CNTs can be regarded as the coiled sheets of graphene and lack of a bandgap. When CNTs were smashed to 0D CQDs through chemical or physical methods, the CQDs would possess a non-zero bandgap and fluorescence, which can be ascribed to quantum confinement and edge effect [34,35]. We prepared luminescent CQDs via the thiol-ene click reaction between CNTs and thiomalic acid (TA). Compared with general approaches, this synthetic method is simple, inexpensive and efficient. Most importantly, the thiol-ene click strategy can prepare devisable functionalized CQDs through employing diversified sulfhydryl compounds. Besides, CQDs prepared by the simple one-step chemical strategy display excellent fluorescent properties, low cytotoxicity and commendable water dispersion. Furthermore, CQDs could possess accordant small particle size, which enhanced cell permeability of CQDs. Moreover, CQDs are covered with abundant carboxyl groups, which improved water dispersibility and biocompatibility. All of these merits endow CQDs tremendous potential in cell imaging and drug delivery.
(Mw: 46.07 Da, 64-17-5, AR) were obtained from Tianjin Damao Chemical Reagent Co. Ltd., All chemical agents were used as received without further purification. 2.2. Characterization X-ray photoelectron spectroscopy (XPS) measurements were performed using a XPS instrument (ESCALAB250Xi) (Thermo Fisher Scientific Inc. USA) with Al Kα X-ray source. Transmission electronic microscope (TEM) were conducted on a JEM-2100 TEM (Japan Electron Optics Laboratory Co., Ltd.) was operated at an accelerating voltage of 200 kV. Fourier Transform Infrared (FT-IR) spectra were recorded on a Nicolet 5700 FT-IR instrument (Nicolet Instrument Company, USA) over a range from 400 to 4000 cm−1. 1H nuclear magnetic resonance (NMR) spectra were recorded on Bruker Avance400 spectrometer with D2O as the solvent. The fluorescent data were obtained from the Fluorescence spectrophotometer (FSP, model: C11367-11), which purchased from Hamamatsu (Japan). The different solution pH for the study was deployed by 0.1 mol/L HCl aqueous solution and 0.1 mol/L NaOH aqueous solution. 2.3. Procedure for preparation of CQDs The procedure for preparation of CQDs from multiwalled carbon nanotubes was shown in Scheme 1. First, carbon nanotubes (200 mg) was dispersed in 25 mL anhydrous DMF and executed with ultrasonicprocessing for 12 min. After that, TA (1.0 g) and dicumyl peroxide (1.0 g) were added into DMF. Then the solution was transferred to reaction flask and heated at 180 °C for 48 h with reflux condition and nitrogen protection. After the reaction, the reaction flask was cooled to room temperature naturally. Reaction product was centrifuged at a high speed (8000 rpm) for 10 min and the yellow liquid can be separated. In order to obtain the pure fluorescent CQDs, the yellow liquid was dialyzed against distilled water with dialysis bag with 1000 Da molecular weight cut-off for 24 h to eliminate the unreacted impurities and we remove the water by reduced pressure distillation and vacuum drying. Finally, we acquired yellow-brown viscous liquid. 2.4. Cytotoxicity evaluation of CQDs The cell viability is an important parameter for application in cell imaging. L929 cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). According to our previous reports, the cytotoxicity of CQDs was evaluated by cell counting kit-8 (CCK-8) assay [36,37]. First, in 160 μL of media (containing 10% fetal bovine serum (FBS)), L929 cells were seeded in 96-well microplates at a density of 5 × 104 cells per mL. After 24 h, L929 cells were incubated with 0–160 μg mL−1 CQDs for 24 h and the cells were washed with phosphate buffered saline (PBS) three times to remove uninternalized nanoparticles. Then, 10 μL of CCK-8 dye and 100 μL of Dulbecco's Modified Eagle's Medium (DMEM) cell culture media were added to each well, followed by incubation for 2 h at 37 °C. The plates were then analyzed with a microplate reader (Victor III, Perkin-Elmer). The values were proportional to the number of live cells. The percent reduction of
2. Experiment sections 2.1. Materials The following materials were used: multiwalled carbon nanotubes were purchased from Chengdu Organic Chemicals Co., Ltd. (Chinese Academy of Sciences). Dicumyl peroxyide (Mw: 270.37 Da, 80-43-3, 98%) and mercaptosuccinic acid (Mw: 150.15 Da, 70-49-5, 98%) were purchased from the Aladdin Industrial Co., Ltd. (Shanghai, China). N, N-Dimethylformamide (DMF, Mw: 73.09 Da, 68-12-2, AR) and ethanol
Scheme 1. Schematic showing the synthesis of CQDs through the thiol-ene click reaction. 2
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TA. Moreover, we also found that the signals about symmetrical stretching vibration and a symmetrical stretching vibration of methylene groups with weak intensities were also emerged at peaks of 2977.6 and 2929.4 cm−1, respectively. The adsorption peak at 675 cm−1 could be assigned to the wagging vibration of –CH2-. All of the above results indicated that carboxyl groups and TA have introduced on surface of CQDs. Therefore, we could confirm that thiol-ene click reaction was successful. XPS techniques were also used for characterization analysis. As shown in Fig. 2A, in the full survey XPS spectrum of the CQDs, three predominant peaks at 163.6 eV (showed in Fig. 2D), 284.3 eV (showed in Fig. 2B) and 532.3 eV (showed in Fig. 2C) are corresponding to the binding energies of S 2p, C 1s and O 1s, respectively. These signals clearly indicated that the CQDs mainly consist of three elements of carbon, oxygen and sulfur. Furthermore, the atomic concentrations of C, O, and S were calculated based on XPS measurement. As shown in Table S1, the contents of three elements in CQDs were 95.97% (C), 3.23% (O) and 0.79% (S), respectively. As is well-known, CNTs are thoroughly composed of carbon, the emergence of sulfur and oxygen in XPS spectra could clearly evidence the successful immobilization of TA on the surface of CNTs and obtained carboxyl groups functionalized CQDs. More importantly, we also found that the ratio of S and O is about 1:4, which is well consistent with ratio of S/O in TA. It is therefore, we could also clearly evidence successful preparation of carboxyl groups functionalized CQDs through the one-step thiol-ene click reaction between CNTs and TA. Based on the above characterization, we could confirm that the thiol-ene click reaction has occurred successfully. The TEM was employed to characterize the size and morphology of samples. Fig. 3A and B clearly show representative TEM images of CQDs. It could be seen that formed CQDs maintain a spherical shape and dispersed uniformly without apparent aggregation on TEM grid surface. Besides, as displayed in Fig. 3C and D, the diameter of individual pristine CQDs was less than 10 nm. The dynamic light scattering (DLS) technique was employed to characterize the hydrodynamic size distribution of CQDs. As shown in Fig. S2, the results indicated the hydrodynamic size of CQDs is 342.0 ± 96.5 nm with polydispersity index (PDI = 0.403). The high surface energy can lead to the aggregation of CQDs and carboxyl groups can form hydrogen bonding between CQDs. Besides, the hydrated particle size includes the nuclei of nanoparticles and expanded micelles while the TEM show the dry particle, these factors above can make the hydrodynamic size of CQDs is much bigger than that of TEM characterization. According to all the above characterization data, we could demonstrate CQDs have been successfully synthesized by the thiol-ene click reaction. Hence, in the following section, the optical properties and biocompatibility of CQDs will be evaluated. The small particle size and abundant surface functional groups of CQDs not only render them good water dispersibility but also endow their
CCK-8 was compared to the control (cells not exposed to nanoparticles), which represented a 100% CCK-8 reduction. Three replicate wells were used for each control and test concentration per microplate, and the experiment was repeated three times. Cell survival is expressed as the absorbance relative to that of untreated controls. The results are presented as the mean ± standard deviation (SD). 2.5. Confocal microscopic imaging L929 cells were cultured on a culture dish maintained at 37 °C under a humidified condition of 5% CO2 in culture medium and until cells reached 60–70% confluence. L929 cells were treated with a final concentration of 20 μg mL−1 of CQDs for 3 h. Cells were then washed three times with PBS to remove uninternalized nanoparticles and fixed with 4% paraformaldehyde for 10 min at room temperature. Cell images were taken with a confocal laser scanning microscope (CLSM) Zeiss 710 3-channel (Zeiss, Germany) at an excitation wavelength of 405 nm. 3. Results and discussion Owing to the admirable properties such as low cell toxicity, high water dispersibility and excellent fluorescent properties, CQDs are considered as enormous potential materials in biological applications. However, the existing synthesis methods are often unsatisfactory, which requires tedious steps or expensive instruments and poor designability. Besides, proper particle size and water dispersion are also important. Therefore, a simple, cheap and efficient synthetic method is necessary. In this work, we developed a one-step strategy for synthesis of CQDs. The evidence of success consists of a series of characterization methods. 1H NMR spectrum was first introduced to prove the successful introduction of TA on the surface of CQDs. As shown in Fig. 1A, a number of chemical shift signals at 2–3 ppm could be observed and two chemical shifts appeared at about 3.3 ppm, compared with the spectrum of unadorned TA (shown in Fig. S1), we can find the similar chemical shifts also existed in the 1H NMR spectrum of TA. This illustrated that TA was introduced on the surface of CNTs successfully via the thiol-ene click chemistry and formation of CQDs with carboxyl groups. In order to gain further structural characterization, the FT-IR spectroscopy was performed to evidence the surface functional groups on CQDs. The FT-IR spectra of CNTs and CQDs are shown in Fig. 1B, it is clear that almost no obvious peaks were appeared in the FT-IR spectrum of CNTs, which indicated less functional groups existence on the surface of CNTs. Whereas several obvious peaks could be observed on the FT-IR spectrum of CQDs. Apparently, the peak at 3450 cm−1 could be ascribed to –OH and peak at 1700 cm−1 could be assigned to –C]O. Correspondingly, the COO− symmetric stretching vibration (vs COO−) at 1421 cm−1 was also observed in FT-IR spectrum. Obviously, the –OH and the –C]O groups of CQDs were originated from introduction of
Fig. 1. The 1H NMR spectrum of the CQDs in D2O (A). The FT-IR spectra of pristine CNTs and CQDs samples (B). Many characteristic signals have indicated that carboxyl groups have been immobilized on the surface of CQDs. 3
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Fig. 2. XPS spectra of the CQDs. (A) Survey scan ranging from 0 to 1200 eV, (B) C 1s, (C) O 1s and (D) S 2p spectra of the CQDs.
wavelength at 410 nm. On the other hand, when the emission wavelength was set at 500 nm, the excitation peak was found at 410 nm. The Stoke shift is 90 nm, which makes CQDs great potential for biological imaging owing to the few overlap between the excitation and emission wavelengths. Besides, the uniform CQDs aqueous solution was exhibited in insert of Fig. 4B (left cuvette) and exhibits light yellow in natural light. Especially, the mark “Q” as background could be observed clearly through the cuvette containing the samples, which displayed the great water dispersibility of CQDs. Moreover, in insert of Fig. 4B (right cuvette), the water suspension of CQDs emit strong blue fluorescence under irradiated by UV lamp (365 nm), which emerged the good fluorescence properties of CQDs. In order to further evaluate the
capability be internalized by cells. More importantly, the carboxyl groups introduced on the surface CQDs could be used for loading drugs and further conjugation reactions. In order to further evaluate optical properties, UV–Vis spectrum was employed. As displayed in Fig. 4A, it is clearly seen that two adsorption peaks are located at 208 and 266 nm, respectively. The adsorption peak at 208 nm could be ascribed to π→π* transition of aromatic rings and the adsorption peak at 208 nm could be attributed to n→π* transition of C]O resulting from TA. The UV–Vis spectrum further proved the TA was conjugated on the surface of CNTs and formation of CQDs. Fig. 4B shows typical fluorescent spectra of CQDs, the maximum emission wavelength was settled at 500 nm when the CQDs were excited with the
Fig. 3. (A) TEM image of CQDs, scale bar = 500 nm; (B) enlarged TEM image of CQDs, scale bar = 100 nm; (C) and (D) enlarged TEM images of CQDs, scale bar = 10 nm. 4
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Fig. 4. (A) UV–Vis spectrum of the CQDs in ethanol solution. (B) Photoluminescence excitation and emission spectra of CQDs in aqueous solutions. The excitation spectrum was obtained by set the emission wavelength at 500 nm while the emission spectrum was obtained via a 410 nm excitation wavelength. Insert shows the photographs of the CQDs in aqueous solution under natural light (left cuvette) and UV light (λ = 365 nm, right cuvette). (C) Fluorescence spectra of the CQDs in aqueous solution excited by different wavelengths of light. (D) The effects of excitation wavelengths on the fluorescence emission intensity.
fluorescence property of CQDs, the influence of excitation wavelengths on the emission of CQDs was also investigated (Fig. 4C). For example, when the excitation wavelengths were increased from 320 to 520 nm with 20 nm escalation, the emission peaks of CQDs were gradually red shift correspondingly. On the other hand, we also found that the fluorescence intensities of CQDs were also changed with the increasing of excitation wavelengths. As shown in Fig. 4D, the fluorescence intensities were increased when the excitation wavelengths were increased from 320 to 410 nm and reached the maximum value at 410 nm. After that, the intensities were significantly decreased after when the excitation wavelengths were further increased from 410 to 530 nm. This fluorescence phenomenon is well similar with other CQDs. This is possibly ascribed to the heterogeneity of CQDs. The above results demonstrated that the CQDs could be existed with various excitation wavelength and displayed different emission color. When the excitation wavelength was set at 405 nm, green fluorescence will emit. However, when the excitation wavelength was changed to 488 nm, the yellow fluorescence will emit. This provide multi-color imaging using CQDs when the excitation lasers were changed. Great photostability is also important for potential bioimaging applications. To confirm the stability of the CQDs under high-energy ray, we measured the changes of fluorescence intensity of CQDs under ultraviolet light (λ = 365 nm) at different times. As shown in Fig. S3, the fluorescence intensity of CQDs aqueous solution is 806.87 a.u. After being exposed in UV light for 1 h, the fluorescence intensity of CQDs aqueous solution is decreased to 735.69 a.u. from 806.87 a.u. The fluorescence intensity decreases only by 8.83%. Even after irradiating for 3 h, the fluorescence intensity can still keep at 80.8% of initial intensity (651.98 a.u.), which could demonstrate CQDs possess decent fluorescence stability. This guarantees the applications of CQDs in cell imaging and other fields. Besides fluorescence stability, the pH effects on fluorescence are also worth to exploring. A series of different pH values of CQDs aqueous solution were prepared. The changes of fluorescence in different pH conditions are displayed in Fig. 5, it should be easy to discover that the emission wavelength is almost impervious to pH values. However, fluorescence intensity of CQDs in aqueous
Fig. 5. The fluorescence intensity of CQDs in the aqueous solution with different pH values.
solution would be influenced by different pH values. As shown in Fig. S4, the fluorescence intensity heightens gradually with the changes of pH values from pH 1.0 to pH 5.0, the fluorescence intensity reaches the strongest at the pH 5.0 and slowly decreases with the pH values improved. This character endows CQDs great potential application in pH measurement as highly effective fluorescent probes. Excellent water dispersion is also necessary property for biological imaging. However, the pristine CNTs possess bad water dispersion, which was a serious obstacle to biological application. In this work, we employed hydrophilic TA to ameliorate its dispersibility. Therefore, we assessed the dispersibility in water and alcohol by comparing the dispersion effect under different time conditions. As shown in Fig. 6, it is obvious that the most pristine CNTs sink to the bottom quickly within 10 min whether in an aqueous solution or an ethanol solution. On the contrary, the CQDs could maintain uniform distribution in water 5
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CQDs at concentration of 160 μg mL−1 for 24 h, the cell viability values still kept above 90%. The results above could deduce that the CQDs possess great biocompatibility and extensive application prospect in biomedicine fields. Previously, fluorescent polymeric nanoparticles from the self-assembly of amphiphilic copolymers that contain hydrophobic fluorescent organic dyes and hydrophilic components have also be reported by some groups [38–40]. Especially, the preparation of fluorescent polymeric nanoparticles with aggregation-induced emission (AIE) feature has attracted the most research interest because the unique AIE characteristic of these fluorescent nanoparticles could effective overcome the fluorescence quenching of conventional organic dyes [41–44]. Some groups have demonstrated that these AIE-active fluorescent polymeric nanoparticles could be synthesized by a number of methods and could be utilized for different biomedical applications such as biological imaging, cancer treatment, bio/chem sensors [45–52]. Although these fluorescent polymeric nanoparticles would be well designed, the synthesis of AIE-active organic dyes is complex, expensive and time consuming. On the other hand, the size and morphology of final self-assemblies is not controllable and final particle size is relatively large. In this work, we demonstrated the preparation of CQDs through the one-pot thiol-ene click reaction between CNTs with TA. This procedure is rather simple and effective. The particle size is less than 10 nm and the surface functional groups could be well adjusted through choosing thiol-containing molecules. Therefore, we believe that the method described in this work should be a general and useful route to prepare a functionalized CQDs [35]. We have confirmed that CQDs has good biocompatibility above. In the following section, the cell uptake behavior of CQDs would be evaluated via CLSM to explore their potential in cell imaging. As shown in Fig. 7B, in the bright field image, L929 cells still could keep their natural size and morphology after they were incubated with 20 μg mL−1 of CQDs for 3 h, which further evaluated the splendid hypotoxicity and biocompatibility. The Fig. 7C exhibited the fluorescence image of L929 cells under 405 nm laser. There are no fluorescence
Fig. 6. Photographs of CNTs in water (A), CNTs in alcohol (B), CQDs in water (C) and CQDs in alcohol (D) for different resting time points.
solution or alcohol solution more than 12 h. Therefore, we could assert the CQDs possess excellent dispersion in water or organic solvent far beyond CNTs, which grant CQDs possess great potential in further biomedical applications. The results above indicated that the CQDs possess suitable size and superb dispersibility. Besides, the strong green fluorescence property in aqueous solution makes CQDs desirable probes for biological imaging. However, assessment of biocompatibility is also important for biological applications. Hence, in this work, we adopted a typical CCK-8 assay to evaluate the cytotoxicity of CQDs. As shown in Fig. 7A, the cells were incubated with different concentrations of CQDs (from 0160 μg mL−1) for 24 h. It could be clearly observed that no significant decrease was found based on cell viability values. Even cultured with
Fig. 7. Cytotoxicity testing results of CQDs via CCK-8 assay in different concentrations of CQDs for 24 h (A). Cell imaging of CQDs, bright field (B), fluorescent image (C), and the overlap image of A and B (D), where the scale bar = 20 μm. The cells were excited with a single laser (λ = 405 nm). 6
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signals in cell nucleus could be observed and some strong fluorescence signal surrounded, which illustrated the CQDs mainly accumulated in cytoplasm through non-specific endocytosis. The Fig. 7D shows the overlapped image established on bright field and fluorescence image. It can be viewed that the fluorescence signals covered on the cell locations and the fluorescence signals mainly distributed in cytoplasm rather than cell nucleus. The results further evaluate the great biocompatibility and demonstrated the CQDs were internalized by L929 cells. Therefore, CQDs could turn into promising biomaterials as fluorescent probes applied in biological imaging.
X. Zhang, Y. Wei, Dyes Pigments 149 (2018) 581–587. [11] R. Jiang, M. Liu, H. Huang, L. Mao, Q. Huang, Y. Wen, Q.Y. Cao, J. Tian, X. Zhang, Y. Wei, Dyes Pigments 153 (2018) 99–105. [12] R. Jiang, M. Liu, H. Huang, L. Mao, Q. Huang, Y. Wen, Q.-y. Cao, J. Tian, X. Zhang, Y. Wei, Dyes Pigments 153 (2018) 99–105. [13] K. B, Z. A, D. C, Z. X, L. B, T. Y, Adv. Mater. 24 (2012) 5844–5848. [14] V. Vaijayanthimala, H.C. Chang, Nanomedicine 4 (2009) 47–55. [15] A. Zhu, Q. Qu, X. Shao, B. Kong, Y. Tian, Angew. Chem. Int. Ed. 124 (2012) 7297–7301. [16] P.G. Luo, S. Sahu, S.-T. Yang, S.K. Sonkar, J. Wang, H. Wang, G.E. LeCroy, L. Cao, Y.-P. Sun, J. Mater. Chem. B 1 (2013) 2116–2127. [17] P.G. Luo, F. Yang, S.-T. Yang, S.K. Sonkar, L. Yang, J.J. Broglie, Y. Liu, Y.-P. Sun, RSC Adv. 4 (2014) 10791–10807. [18] X. Wang, L. Cao, S.-T. Yang, F. Lu, M.J. Meziani, L. Tian, K.W. Sun, M.A. Bloodgood, Y.-P. Sun, Angew. Chem., Int. Ed. Engl. 49 (2010) 5310–5314. [19] H. Ouyang, M. Zhou, Y. Guo, M. He, H. Huang, X. Ye, Y. Feng, X. Zhou, S. Yang, Fitoterapia 96 (2014) 152–158. [20] X. Zhang, J. Li, B. Xie, B. Wu, S. Lei, Y. Yao, M. He, H. Ouyang, Y. Feng, W. Xu, S. Yang, Front. Pharmacol. 9 (2018). [21] X. Zhang, M. He, S. Lei, B. Wu, T. Tan, H. Ouyang, W. Xu, Y. Feng, J. Pharmaceut. Biomed. 156 (2018) 221–231. [22] H. Li, F.Q. Shao, S.Y. Zou, Q.J. Yang, H. Huang, J.J. Feng, A.J. Wang, Microchim. Acta 183 (2016) 1–6. [23] C. Shi, H. Qi, R. Ma, Z. Sun, L. Xiao, G. Wei, Z. Huang, S. Liu, J. Li, M. Dong, J. Fan, Z. Guo, Mater. Sci. Eng. C 105 (2019) 110132. [24] Q. Qu, A. Zhu, X. Shao, G. Shi, Y. Tian, Chem. Commun. 48 (2012) 5473–5475. [25] Z. Yan, J. Shu, Y. Yu, Z. Zhang, Z. Liu, J. Chen, Luminescence 30 (2015) 388–392. [26] H. Li, H. Ming, Y. Liu, H. Yu, X. He, H. Huang, K. Pan, Z. Kang, S.T. Lee, New J. Chem. 35 (2011) 2666–2670. [27] S. Dey, A. Govindaraj, K. Biswas, C.N.R. Rao, Chem. Phys. Lett. 595–596 (2014) 203–208. [28] X. Li, H. Wang, Y. Shimizu, A. Pyatenko, K. Kawaguchi, N. Koshizaki, Chem. Commun. 47 (2010) 932–934. [29] M. Lu, L. Zhou, Mater. Sci. Eng. C 101 (2019) 352–359. [30] C. Cheng, M. Xing, Q. Wu, Mater. Sci. Eng. C 99 (2019) 611–619. [31] X. Su, Y. Xu, Y. Che, X. Liao, Y. Jiang, J. Biomed. Mater. Res. A 103 (2015) 3956–3964. [32] J. Shen, S. Shang, X. Chen, D. Wang, Y. Cai, Mater. Sci. Eng. C 76 (2017) 856–864. [33] C. Ma, X. Zhang, L. Yang, Y. Wu, H. Liu, X. Zhang, Y. Wei, Mater. Sci. Eng. C 68 (2016) 37–42. [34] S. Zhu, J. Zhang, C. Qiao, S. Tang, Y. Li, W. Yuan, B. Li, L. Tian, F. Liu, R. Hu, Chem. Commun. 47 (2011) 6858–6860. [35] S. Zhu, Y. Song, X. Zhao, J. Shao, J. Zhang, B. Yang, Nano Res. 8 (2015) 355–381. [36] X. Zhang, M. Liu, X. Zhang, F. Deng, C. Zhou, J. Hui, W. Liu, Y. Wei, Toxicol. Res. 4 (2015) 160–168. [37] X. Zhang, H. Qi, S. Wang, L. Feng, Y. Ji, L. Tao, S. Li, Y. Wei, Toxicol. Res. 1 (2012) 201–205. [38] X. Zhang, K. Wang, M. Liu, X. Zhang, L. Tao, Y. Chen, Y. Wei, Nanoscale 7 (2015) 11486–11508. [39] Y. Shi, R. Jiang, M. Liu, L. Fu, G. Zeng, Q. Wan, L. Mao, F. Deng, X. Zhang, Y. Wei, Mater. Sci. Eng. C 77 (2017) 972–977. [40] J. Mei, N.L. Leung, R.T. Kwok, J.W. Lam, B.Z. Tang, Chem. Rev. 115 (2015) 11718–11940. [41] X. Zhang, X. Zhang, B. Yang, M. Liu, W. Liu, Y. Chen, Y. Wei, Polym. Chem. 5 (2014) 356–360. [42] X. Zhang, X. Zhang, B. Yang, M. Liu, W. Liu, Y. Chen, Y. Wei, Polym. Chem. 5 (2014) 399–404. [43] Q. Wan, Q. Huang, M. Liu, D. Xu, H. Huang, X. Zhang, Y. Wei, Appl. Mater. Today 9 (2017) 145–160. [44] J. Mei, Y. Hong, J.W. Lam, A. Qin, Y. Tang, B.Z. Tang, Adv. Mater. 26 (2014) 5429–5479. [45] Q.-y. Cao, R. Jiang, M. Liu, Q. Wan, D. Xu, J. Tian, H. Huang, Y. Wen, X. Zhang, Y. Wei, Mater. Sci. Eng. C 80 (2017) 578–583. [46] Q.-y. Cao, R. Jiang, M. Liu, Q. Wan, D. Xu, J. Tian, H. Huang, Y. Wen, X. Zhang, Y. Wei, Mater. Sci. Eng. C 80 (2017) 411–416. [47] H. Huang, D. Xu, M. Liu, R. Jiang, L. Mao, Q. Huang, Q. Wan, Y. Wen, X. Zhang, Y. Wei, Mater. Sci. Eng. C 78 (2017) 862–867. [48] L. Huang, S. Yang, J. Chen, J. Tian, Q. Huang, H. Huang, Y. Wen, F. Deng, X. Zhang, Y. Wei, Mater. Sci. Eng. C 94 (2019) 270–278. [49] R. Jiang, H. Liu, M. Liu, J. Tian, Q. Huang, H. Huang, Y. Wen, Q.-y. Cao, X. Zhang, Y. Wei, Mater. Sci. Eng. C 81 (2017) 416–421. [50] R. Jiang, M. Liu, C. Li, Q. Huang, H. Huang, Q. Wan, Y. Wen, Q.-y. Cao, X. Zhang, Y. Wei, Mater. Sci. Eng. C 80 (2017) 708–714. [51] L. Mao, Y. Liu, S. Yang, Y. Li, X. Zhang, Y. Wei, Dyes Pigments 162 (2019) 611–623. [52] Y. Hong, J.W. Lam, B.Z. Tang, Chem. Soc. Rev. 40 (2011) 5361–5388.
4. Conclusions In conclusion, we have successfully synthesized carboxyl groups functionalized CQDs by a simple, efficient one-pot thiol-ene click approach, which reacted between CNTs and TA. These functionalized CQDs were well isolated from CNTs and emitted strong, stable and excitation independent fluorescence. In addition, the CQDs exhibited wonderful fluorescence properties and great biocompatibility, which permit CQDs to be adopted as efficient fluorescent agents for in vitro/in vivo imaging. Besides, superb pH adaptability and good photostability guarantee extensive application prospects of fluorescent CQDs. Taken together, this work provides a facile, inexpensive approach for synthesis of carboxyl groups functionalized CQDs and they possess great potential in numerous biomedical applications. More importantly, this method could also open a novel avenue for preparation of other functionalized CQDs with good designability and these CQDs could be of great interest for different biomedical applications. Declaration of competing interest The authors declared that there is no conflict of interest. Acknowledgments This research was supported by the National Natural Science Foundation of China (Nos. 21788102, 21865016, 51363016, 21474057, 21564006, 21561022, 21644014). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.msec.2019.110376. References [1] X. Gao, Y. Cui, R.M. Levenson, L.W. Chung, S. Nie, Nat. Biotechnol. 22 (2004) 969–976. [2] L.C. Mattheakis, J.M. Dias, Y.J. Choi, J. Gong, M.P. Bruchez, J. Liu, E. Wang, Anal. Biochem. 327 (2004) 200–208. [3] G. R, Z. M, W. I, Angew. Chem. Int. Ed. 47 (2010) 7602–7625. [4] M. X, P. FF, B. LA, T. JM, D. S, L. JJ, S. G, W. AM, G. SS, W. S, Science 307 (2005) 538–544. [5] R. E.B, S. Nie, J. Am. Chem. Soc. 125 (2003) 7100–7106. [6] A.D. Yoffe, Adv. Phys. 50 (2001) 1–208. [7] J. Chen, M. Liu, Q. Huang, L. Huang, H. Huang, F. Deng, Y. Wen, J. Tian, X. Zhang, Y. Wei, Chem. Eng. J. 337 (2018) 82–90. [8] H. Huang, M. Liu, S. Luo, K. Wang, Q. Wan, F. Deng, D. Xu, X. Zhang, Y. Wei, Chem. Eng. J. 304 (2016) 149–155. [9] R. Jiang, M. Liu, T. Chen, H. Huang, Q. Huang, J. Tian, Y. Wen, Q.-y. Cao, X. Zhang, Y. Wei, Dyes Pigments 148 (2018) 52–60. [10] R. Jiang, M. Liu, H. Huang, L. Huang, Q. Huang, Y. Wen, Q.-y. Cao, J. Tian,
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