Materials Science and Engineering C 58 (2016) 730–736
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Easy synthesis of highly fluorescent carbon dots from albumin and their photoluminescent mechanism and biological imaging applications Xiaohua Hu, Xueqin An ⁎, Lielie Li School of Chemistry and Molecular Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China
a r t i c l e
i n f o
Article history: Received 19 January 2015 Received in revised form 8 June 2015 Accepted 16 September 2015 Available online 18 September 2015 Keywords: Carbon dots Novel synthesis method Photoluminescent mechanism Biological imaging
a b s t r a c t A simple and green approach was developed to synthesize highly fluorescent carbon dots (CDs) using albumin as a carbon source in aqueous solution at room temperature. The CDs were characterized by excellent monodispersion, superior photostability, pH-independent emission, long fluorescence lifetime and high quantum yield (QY). The photoluminescent (PL) mechanism of CDs was explored by means of time-resolved PL decay, and the results revealed that PL originated from the emission of both defect state and intrinsic state. In addition, biological imaging with the application of CDs was carried out in human breast cancer Bcap-37 cell, which demonstrated that CDs were provided with an excellent biocompatiblity, low cytotoxicity and good transmembrane ability. Besides, CDs could be considered as a potential substitute for organic dyes or semiconductor quantum dots (SQDs) in biological imaging. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Fluorescence remains one of the most powerful and common tools for biological research [1]. As fluorescent materials, semiconductor quantum dots (SQDs) and organic dyes were employed in biological application during the past decade [2]. However, the photostability of organic dyes is poor, and SQDs with heavy metal ions may lead to potential cytotoxicity [2]. SQDs without cadmium are considered to be low toxicity, but their shortcomings of complicated synthesis and purification process are observed [3], which restrict their biotechnological and medical applications. With the advancement in fluorescent nanomaterials, photoluminescent carbon dots (PL CDs) as new nanoparticles are emerging, which possess unique properties such as low toxicity, good aqueous solubility, great biocompatibility, excellent optical performance and low photobleaching [4–6]. The CDs have been widely used in biochemical sensing [7], fluorescent probes [8], biological imaging [9,10], photocatalytic technology [11], drug carriers [12], light emitting devices [13], energy conversion/storage devices [14] and so on. So far, a number of approaches have been developed to prepare CDs, such as laser irradiation [15], arc discharge [16], electrochemical synthesis [17], hydrothermal method [5], chemical oxidation [18], ultrasonic treatment [19] and microwave synthesis [20]. However, complicated experimental condition and post-treatment process are involved in the majority of the methods. An energy efficient method to synthesize CDs has been reported utilizing saccharide as carbon source, but the
significant drawback is low quantum yield (1.2%) [21]. In our group, a combination method of hydrothermal and microwave digestion was used to synthesize CDs using ascorbic acid as carbon source [22], nevertheless expensive apparatus and complex procedures are required in the method. On the other hand, carbon source plays a significant role in CDs preparation. Currently, various carbon sources such as fullerene [23], polyacrylamide [24], polyethylene glycol [6], polyacrylic acid [25], polyethylenimine [26] and albumin [27,28], have been reported to synthesize CDs by different methods. Up to now, further investigations which could help understand CDs in depth are quite deficient. More facile methods are required because of the easy fluorescence quenching during multi-step chemical reactions. Some important information about CDs, such as the PL mechanism of CDs, cell imaging and biological labeling, is still rare. As a consequence, further investigations based on CDs are not only challenging, but also necessary. In this article, a novel one-step alkaline hydrolysis (AH) method is presented to fabricate CDs using albumin as source material in NaOH solution at room temperature. Comparisons among the morphology, size and optical properties of CDs prepared with different approaches were made. The PL mechanism of CDs was explored by means of time-resolved PL decay. Cytotoxicity, biocompatibility and transmembrane ability of the CDs were demonstrated.
2. Experimental 2.1. Materials
⁎ Corresponding author. E-mail address: [email protected] (X. An).
http://dx.doi.org/10.1016/j.msec.2015.09.066 0928-4931/© 2015 Elsevier B.V. All rights reserved.
Fresh eggs were purchased from the local market. Sodium hydroxide, polyacrylic acid, polyethylenimine, polyacrylamide, polyethylene
X. Hu et al. / Materials Science and Engineering C 58 (2016) 730–736
731
glycol and hydrochloric acid were purchased from Aladdin (Shanghai, China). Besides, all chemicals were of analytical grade, which could be applied without further purification. In addition, double deionized water was employed throughout all the experiments.
sample containing CDs. All experiments were conducted in triplicate and mean values were taken into consideration.
2.2. Synthesis of CDs
2.0 mL of human breast cancer Bcap-37 cells was seeded in a cover glass bottom dish and cultured at 37 °C for 24 h, which were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 1% penicillin streptomycin. And then, the culture medium was changed to 2.0 mL of fresh medium containing 2.0 mg/mL CDs and cells were incubated at 37 °C for 24 h. Finally, the culture medium was removed and cells were washed with PBS three times. Under a laser scanning confocal microscopy, cell imaging was conducted with laser excitation wavelengths of 405 and 488 nm with the collection of fluorescence in blue and green regions.
During a typical preparation, albumin (egg white) was well separated from egg with the aid of an egg separator and freeze-dried by means of a freeze drier, which served as a carbon source to produce CDs without any further purification. To prepare various CDs samples, 6.0 mL of albumin solution (25 g/L) was mixed with 1.0 mL of NaOH solution (1.0 mol/L), and then the mixture was incubated for different times at room temperature. After CDs preparation, remaining alkali was neutralized with HCl to adjust the pH of CDs solution to 7. Then the solution was purified to remove NaCl by dialysis for 24 h. The comparison between PL intensities of CDs was conducted under different incubation times in Fig. S1a, and the optimal incubation time was determined as 80 h. To determine optimum NaOH concentration (CNaOH), CDs were prepared in solutions of different CNaOH at room temperature for 80 h. The PL intensities of CDs prepared in different CNaOH were presented in Fig. S1b, and the optimal CNaOH was chosen as 1.0 mol/L. Taking albumin as a carbon source, CDs were synthesized by making use of microwave (MW) and ultrasonic manipulation (UM) methods for comparison. In the MW method, 0.75 g of albumin was dissolved in 30 mL of water, and the albumin solution was heated in a domestic microwave oven (700 W) for 5 min. After that, the solution was cooled down to room temperature, and CDs suspension was obtained. The CDs solution was purified by centrifugation at 14,000 rpm for 15 min to remove precipitate. While in the UM method, 0.75 g of albumin was dissolved in 30 mL of water and then 5.0 mL of NaOH solution (1.0 mol/L) was added. The mixture was treated by ultrasound (180 W) for 5 h.
2.5. Biological imaging
2.6. Characterization Transmission electron microscopy (TEM) image of CDs was obtained by a JEOL JEM 2100 microscope. Dynamic light scattering (DLS) measurement was performed by using Zetasizer Nano ZS90 (Malvern, UK) to obtain CDs size. Crystal structure of CDs was studied by using X-ray diffraction (Rigaku, Japan). Fourier transform infrared (FT-IR) spectra of CDs were recorded on a Perkin Elmer (FT-IR spectrum BX, Germany). X-ray photoelectron spectroscopy (XPS, ESCALAB250Xi) was adopted for the measurement of elemental composition of CDs. UV2450 spectrophotometer (Hitachi, Japan) was used to measure UV– visible spectra of CDs. F900 fluorescence spectrometer (Edinburgh, UK) was adopted to conduct fluorescence spectra of CDs. Taking CDs as fluorescence labels, cell imaging was investigated under laser scanning confocal microscopy (Leica DM6000 CS). 3. Results and discussion
2.3. Quantum yield measurements The QY of CDs was calculated with the following equation:
ϕx ¼ ϕS
mx η 2x mS η 2S
ð1Þ
Quinine sulfate (QY = 54%) in 0.1 mol/L H2SO4 solution was regarded as the standard reference. In the equation, the subscripts S and X denote the standard and tested sample, respectively. ϕ is the QY, m is the slope from the plot of the integrated PL intensity vs. the absorbance of the sample or the standard at different concentrations, and η is the refractive index of the solvent. 2.4. Cytotoxicity assay 100 μL of human breast cancer Bcap-37 cell suspension was added to the well and incubated in a 5% CO2 humidified incubator at 37 °C for 24 h, which were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 1% penicillin streptomycin. And then, the culture medium was changed to fresh medium containing CDs (0.4–2.0 mg/mL) and cells were incubated in various CDs concentrations for 24 h. The culture medium was removed and phosphate buffered saline (PBS) was adopted to wash cells. 20 μL of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5.0 mg/mL) was added per well and incubated for another 4 h, so that violet colored formazan could be formed. After the culture medium was removed by employing MTT, 150 μL of dimethyl sulfoxide was added. After shaking the resulting mixture for 10 min, the mixture absorbance at 490 nm was measured through the application of Bio-Rad model-680 microplate reader. The cell viability was obtained by means of calculating the absorbance percentage relative to control
TEM and high-resolution TEM images of CDs are shown in Fig. 1a and Fig. S2, respectively. The CDs with spherical shape are excellent dispersion in aqueous solution, and the average diameter is 3.2 ± 1.1 nm (Fig. 1d). Average hydrodynamic diameter of the CDs is determined as 3.6 ± 1.5 nm by DLS, which is accorded with the result obtained by TEM in the experimental errors (in Fig. 1e). To verify reliability and efficiency of this method, CDs were synthesized by employing MW and UM methods and the same carbon source. The morphology and sizes of CDs are compared in Fig. 1. Average diameters of CDs prepared with UM and MW methods are 4.1 ± 2.8 nm and 13.4 ± 8.5 nm (Fig. 1b and c), respectively. Size distributions of CDs synthesized by different methods are shown in Fig 1d. The monodispersion of CDs prepared by AH method is much better than that synthesized by UM and MW methods. XRD was adopted to investigate crystallinity of the CDs, and there is a broad diffraction peak centered at about 2θ = 24° (Fig. 2a). Considering the interlayer space in graphite (0.34 nm), these CDs show a graphitic nature with highly disordered carbon atoms [4,29,30, 31]. FT-IR spectroscopy was employed to investigate the surface functional groups of CDs. In the FT-IR spectra of the CDs, there are respectively peaks at 3421, 2962, 2823, 1653 (1545), 1447 (1401) and 1239 (1076) cm−1 in the FT-IR spectra of CDs for O\\H/N\\H, C\\H, S\\H, C_O, C\\H/C\\N and C\\O/C\\S, as shown in Fig. 2b [21,22]. Besides, XPS was carried out to determine the elemental composition of CDs and the XPS spectrum presented four peaks of C1s, N1s, O1s and S2p (Fig. 2c), and which indicates that CDs are mainly composed of C, N, O and S. In addition, the high-resolution spectrum of C1s (Fig. 2d) displays four main peaks at 284.11, 284.56, 287.02 and 288.33 eV, which are attributed to C_C/C\\C, C\\N, C\\O and C_O/C_N bonds, respectively. Other elements (O1s, N1s and S2p) of the high-resolution spectrum are shown in Fig. S3. The graphitic structure (C_C/C\\C) of CDs was
732
X. Hu et al. / Materials Science and Engineering C 58 (2016) 730–736
Fig. 1. (a, b and c) TEM images of CDs obtained by AH, UM and MW methods, respectively. (d) Size distributions of CDs synthesized by different methods (TEM). (e) Size of CDs synthesized by AH method (DLS).
confirmed by the peak of binding energy at 284.11 eV and this was consistent with findings in XRD. In a word, these results reveal that the CDs surface may be provided with carbonyl unit, amide and hydroxyl groups. The surface hydrophilic groups not only significantly improve
the solubility and stability of CDs in an aqueous system, but also greatly promote applications in biology and medicine. For CDs synthesized by AH method, UV–visible absorption, fluorescence excitation and emission spectra are presented in Fig. 3a. Two
Fig. 2. (a) XRD pattern of CDs. (b) FT-IR spectrum of CDs. (c) XPS spectrum of CDs. (d) High-resolution C1s spectrum of CDs.
X. Hu et al. / Materials Science and Engineering C 58 (2016) 730–736
733
Fig. 3. (a) UV–visible absorption (Abs), fluorescence excitation (Ex) and emission (Em) spectra of CDs. (b) Normalized PL spectra at excitation wavelenths from 320 to 520 nm in a 20 nm increment. (c) PL spectra of CDs obtained by different methods at an excitation wavelength of 333 nm, taking albumin as a carbon source. (d) PL spectra of CDs obtained by AH method at an excitation wavelength of 333 nm, taking different carbon sources.
peaks occur at 218 and 276 nm, which are similar to that previously reported [30,32]. It is likely that the former peak (218 nm) is attributed to π–π* transition of aromatic sp2 domains, while the latter (276 nm) may be derived from n–π* transition of C_O band [30,33,34]. Besides, comparison between UV–visible absorption of CDs synthesized by different methods is conducted in Fig. S4. At an excitation wavelength of 333 nm, maximum emission wavelength of 412 nm was observed as indicated by green line in Fig. 3a. Moreover, the emission spectrum is characterized by good symmetry and narrow full width at half maximum (FWHM) of 87 nm. It may mean that the size distribution of CDs is narrow [22,35]. While the FWHMs of CDs prepared with UM and MW methods are 101 nm and 112 nm, respectively (Fig. S5). Among the three methods, AH method is probably the best one to obtain monodisperse CDs, and this is consistent with TEM analysis in Fig. 1d. The PL spectra of CDs were investigated under different excitation wavelengths (Fig. 3b) and the excitation-dependent emission wavelength behavior was observed. When excitation wavelengths changed from 320 to 520 nm, the emission wavelengths shifted from 405 to 576 nm correspondingly. The excitation-dependent emission behavior may be attributed to different particle sizes and a distribution of various emissive sites on CDs [4,30,34]. The excitation-dependent emission behavior could be useful in multicolor imaging applications [7]. A comparison of PL intensity of CDs synthesized by different methods is shown in Fig. 3c, and the results demonstrate that PL intensity of CDs obtained by AH method is stronger than those prepared by other methods. It is well known that carbon source plays a key role in CDs preparation and different carbon sources are required in different methods. In order to choose the optimum carbon source for AH method in CDs synthesis, PL intensities of CDs synthesized with various carbon sources are shown in Fig. 3d. When compared with other sources, the PL intensity of CDs using albumin as carbon source is better. Thus, albumin was considered as optimum carbon source for AH method in CDs preparation. In order to compare the QY of CDs prepared with various methods, the QY of CDs was measured by taking quinine sulfate as fluorescence
standard [6,22]. The QY of CDs prepared by AH, MW and UM methods are 16.8% ± 0.1%, 6.6% ± 0.1% and 2.3% ± 0.2%, respectively (details in Fig. S6 and Table S1). It is worth noticing that the QY of CDs prepared by AH method is significantly higher than that prepared by MW and UM methods. Furthermore, in the previous report, the QY of CDs prepared by AH method is higher than that prepared by the plasmainduced method with albumin being taken as the precursor [27]. The carbonyl groups of the CDs can serve as non-radiative electron–hole recombination centers. The higher QY of CDs synthesized by AH method probably originates from removing these carbonyl groups in NaOH solution [36], which results in the stabilization effect of excitons [30]. Moreover, gentle surface self-passivation process (about 80 h) is involved in AH method of synthesizing CDs [30]. However, the CDs preparation process using MW and UM methods is too fast to complete the passivation process. To probe the effect of pH value on PL intensity of CDs, photobleaching properties of CDs were studied in various pH value solutions. Surprisingly, high resistance to photobleaching of CDs is observed in pH range of values from 1 to 13 (Fig. S7), which means that CDs synthesized by AH method present an excellent photostability in various acidic/basic media. In addition, the pH-independent emission behavior has also been observed in CDs [27] and graphene quantum dots as well [37,38]. The behavior could be attributed to highly stable structure morphology of CDs [38]. Light irradiation (UV-lamp of 365 nm) was adopted to explore the photostability of CDs. The results suggest that PL intensity of CDs still maintains at about 76.6% after 20 h (Fig. S8), which indicates that the CDs possess better photostability. Besides, a similar result has been reported [39]. The radiative lifetime of emission is an important characteristic of light-emitting nanoparticles. Different radiative lifetimes may correspond to different electron–hole recombination mechanisms. Timeresolved, pulsed laser excitation techniques are most suitable for probing the lifetime. In order to further characterize the fluorescence origin of CDs, PL decay curves are analyzed and the results are shown
734
X. Hu et al. / Materials Science and Engineering C 58 (2016) 730–736
Fig. 4. PL decay and fitting curves of CDs under different emission wavelengths (excitation wavelength of 333 nm).
Table 1 Fitting parameters of the corresponding PL decay curve. λex/nm
λem/nm
τ1/ns
A1/%
τ2/ns
A2/%
τavg/ns
χ2
333
550 500 450 410
1.98 2.08 1.91 1.97
35.03 37.48 35.66 36.71
9.41 8.94 8.58 7.98
64.97 62.52 64.34 63.29
8.7 8.1 7.8 7.2
1.002 1.002 1.003 1.002
in Fig. 4 (details in Fig. S9). Based on a non-linear least square analysis, the decay trace is fitted through the application of biexponential functions I(t) with the equation below: Iðt Þ ¼ A1 exp −t τ1 þ A2 exp −t τ2
ð2Þ
where τ1 and τ2 represent the decay time constants, A1 and A2 are the normalized amplitude of the time-resolved decay lifetime of τ1 and τ2[6,40]. The average lifetime (τavg) of CDs was calculated with equation below: τavg ¼
A1 τ21 þ A2 τ 22 ðA1 τ1 þ A2 τ 2 Þ
ð3Þ
The biexponential behavior of lifetime can imply two different emissive sites (fast and slow decay components), which correspond to the recombination from intrinsic state and defect state, respectively [37, 41]. When compared with defect state, the emission originating from intrinsic state (sp2 carbon nano domains embedded in sp3 carbon matrix consist of carbogenic core) generally exhibits a shorter recombination lifetime [40,41,42]. The decay fitting results are listed in Table 1.
The short lifetime (τ1) of ~ 2.0 ns may be attributed to intrinsic state, while the long lifetime (τ2) of 8.0–9.0 ns may be corresponded to defect state. Moreover, the PL decay of CDs is demonstrated to depend on emission wavelength. With the increase of emission wavelength, short lifetime (τ1) does not change much, while long lifetime (τ2) is gradually increased, which indicates that the lifetime change could have roots in the defect state of CDs and it changes with emission wavelength. λexis the excitation wavelength, λemis the emission wavelength, χ2is the value of the goodness-of-fitting parameter. The possible forming mechanism of CDs is shown in Scheme 1. During the synthesis process of CDs, albumins were hydrolyzed into amino acids under strong alkaline condition [43,44]. In the system, the concentration of individual amino acids was increased sharply when albumins were fully hydrolyzed into individual amino acids. In the next step, amino acids act in situ as molecular precursors for carbon nanomaterials [5,45,46]. Consequently, organic aggregates were obtained by means of primary dehydration and polymerization of amino acids, and finally CDs were formed after intermolecular and intramolecular dehydration. MTT assay was carried out to evaluate the cytotoxicity of CDs [5,22, 30]. The Bcap-37 cell viability was tested in different CDs concentrations after incubation for 24 h (Fig. 5a). The cell viabilities are about 98.0%, 96.2%, and 94.0% for CDs concentrations of 0.4 mg/mL, 1.2 mg/mL and 2.0 mg/mL, respectively. It suggests that the CDs have good biocompatibility and extremely low cytotoxicity, and could be safe for in vivo applications. In order to extend the application potential of CDs as fluorescent imaging probes, CDs as in vivo cell imaging probes for Bcap-37 cell have been investigated. Through the application of confocal laser scanning microscopy, intracellular fluorescence is observed after cells are incubated with CDs. In the cell cytoplasm region, the bright blue and green fluorescences of CDs are observed at excitation wavelengths of 405 and 488 nm, respectively (Fig. 5c, d and S10). In addition, the excitation-dependent emission behavior of CDs was proved by biological imaging. The results clearly reveal that CDs are provided with good biocompatibility and can be effectively applied in multiple fluorescence emission for in vivo cell imaging and biological labeling.
4. Conclusions A new green method has been developed to synthesize fluorescent CDs using albumin as a carbon source at room temperature. This method is simple and convenient, and it is also energy, material and labor efficient. The PL mechanism of CDs was explored by making a timeresolved PL decay, which indicated that the PL originated from the emission of both defect state and intrinsic state. Besides, CDs were characterized by strong and tunable fluorescence, good monodispersion, superior solubility, excellent chemical stability and photostability, pHindependent emission, high QY and long fluorescence lifetime. Furthermore, CDs that are of low cytotoxicity and good biocompatibility could be considered as a potential biological imaging agent.
Scheme 1. Possible mechanism of forming CDs.
X. Hu et al. / Materials Science and Engineering C 58 (2016) 730–736
735
Fig. 5. (a) The effect of CDs on Bcap-37 cell viability (MTT assay). Confocal fluorescence microphotographs of CDs labeled Bcap-37 cells under bright field (b), at excitation wavelengths of 405 nm (c) and 488 nm (d). All scale bars are 20 μm.
Acknowledgments This research was supported by the National High-Tech Research and Development Plan of China (“863” plan, No.2011AA06A107) and the National Nature Science Foundation of China (21473055 and 21273073).
[6]
[7]
[8]
Appendix A. Supplementary data [9]
Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2015.09.066.
[10]
References
[11]
[1] M. Massey, M. Wu, E.M. Conroy, W.R. Algar, Mind your P's and Q's: the coming of age of semiconducting polymer dots and semiconductor quantum dots in biological applications, Curr. Opin. Biotechnol. 34 (2015) 30–40. [2] A. Mewada, S. Pandey, S. Shinde, N. Mishra, G. Oza, M. Thakur, M. Sharon, M. Sharon, Green synthesis of biocompatible carbon dots using aqueous extract of Trapa bispinosa peel, Mater. Sci. Eng. C 33 (2013) 2914–2917. [3] M. Geszke, M. Murias, L. Balan, G. Medjahdi, J. Korczynski, M. Moritz, J. Lulek, R. Schneider, Folic acid-conjugated core/shell ZnS: Mn/ZnS quantum dots as targeted probes for two photon fluorescence imaging of cancer cells, Acta Biomater. 7 (2011) 1327–1338. [4] A. Barati, M. Shamsipur, E. Arkan, L. Hosseinzadeh, H. Abdollahi, Synthesis of biocompatible and highly photoluminescent nitrogen doped carbon dots from lime: analytical applications and optimization using response surface methodology, Mater. Sci. Eng. C 47 (2015) 325–332. [5] W. Li, Z.H. Zhang, B. Kong, S.S. Feng, J.X. Wang, L.Z. Wang, J.P. Yang, F. Zhang, P.Y. Wu, D.Y. Zhao, Simple and green synthesis of nitrogen-doped photoluminescent
[12] [13] [14]
[15] [16]
[17]
carbonaceous nanospheres for biological imaging, Angew. Chem. Int. Ed. 52 (2013) 8151–8155. R.J. Fan, Q. Sun, L. Zhang, Y. Zhang, A.H. Lu, Photoluminescent carbon dots directly derived from polyethylene glycol and their application for cellular imaging, Carbon 71 (2014) 87–93. S.J. Zhu, Q.N. Meng, L. Wang, J.H. Zhang, Y.B. Song, H. Jin, K. Zhang, H.C. Sun, H.Y. Wang, B. Yang, Highly photoluminescent carbon dots for multicolor patterning, sensors, and biological imaging, Angew. Chem. Int. Ed. 52 (2013) 3953–3957. Y.Q. Dong, R.X. Wang, G.L. Li, C.Q. Chen, Y.W. Chi, G.N. Chen, Polyaminefunctionalized carbon quantum dots as fluorescent probes for selective and sensitive detection of copper ions, Anal. Chem. 48 (2012) 6220–6224. S.T. Yang, L. Cao, P.J.G. Luo, F.S. Lu, X. Wang, H.F. Wang, M.J. Meziani, Y.F. Liu, G. Qi, Y.P. Sun, Carbon dots for optical imaging in vivo, J. Am. Chem. Soc. 131 (2009) 11308–11309. P.J.G. Luo, S. Sahu, S.T. Yang, S.K. Sonkar, J.P. Wang, H.F. Wang, G.E. LeCroy, L. Cao, Y.P. Sun, Carbon “quantum” dots for optical biological imaging, J. Mater. Chem. B 1 (2013) 2116–2127. H.T. Li, X.D. He, Z.H. Kang, H. Huang, Y. Liu, J.L. Liu, S.Y. Lian, C.H.A. Tsang, X.B. Yang, S.T. Lee, Water-soluble fluorescent carbon quantum dots and photocatalyst design, Angew. Chem. Int. Ed. 49 (2010) 4430–4434. Q.L. Wang, X.X. Huang, Y.J. Long, X.L. Wang, H.J. Zhang, R. Zhu, L.P. Liang, P. Teng, H.Z. Zheng, Hollow luminescent carbon dots for drug delivery, Carbon 59 (2013) 192–199. F. Wang, Y.H. Chen, C.Y. Liu, D.G. Ma, White light-emitting devices based on carbon dots' electroluminescence, Chem. Commun. 47 (2011) 3502–3504. I.V. Lightcap, P.V. Kamat, Graphitic design: prospects of graphene-based nanocomposites for solar energy conversion, storage, and sensing, Accounts Chem. Res. 46 (2013) 2235–2243. S.L. Hu, K.Y. Niu, J. Sun, J. Yang, N.Q. Zhao, X.W. Du, One-step synthesis of fluorescent carbon nanoparticles by laser irradiation, J. Mater. Chem. 19 (2009) 484–488. X.Y. Xu, R. Ray, Y.L. Gu, H.J. Ploehn, L. Gearheart, K. Raker, W.A. Scrivens, Electrophoretic analysis and purification of fluorescent single-walled carbon nanotube fragments, J. Am. Chem. Soc. 126 (2004) 12736–12737. J.G. Zhou, C. Booker, R.Y. Li, X.T. Zhou, T.K. Sham, X.L. Sun, Z.F. Ding, An electrochemical avenue to blue luminescent nanocrystals from multi-walled carbon nanotubes (MWCNTs), J. Am. Chem. Soc. 129 (2007) 744–745.
736
X. Hu et al. / Materials Science and Engineering C 58 (2016) 730–736
[18] Z.A. Qiao, Y.F. Wang, Y. Gao, H.W. Li, T.Y. Dai, Y.L. Liu, Q.S. Huo, Commercially activated carbon as the source for producing multicolor photoluminescent carbon dots by chemical oxidation, Chem. Commun. 26 (2010) 8812–8814. [19] H.T. Li, X.D. He, Y. Liu, H. Huang, S.Y. Lian, S.T. Lee, Z.H. Kang, One-step ultrasonic synthesis of water-soluble carbon nanoparticles with excellent photoluminescent properties, Carbon 49 (2011) 605–609. [20] X.H. Wang, K.G. Qu, B.L. Xu, J.S. Ren, X.G. Qu, Microwave assisted one-step green synthesis of cell-permeable multicolor photoluminescent carbon dots without surface passivation reagents, J. Mater. Chem. 21 (2011) 2445–2450. [21] Y.S. Li, X.X. Zhong, A.E. Rider, S.A. Furman, K.(.K.). Ostrikov, Fast, energy-efficient synthesis of luminescent carbon quantum dots, Green Chem. 16 (2014) 2566–2570. [22] J. Gong, X.Q. An, X.J. Yan, A novel rapid and green synthesis of highly luminescent carbon dots with good biocompatibility for cell imaging, New J. Chem. 38 (2014) 1376–1379. [23] J. Lu, P.S.E. Yeo, C.K. Gan, P. Wu, K.P. Loh, Transforming C60 molecules into graphene quantum dots, Nat. Nanotechnol. 6 (2011) 247–252. [24] J.J. Gu, W.N. Wang, Q.H. Zhang, Z. Meng, X.D. Jia, K. Xi, Synthesis of fluorescent carbon nanoparticles from polyacrylamide for fast cellular endocytosis, RSC Adv. 3 (2013) 15589–15591. [25] L.H. Mao, W.Q. Tang, Z.Y. Deng, S.S. Liu, C.F. Wang, S. Chen, Facile access to white fluorescent carbon dots toward light-emitting devices, Ind. Eng. Chem. Res. 53 (2014) 6417–6425. [26] J.Y. Yin, H.J. Liu, S.Z. Jiang, Y. Chen, Y.F. Yao, Hyperbranched polymer functionalized carbon dots with multistimuli-responsive property, ACS Macro Lett. 2 (2013) 1033–1037. [27] J. Wang, C.F. Wang, S. Chen, Amphiphilic egg-derived carbon dots: rapid plasma fabrication, pyrolysis process, and multicolor printing patterns, Angew. Chem. Int. Ed. 51 (2012) 9297–9301. [28] S.S. Wee, Y.H. Ng, S.M. Ng, Synthesis of fluorescent carbon dots via simple acid hydrolysis of bovine serum albumin and its potential as sensitive sensing probe for lead (II) ions, Talanta 116 (2013) 71–76. [29] W.J. Wang, S. Chakrabarti, Z.G. Chen, Z.F. Yan, M.O. Tade, J. Zou, Q. Li, A novel bottom-up solvothermal synthesis of carbon nanosheets, J. Mater. Chem. A 2 (2014) 2390–2396. [30] L.L. Li, G.H. Wu, G.H. Yang, J. Peng, J.W. Zhao, J.J. Zhu, Focusing on luminescent graphene quantum dots: current status and future perspectives, Nanoscale 5 (2013) 4015–4039. [31] V.N. Mehta, S. Jha, S.K. Kailasa, One-pot green synthesis of carbon dots by using Saccharum officinarum juice for fluorescent imaging of bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae) cells, Mater. Sci. Eng. C 38 (2014) 20–27. [32] X.M. Wen, P. Yu, Y.R. Toh, X.Q. Ma, J. Tang, On the upconversion fluorescence in carbon nanodots and graphene quantum dots, Chem. Commun. 50 (2014) 4703–4706.
[33] A. Talib, S. Pandey, M. Thakur, H.F. Wu, Synthesis of highly fluorescent hydrophobic carbon dots by hot injection method using paraplast as precursor, Mater. Sci. Eng. C 48 (2015) 700–703. [34] Y. Wang, S. Kalytchuk, Y. Zhang, H.C. Shi, S.V. Kershaw, A.L. Rogach, Thickness dependent full-color emission tunability in a flexible carbon dot ionogel, J. Phys. Chem. Lett. 5 (2014) 1412–1420. [35] B. Zhang, C.Y. Liu, Y. Liu, A novel one-step approach to synthesize fluorescent carbon nanoparticles, Eur. J. Inorg. Chem. 4411-4414 (2010). [36] L.X. Zhao, F. Di, D.B. Wang, L.H. Guo, Y. Yang, B. Wan, H. Zhang, Chemiluminescence of carbon dots under strong alkaline solutions: a novel insight into carbon dot optical properties, Nanoscale 5 (2013) 2655–2658. [37] S.J. Zhu, J.H. Zhang, S.J. Tang, C.Y. Qiao, L. Wang, H.Y. Wang, X. Liu, B. Li, Y.F. Li, W.L. Yu, X.F. Wang, H.C. Sun, B. Yang, Surface chemistry routes to modulate the photoluminescence of graphene quantum dots: from fluorescence mechanism to up-conversion biological imaging applications, Adv. Funct. Mater. 22 (2012) 4732–4740. [38] P. Roy, A.P. Periasamy, C. Chuang, Y.R. Liou, Y.F. Chen, J. Joly, C.T. Liang, H.T. Chang, Plant leaf-derived graphene quantum dots and applications for white LEDs, New J. Chem. 38 (2014) 4946–4951. [39] Z. Zhang, J.H. Hao, J. Zhang, B.L. Zhang, J.L. Tang, Protein as the source for synthesizing fluorescent carbon dots by a one-pot hydrothermal route, RSC Adv. 2 (2012) 8599–8601. [40] X.Y. Zhang, Y. Zhang, Y. Wang, S. Kalytchuk, S.V. Kershaw, Y.H. Wang, P. Wang, T.Q. Zhang, Y. Zhao, H.Z. Zhang, T. Cui, Y.D. Wang, J. Zhao, W.W. Yu, A.L. Rogach, Colorswitchable electroluminescence of carbon dot light-emitting diodes, ACS Nano 7 (2013) 11234–11241. [41] F. Liu, M.H. Jang, H.D. Ha, J.H. Kim, Y.H. Cho, T.S. Seo, Facile synthetic method for pristine graphene quantum dots and graphene oxide quantum dots: origin of blue and green luminescence, Adv. Mater. 25 (2013) 3657–3662. [42] X.M. Wen, P. Yu, Y.R. Toh, X.T. Hao, J. Tang, Intrinsic and extrinsic fluorescence in carbon nanodots: ultrafast time-resolved fluorescence and carrier dynamics, Adv. Opt. Mater. 1 (2013) 173–178. [43] R.L. Levine, Rapid benchtop method of alkaline hydrolysis of proteins, J. Chromatogr. A 236 (1982) 499–502. [44] H.K. Nielsen, R.F. Hurrell, Tryptophan determination of food proteins by HPLC after alkaline hydrolysis, J. Sci. Food Agric. 36 (1985) 893–907. [45] M.M. Titirici, M. Antonietti, Chemistry and materials options of sustainable carbon materials made by hydrothermal carbonization, Chem. Soc. Rev. 39 (2010) 103–116. [46] B. Hu, K. Wang, L. Wu, S.H. Yu, M. Antonietti, M.M. Titirici, Engineering carbon materials from the hydrothermal carbonization process of biomass, Adv. Mater. 22 (2010) 813–828.
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Easy synthesis of highly fluorescent carbon dots from albumin and their photoluminescent mechanism and biological imaging applications Xiaohua Hu, Xueqin An ⁎, Lielie Li School of Chemistry and Molecular Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China
a r t i c l e
i n f o
Article history: Received 19 January 2015 Received in revised form 8 June 2015 Accepted 16 September 2015 Available online 18 September 2015 Keywords: Carbon dots Novel synthesis method Photoluminescent mechanism Biological imaging
a b s t r a c t A simple and green approach was developed to synthesize highly fluorescent carbon dots (CDs) using albumin as a carbon source in aqueous solution at room temperature. The CDs were characterized by excellent monodispersion, superior photostability, pH-independent emission, long fluorescence lifetime and high quantum yield (QY). The photoluminescent (PL) mechanism of CDs was explored by means of time-resolved PL decay, and the results revealed that PL originated from the emission of both defect state and intrinsic state. In addition, biological imaging with the application of CDs was carried out in human breast cancer Bcap-37 cell, which demonstrated that CDs were provided with an excellent biocompatiblity, low cytotoxicity and good transmembrane ability. Besides, CDs could be considered as a potential substitute for organic dyes or semiconductor quantum dots (SQDs) in biological imaging. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Fluorescence remains one of the most powerful and common tools for biological research [1]. As fluorescent materials, semiconductor quantum dots (SQDs) and organic dyes were employed in biological application during the past decade [2]. However, the photostability of organic dyes is poor, and SQDs with heavy metal ions may lead to potential cytotoxicity [2]. SQDs without cadmium are considered to be low toxicity, but their shortcomings of complicated synthesis and purification process are observed [3], which restrict their biotechnological and medical applications. With the advancement in fluorescent nanomaterials, photoluminescent carbon dots (PL CDs) as new nanoparticles are emerging, which possess unique properties such as low toxicity, good aqueous solubility, great biocompatibility, excellent optical performance and low photobleaching [4–6]. The CDs have been widely used in biochemical sensing [7], fluorescent probes [8], biological imaging [9,10], photocatalytic technology [11], drug carriers [12], light emitting devices [13], energy conversion/storage devices [14] and so on. So far, a number of approaches have been developed to prepare CDs, such as laser irradiation [15], arc discharge [16], electrochemical synthesis [17], hydrothermal method [5], chemical oxidation [18], ultrasonic treatment [19] and microwave synthesis [20]. However, complicated experimental condition and post-treatment process are involved in the majority of the methods. An energy efficient method to synthesize CDs has been reported utilizing saccharide as carbon source, but the
significant drawback is low quantum yield (1.2%) [21]. In our group, a combination method of hydrothermal and microwave digestion was used to synthesize CDs using ascorbic acid as carbon source [22], nevertheless expensive apparatus and complex procedures are required in the method. On the other hand, carbon source plays a significant role in CDs preparation. Currently, various carbon sources such as fullerene [23], polyacrylamide [24], polyethylene glycol [6], polyacrylic acid [25], polyethylenimine [26] and albumin [27,28], have been reported to synthesize CDs by different methods. Up to now, further investigations which could help understand CDs in depth are quite deficient. More facile methods are required because of the easy fluorescence quenching during multi-step chemical reactions. Some important information about CDs, such as the PL mechanism of CDs, cell imaging and biological labeling, is still rare. As a consequence, further investigations based on CDs are not only challenging, but also necessary. In this article, a novel one-step alkaline hydrolysis (AH) method is presented to fabricate CDs using albumin as source material in NaOH solution at room temperature. Comparisons among the morphology, size and optical properties of CDs prepared with different approaches were made. The PL mechanism of CDs was explored by means of time-resolved PL decay. Cytotoxicity, biocompatibility and transmembrane ability of the CDs were demonstrated.
2. Experimental 2.1. Materials
⁎ Corresponding author. E-mail address: [email protected] (X. An).
http://dx.doi.org/10.1016/j.msec.2015.09.066 0928-4931/© 2015 Elsevier B.V. All rights reserved.
Fresh eggs were purchased from the local market. Sodium hydroxide, polyacrylic acid, polyethylenimine, polyacrylamide, polyethylene
X. Hu et al. / Materials Science and Engineering C 58 (2016) 730–736
731
glycol and hydrochloric acid were purchased from Aladdin (Shanghai, China). Besides, all chemicals were of analytical grade, which could be applied without further purification. In addition, double deionized water was employed throughout all the experiments.
sample containing CDs. All experiments were conducted in triplicate and mean values were taken into consideration.
2.2. Synthesis of CDs
2.0 mL of human breast cancer Bcap-37 cells was seeded in a cover glass bottom dish and cultured at 37 °C for 24 h, which were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 1% penicillin streptomycin. And then, the culture medium was changed to 2.0 mL of fresh medium containing 2.0 mg/mL CDs and cells were incubated at 37 °C for 24 h. Finally, the culture medium was removed and cells were washed with PBS three times. Under a laser scanning confocal microscopy, cell imaging was conducted with laser excitation wavelengths of 405 and 488 nm with the collection of fluorescence in blue and green regions.
During a typical preparation, albumin (egg white) was well separated from egg with the aid of an egg separator and freeze-dried by means of a freeze drier, which served as a carbon source to produce CDs without any further purification. To prepare various CDs samples, 6.0 mL of albumin solution (25 g/L) was mixed with 1.0 mL of NaOH solution (1.0 mol/L), and then the mixture was incubated for different times at room temperature. After CDs preparation, remaining alkali was neutralized with HCl to adjust the pH of CDs solution to 7. Then the solution was purified to remove NaCl by dialysis for 24 h. The comparison between PL intensities of CDs was conducted under different incubation times in Fig. S1a, and the optimal incubation time was determined as 80 h. To determine optimum NaOH concentration (CNaOH), CDs were prepared in solutions of different CNaOH at room temperature for 80 h. The PL intensities of CDs prepared in different CNaOH were presented in Fig. S1b, and the optimal CNaOH was chosen as 1.0 mol/L. Taking albumin as a carbon source, CDs were synthesized by making use of microwave (MW) and ultrasonic manipulation (UM) methods for comparison. In the MW method, 0.75 g of albumin was dissolved in 30 mL of water, and the albumin solution was heated in a domestic microwave oven (700 W) for 5 min. After that, the solution was cooled down to room temperature, and CDs suspension was obtained. The CDs solution was purified by centrifugation at 14,000 rpm for 15 min to remove precipitate. While in the UM method, 0.75 g of albumin was dissolved in 30 mL of water and then 5.0 mL of NaOH solution (1.0 mol/L) was added. The mixture was treated by ultrasound (180 W) for 5 h.
2.5. Biological imaging
2.6. Characterization Transmission electron microscopy (TEM) image of CDs was obtained by a JEOL JEM 2100 microscope. Dynamic light scattering (DLS) measurement was performed by using Zetasizer Nano ZS90 (Malvern, UK) to obtain CDs size. Crystal structure of CDs was studied by using X-ray diffraction (Rigaku, Japan). Fourier transform infrared (FT-IR) spectra of CDs were recorded on a Perkin Elmer (FT-IR spectrum BX, Germany). X-ray photoelectron spectroscopy (XPS, ESCALAB250Xi) was adopted for the measurement of elemental composition of CDs. UV2450 spectrophotometer (Hitachi, Japan) was used to measure UV– visible spectra of CDs. F900 fluorescence spectrometer (Edinburgh, UK) was adopted to conduct fluorescence spectra of CDs. Taking CDs as fluorescence labels, cell imaging was investigated under laser scanning confocal microscopy (Leica DM6000 CS). 3. Results and discussion
2.3. Quantum yield measurements The QY of CDs was calculated with the following equation:
ϕx ¼ ϕS
mx η 2x mS η 2S
ð1Þ
Quinine sulfate (QY = 54%) in 0.1 mol/L H2SO4 solution was regarded as the standard reference. In the equation, the subscripts S and X denote the standard and tested sample, respectively. ϕ is the QY, m is the slope from the plot of the integrated PL intensity vs. the absorbance of the sample or the standard at different concentrations, and η is the refractive index of the solvent. 2.4. Cytotoxicity assay 100 μL of human breast cancer Bcap-37 cell suspension was added to the well and incubated in a 5% CO2 humidified incubator at 37 °C for 24 h, which were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 1% penicillin streptomycin. And then, the culture medium was changed to fresh medium containing CDs (0.4–2.0 mg/mL) and cells were incubated in various CDs concentrations for 24 h. The culture medium was removed and phosphate buffered saline (PBS) was adopted to wash cells. 20 μL of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5.0 mg/mL) was added per well and incubated for another 4 h, so that violet colored formazan could be formed. After the culture medium was removed by employing MTT, 150 μL of dimethyl sulfoxide was added. After shaking the resulting mixture for 10 min, the mixture absorbance at 490 nm was measured through the application of Bio-Rad model-680 microplate reader. The cell viability was obtained by means of calculating the absorbance percentage relative to control
TEM and high-resolution TEM images of CDs are shown in Fig. 1a and Fig. S2, respectively. The CDs with spherical shape are excellent dispersion in aqueous solution, and the average diameter is 3.2 ± 1.1 nm (Fig. 1d). Average hydrodynamic diameter of the CDs is determined as 3.6 ± 1.5 nm by DLS, which is accorded with the result obtained by TEM in the experimental errors (in Fig. 1e). To verify reliability and efficiency of this method, CDs were synthesized by employing MW and UM methods and the same carbon source. The morphology and sizes of CDs are compared in Fig. 1. Average diameters of CDs prepared with UM and MW methods are 4.1 ± 2.8 nm and 13.4 ± 8.5 nm (Fig. 1b and c), respectively. Size distributions of CDs synthesized by different methods are shown in Fig 1d. The monodispersion of CDs prepared by AH method is much better than that synthesized by UM and MW methods. XRD was adopted to investigate crystallinity of the CDs, and there is a broad diffraction peak centered at about 2θ = 24° (Fig. 2a). Considering the interlayer space in graphite (0.34 nm), these CDs show a graphitic nature with highly disordered carbon atoms [4,29,30, 31]. FT-IR spectroscopy was employed to investigate the surface functional groups of CDs. In the FT-IR spectra of the CDs, there are respectively peaks at 3421, 2962, 2823, 1653 (1545), 1447 (1401) and 1239 (1076) cm−1 in the FT-IR spectra of CDs for O\\H/N\\H, C\\H, S\\H, C_O, C\\H/C\\N and C\\O/C\\S, as shown in Fig. 2b [21,22]. Besides, XPS was carried out to determine the elemental composition of CDs and the XPS spectrum presented four peaks of C1s, N1s, O1s and S2p (Fig. 2c), and which indicates that CDs are mainly composed of C, N, O and S. In addition, the high-resolution spectrum of C1s (Fig. 2d) displays four main peaks at 284.11, 284.56, 287.02 and 288.33 eV, which are attributed to C_C/C\\C, C\\N, C\\O and C_O/C_N bonds, respectively. Other elements (O1s, N1s and S2p) of the high-resolution spectrum are shown in Fig. S3. The graphitic structure (C_C/C\\C) of CDs was
732
X. Hu et al. / Materials Science and Engineering C 58 (2016) 730–736
Fig. 1. (a, b and c) TEM images of CDs obtained by AH, UM and MW methods, respectively. (d) Size distributions of CDs synthesized by different methods (TEM). (e) Size of CDs synthesized by AH method (DLS).
confirmed by the peak of binding energy at 284.11 eV and this was consistent with findings in XRD. In a word, these results reveal that the CDs surface may be provided with carbonyl unit, amide and hydroxyl groups. The surface hydrophilic groups not only significantly improve
the solubility and stability of CDs in an aqueous system, but also greatly promote applications in biology and medicine. For CDs synthesized by AH method, UV–visible absorption, fluorescence excitation and emission spectra are presented in Fig. 3a. Two
Fig. 2. (a) XRD pattern of CDs. (b) FT-IR spectrum of CDs. (c) XPS spectrum of CDs. (d) High-resolution C1s spectrum of CDs.
X. Hu et al. / Materials Science and Engineering C 58 (2016) 730–736
733
Fig. 3. (a) UV–visible absorption (Abs), fluorescence excitation (Ex) and emission (Em) spectra of CDs. (b) Normalized PL spectra at excitation wavelenths from 320 to 520 nm in a 20 nm increment. (c) PL spectra of CDs obtained by different methods at an excitation wavelength of 333 nm, taking albumin as a carbon source. (d) PL spectra of CDs obtained by AH method at an excitation wavelength of 333 nm, taking different carbon sources.
peaks occur at 218 and 276 nm, which are similar to that previously reported [30,32]. It is likely that the former peak (218 nm) is attributed to π–π* transition of aromatic sp2 domains, while the latter (276 nm) may be derived from n–π* transition of C_O band [30,33,34]. Besides, comparison between UV–visible absorption of CDs synthesized by different methods is conducted in Fig. S4. At an excitation wavelength of 333 nm, maximum emission wavelength of 412 nm was observed as indicated by green line in Fig. 3a. Moreover, the emission spectrum is characterized by good symmetry and narrow full width at half maximum (FWHM) of 87 nm. It may mean that the size distribution of CDs is narrow [22,35]. While the FWHMs of CDs prepared with UM and MW methods are 101 nm and 112 nm, respectively (Fig. S5). Among the three methods, AH method is probably the best one to obtain monodisperse CDs, and this is consistent with TEM analysis in Fig. 1d. The PL spectra of CDs were investigated under different excitation wavelengths (Fig. 3b) and the excitation-dependent emission wavelength behavior was observed. When excitation wavelengths changed from 320 to 520 nm, the emission wavelengths shifted from 405 to 576 nm correspondingly. The excitation-dependent emission behavior may be attributed to different particle sizes and a distribution of various emissive sites on CDs [4,30,34]. The excitation-dependent emission behavior could be useful in multicolor imaging applications [7]. A comparison of PL intensity of CDs synthesized by different methods is shown in Fig. 3c, and the results demonstrate that PL intensity of CDs obtained by AH method is stronger than those prepared by other methods. It is well known that carbon source plays a key role in CDs preparation and different carbon sources are required in different methods. In order to choose the optimum carbon source for AH method in CDs synthesis, PL intensities of CDs synthesized with various carbon sources are shown in Fig. 3d. When compared with other sources, the PL intensity of CDs using albumin as carbon source is better. Thus, albumin was considered as optimum carbon source for AH method in CDs preparation. In order to compare the QY of CDs prepared with various methods, the QY of CDs was measured by taking quinine sulfate as fluorescence
standard [6,22]. The QY of CDs prepared by AH, MW and UM methods are 16.8% ± 0.1%, 6.6% ± 0.1% and 2.3% ± 0.2%, respectively (details in Fig. S6 and Table S1). It is worth noticing that the QY of CDs prepared by AH method is significantly higher than that prepared by MW and UM methods. Furthermore, in the previous report, the QY of CDs prepared by AH method is higher than that prepared by the plasmainduced method with albumin being taken as the precursor [27]. The carbonyl groups of the CDs can serve as non-radiative electron–hole recombination centers. The higher QY of CDs synthesized by AH method probably originates from removing these carbonyl groups in NaOH solution [36], which results in the stabilization effect of excitons [30]. Moreover, gentle surface self-passivation process (about 80 h) is involved in AH method of synthesizing CDs [30]. However, the CDs preparation process using MW and UM methods is too fast to complete the passivation process. To probe the effect of pH value on PL intensity of CDs, photobleaching properties of CDs were studied in various pH value solutions. Surprisingly, high resistance to photobleaching of CDs is observed in pH range of values from 1 to 13 (Fig. S7), which means that CDs synthesized by AH method present an excellent photostability in various acidic/basic media. In addition, the pH-independent emission behavior has also been observed in CDs [27] and graphene quantum dots as well [37,38]. The behavior could be attributed to highly stable structure morphology of CDs [38]. Light irradiation (UV-lamp of 365 nm) was adopted to explore the photostability of CDs. The results suggest that PL intensity of CDs still maintains at about 76.6% after 20 h (Fig. S8), which indicates that the CDs possess better photostability. Besides, a similar result has been reported [39]. The radiative lifetime of emission is an important characteristic of light-emitting nanoparticles. Different radiative lifetimes may correspond to different electron–hole recombination mechanisms. Timeresolved, pulsed laser excitation techniques are most suitable for probing the lifetime. In order to further characterize the fluorescence origin of CDs, PL decay curves are analyzed and the results are shown
734
X. Hu et al. / Materials Science and Engineering C 58 (2016) 730–736
Fig. 4. PL decay and fitting curves of CDs under different emission wavelengths (excitation wavelength of 333 nm).
Table 1 Fitting parameters of the corresponding PL decay curve. λex/nm
λem/nm
τ1/ns
A1/%
τ2/ns
A2/%
τavg/ns
χ2
333
550 500 450 410
1.98 2.08 1.91 1.97
35.03 37.48 35.66 36.71
9.41 8.94 8.58 7.98
64.97 62.52 64.34 63.29
8.7 8.1 7.8 7.2
1.002 1.002 1.003 1.002
in Fig. 4 (details in Fig. S9). Based on a non-linear least square analysis, the decay trace is fitted through the application of biexponential functions I(t) with the equation below: Iðt Þ ¼ A1 exp −t τ1 þ A2 exp −t τ2
ð2Þ
where τ1 and τ2 represent the decay time constants, A1 and A2 are the normalized amplitude of the time-resolved decay lifetime of τ1 and τ2[6,40]. The average lifetime (τavg) of CDs was calculated with equation below: τavg ¼
A1 τ21 þ A2 τ 22 ðA1 τ1 þ A2 τ 2 Þ
ð3Þ
The biexponential behavior of lifetime can imply two different emissive sites (fast and slow decay components), which correspond to the recombination from intrinsic state and defect state, respectively [37, 41]. When compared with defect state, the emission originating from intrinsic state (sp2 carbon nano domains embedded in sp3 carbon matrix consist of carbogenic core) generally exhibits a shorter recombination lifetime [40,41,42]. The decay fitting results are listed in Table 1.
The short lifetime (τ1) of ~ 2.0 ns may be attributed to intrinsic state, while the long lifetime (τ2) of 8.0–9.0 ns may be corresponded to defect state. Moreover, the PL decay of CDs is demonstrated to depend on emission wavelength. With the increase of emission wavelength, short lifetime (τ1) does not change much, while long lifetime (τ2) is gradually increased, which indicates that the lifetime change could have roots in the defect state of CDs and it changes with emission wavelength. λexis the excitation wavelength, λemis the emission wavelength, χ2is the value of the goodness-of-fitting parameter. The possible forming mechanism of CDs is shown in Scheme 1. During the synthesis process of CDs, albumins were hydrolyzed into amino acids under strong alkaline condition [43,44]. In the system, the concentration of individual amino acids was increased sharply when albumins were fully hydrolyzed into individual amino acids. In the next step, amino acids act in situ as molecular precursors for carbon nanomaterials [5,45,46]. Consequently, organic aggregates were obtained by means of primary dehydration and polymerization of amino acids, and finally CDs were formed after intermolecular and intramolecular dehydration. MTT assay was carried out to evaluate the cytotoxicity of CDs [5,22, 30]. The Bcap-37 cell viability was tested in different CDs concentrations after incubation for 24 h (Fig. 5a). The cell viabilities are about 98.0%, 96.2%, and 94.0% for CDs concentrations of 0.4 mg/mL, 1.2 mg/mL and 2.0 mg/mL, respectively. It suggests that the CDs have good biocompatibility and extremely low cytotoxicity, and could be safe for in vivo applications. In order to extend the application potential of CDs as fluorescent imaging probes, CDs as in vivo cell imaging probes for Bcap-37 cell have been investigated. Through the application of confocal laser scanning microscopy, intracellular fluorescence is observed after cells are incubated with CDs. In the cell cytoplasm region, the bright blue and green fluorescences of CDs are observed at excitation wavelengths of 405 and 488 nm, respectively (Fig. 5c, d and S10). In addition, the excitation-dependent emission behavior of CDs was proved by biological imaging. The results clearly reveal that CDs are provided with good biocompatibility and can be effectively applied in multiple fluorescence emission for in vivo cell imaging and biological labeling.
4. Conclusions A new green method has been developed to synthesize fluorescent CDs using albumin as a carbon source at room temperature. This method is simple and convenient, and it is also energy, material and labor efficient. The PL mechanism of CDs was explored by making a timeresolved PL decay, which indicated that the PL originated from the emission of both defect state and intrinsic state. Besides, CDs were characterized by strong and tunable fluorescence, good monodispersion, superior solubility, excellent chemical stability and photostability, pHindependent emission, high QY and long fluorescence lifetime. Furthermore, CDs that are of low cytotoxicity and good biocompatibility could be considered as a potential biological imaging agent.
Scheme 1. Possible mechanism of forming CDs.
X. Hu et al. / Materials Science and Engineering C 58 (2016) 730–736
735
Fig. 5. (a) The effect of CDs on Bcap-37 cell viability (MTT assay). Confocal fluorescence microphotographs of CDs labeled Bcap-37 cells under bright field (b), at excitation wavelengths of 405 nm (c) and 488 nm (d). All scale bars are 20 μm.
Acknowledgments This research was supported by the National High-Tech Research and Development Plan of China (“863” plan, No.2011AA06A107) and the National Nature Science Foundation of China (21473055 and 21273073).
[6]
[7]
[8]
Appendix A. Supplementary data [9]
Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2015.09.066.
[10]
References
[11]
[1] M. Massey, M. Wu, E.M. Conroy, W.R. Algar, Mind your P's and Q's: the coming of age of semiconducting polymer dots and semiconductor quantum dots in biological applications, Curr. Opin. Biotechnol. 34 (2015) 30–40. [2] A. Mewada, S. Pandey, S. Shinde, N. Mishra, G. Oza, M. Thakur, M. Sharon, M. Sharon, Green synthesis of biocompatible carbon dots using aqueous extract of Trapa bispinosa peel, Mater. Sci. Eng. C 33 (2013) 2914–2917. [3] M. Geszke, M. Murias, L. Balan, G. Medjahdi, J. Korczynski, M. Moritz, J. Lulek, R. Schneider, Folic acid-conjugated core/shell ZnS: Mn/ZnS quantum dots as targeted probes for two photon fluorescence imaging of cancer cells, Acta Biomater. 7 (2011) 1327–1338. [4] A. Barati, M. Shamsipur, E. Arkan, L. Hosseinzadeh, H. Abdollahi, Synthesis of biocompatible and highly photoluminescent nitrogen doped carbon dots from lime: analytical applications and optimization using response surface methodology, Mater. Sci. Eng. C 47 (2015) 325–332. [5] W. Li, Z.H. Zhang, B. Kong, S.S. Feng, J.X. Wang, L.Z. Wang, J.P. Yang, F. Zhang, P.Y. Wu, D.Y. Zhao, Simple and green synthesis of nitrogen-doped photoluminescent
[12] [13] [14]
[15] [16]
[17]
carbonaceous nanospheres for biological imaging, Angew. Chem. Int. Ed. 52 (2013) 8151–8155. R.J. Fan, Q. Sun, L. Zhang, Y. Zhang, A.H. Lu, Photoluminescent carbon dots directly derived from polyethylene glycol and their application for cellular imaging, Carbon 71 (2014) 87–93. S.J. Zhu, Q.N. Meng, L. Wang, J.H. Zhang, Y.B. Song, H. Jin, K. Zhang, H.C. Sun, H.Y. Wang, B. Yang, Highly photoluminescent carbon dots for multicolor patterning, sensors, and biological imaging, Angew. Chem. Int. Ed. 52 (2013) 3953–3957. Y.Q. Dong, R.X. Wang, G.L. Li, C.Q. Chen, Y.W. Chi, G.N. Chen, Polyaminefunctionalized carbon quantum dots as fluorescent probes for selective and sensitive detection of copper ions, Anal. Chem. 48 (2012) 6220–6224. S.T. Yang, L. Cao, P.J.G. Luo, F.S. Lu, X. Wang, H.F. Wang, M.J. Meziani, Y.F. Liu, G. Qi, Y.P. Sun, Carbon dots for optical imaging in vivo, J. Am. Chem. Soc. 131 (2009) 11308–11309. P.J.G. Luo, S. Sahu, S.T. Yang, S.K. Sonkar, J.P. Wang, H.F. Wang, G.E. LeCroy, L. Cao, Y.P. Sun, Carbon “quantum” dots for optical biological imaging, J. Mater. Chem. B 1 (2013) 2116–2127. H.T. Li, X.D. He, Z.H. Kang, H. Huang, Y. Liu, J.L. Liu, S.Y. Lian, C.H.A. Tsang, X.B. Yang, S.T. Lee, Water-soluble fluorescent carbon quantum dots and photocatalyst design, Angew. Chem. Int. Ed. 49 (2010) 4430–4434. Q.L. Wang, X.X. Huang, Y.J. Long, X.L. Wang, H.J. Zhang, R. Zhu, L.P. Liang, P. Teng, H.Z. Zheng, Hollow luminescent carbon dots for drug delivery, Carbon 59 (2013) 192–199. F. Wang, Y.H. Chen, C.Y. Liu, D.G. Ma, White light-emitting devices based on carbon dots' electroluminescence, Chem. Commun. 47 (2011) 3502–3504. I.V. Lightcap, P.V. Kamat, Graphitic design: prospects of graphene-based nanocomposites for solar energy conversion, storage, and sensing, Accounts Chem. Res. 46 (2013) 2235–2243. S.L. Hu, K.Y. Niu, J. Sun, J. Yang, N.Q. Zhao, X.W. Du, One-step synthesis of fluorescent carbon nanoparticles by laser irradiation, J. Mater. Chem. 19 (2009) 484–488. X.Y. Xu, R. Ray, Y.L. Gu, H.J. Ploehn, L. Gearheart, K. Raker, W.A. Scrivens, Electrophoretic analysis and purification of fluorescent single-walled carbon nanotube fragments, J. Am. Chem. Soc. 126 (2004) 12736–12737. J.G. Zhou, C. Booker, R.Y. Li, X.T. Zhou, T.K. Sham, X.L. Sun, Z.F. Ding, An electrochemical avenue to blue luminescent nanocrystals from multi-walled carbon nanotubes (MWCNTs), J. Am. Chem. Soc. 129 (2007) 744–745.
736
X. Hu et al. / Materials Science and Engineering C 58 (2016) 730–736
[18] Z.A. Qiao, Y.F. Wang, Y. Gao, H.W. Li, T.Y. Dai, Y.L. Liu, Q.S. Huo, Commercially activated carbon as the source for producing multicolor photoluminescent carbon dots by chemical oxidation, Chem. Commun. 26 (2010) 8812–8814. [19] H.T. Li, X.D. He, Y. Liu, H. Huang, S.Y. Lian, S.T. Lee, Z.H. Kang, One-step ultrasonic synthesis of water-soluble carbon nanoparticles with excellent photoluminescent properties, Carbon 49 (2011) 605–609. [20] X.H. Wang, K.G. Qu, B.L. Xu, J.S. Ren, X.G. Qu, Microwave assisted one-step green synthesis of cell-permeable multicolor photoluminescent carbon dots without surface passivation reagents, J. Mater. Chem. 21 (2011) 2445–2450. [21] Y.S. Li, X.X. Zhong, A.E. Rider, S.A. Furman, K.(.K.). Ostrikov, Fast, energy-efficient synthesis of luminescent carbon quantum dots, Green Chem. 16 (2014) 2566–2570. [22] J. Gong, X.Q. An, X.J. Yan, A novel rapid and green synthesis of highly luminescent carbon dots with good biocompatibility for cell imaging, New J. Chem. 38 (2014) 1376–1379. [23] J. Lu, P.S.E. Yeo, C.K. Gan, P. Wu, K.P. Loh, Transforming C60 molecules into graphene quantum dots, Nat. Nanotechnol. 6 (2011) 247–252. [24] J.J. Gu, W.N. Wang, Q.H. Zhang, Z. Meng, X.D. Jia, K. Xi, Synthesis of fluorescent carbon nanoparticles from polyacrylamide for fast cellular endocytosis, RSC Adv. 3 (2013) 15589–15591. [25] L.H. Mao, W.Q. Tang, Z.Y. Deng, S.S. Liu, C.F. Wang, S. Chen, Facile access to white fluorescent carbon dots toward light-emitting devices, Ind. Eng. Chem. Res. 53 (2014) 6417–6425. [26] J.Y. Yin, H.J. Liu, S.Z. Jiang, Y. Chen, Y.F. Yao, Hyperbranched polymer functionalized carbon dots with multistimuli-responsive property, ACS Macro Lett. 2 (2013) 1033–1037. [27] J. Wang, C.F. Wang, S. Chen, Amphiphilic egg-derived carbon dots: rapid plasma fabrication, pyrolysis process, and multicolor printing patterns, Angew. Chem. Int. Ed. 51 (2012) 9297–9301. [28] S.S. Wee, Y.H. Ng, S.M. Ng, Synthesis of fluorescent carbon dots via simple acid hydrolysis of bovine serum albumin and its potential as sensitive sensing probe for lead (II) ions, Talanta 116 (2013) 71–76. [29] W.J. Wang, S. Chakrabarti, Z.G. Chen, Z.F. Yan, M.O. Tade, J. Zou, Q. Li, A novel bottom-up solvothermal synthesis of carbon nanosheets, J. Mater. Chem. A 2 (2014) 2390–2396. [30] L.L. Li, G.H. Wu, G.H. Yang, J. Peng, J.W. Zhao, J.J. Zhu, Focusing on luminescent graphene quantum dots: current status and future perspectives, Nanoscale 5 (2013) 4015–4039. [31] V.N. Mehta, S. Jha, S.K. Kailasa, One-pot green synthesis of carbon dots by using Saccharum officinarum juice for fluorescent imaging of bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae) cells, Mater. Sci. Eng. C 38 (2014) 20–27. [32] X.M. Wen, P. Yu, Y.R. Toh, X.Q. Ma, J. Tang, On the upconversion fluorescence in carbon nanodots and graphene quantum dots, Chem. Commun. 50 (2014) 4703–4706.
[33] A. Talib, S. Pandey, M. Thakur, H.F. Wu, Synthesis of highly fluorescent hydrophobic carbon dots by hot injection method using paraplast as precursor, Mater. Sci. Eng. C 48 (2015) 700–703. [34] Y. Wang, S. Kalytchuk, Y. Zhang, H.C. Shi, S.V. Kershaw, A.L. Rogach, Thickness dependent full-color emission tunability in a flexible carbon dot ionogel, J. Phys. Chem. Lett. 5 (2014) 1412–1420. [35] B. Zhang, C.Y. Liu, Y. Liu, A novel one-step approach to synthesize fluorescent carbon nanoparticles, Eur. J. Inorg. Chem. 4411-4414 (2010). [36] L.X. Zhao, F. Di, D.B. Wang, L.H. Guo, Y. Yang, B. Wan, H. Zhang, Chemiluminescence of carbon dots under strong alkaline solutions: a novel insight into carbon dot optical properties, Nanoscale 5 (2013) 2655–2658. [37] S.J. Zhu, J.H. Zhang, S.J. Tang, C.Y. Qiao, L. Wang, H.Y. Wang, X. Liu, B. Li, Y.F. Li, W.L. Yu, X.F. Wang, H.C. Sun, B. Yang, Surface chemistry routes to modulate the photoluminescence of graphene quantum dots: from fluorescence mechanism to up-conversion biological imaging applications, Adv. Funct. Mater. 22 (2012) 4732–4740. [38] P. Roy, A.P. Periasamy, C. Chuang, Y.R. Liou, Y.F. Chen, J. Joly, C.T. Liang, H.T. Chang, Plant leaf-derived graphene quantum dots and applications for white LEDs, New J. Chem. 38 (2014) 4946–4951. [39] Z. Zhang, J.H. Hao, J. Zhang, B.L. Zhang, J.L. Tang, Protein as the source for synthesizing fluorescent carbon dots by a one-pot hydrothermal route, RSC Adv. 2 (2012) 8599–8601. [40] X.Y. Zhang, Y. Zhang, Y. Wang, S. Kalytchuk, S.V. Kershaw, Y.H. Wang, P. Wang, T.Q. Zhang, Y. Zhao, H.Z. Zhang, T. Cui, Y.D. Wang, J. Zhao, W.W. Yu, A.L. Rogach, Colorswitchable electroluminescence of carbon dot light-emitting diodes, ACS Nano 7 (2013) 11234–11241. [41] F. Liu, M.H. Jang, H.D. Ha, J.H. Kim, Y.H. Cho, T.S. Seo, Facile synthetic method for pristine graphene quantum dots and graphene oxide quantum dots: origin of blue and green luminescence, Adv. Mater. 25 (2013) 3657–3662. [42] X.M. Wen, P. Yu, Y.R. Toh, X.T. Hao, J. Tang, Intrinsic and extrinsic fluorescence in carbon nanodots: ultrafast time-resolved fluorescence and carrier dynamics, Adv. Opt. Mater. 1 (2013) 173–178. [43] R.L. Levine, Rapid benchtop method of alkaline hydrolysis of proteins, J. Chromatogr. A 236 (1982) 499–502. [44] H.K. Nielsen, R.F. Hurrell, Tryptophan determination of food proteins by HPLC after alkaline hydrolysis, J. Sci. Food Agric. 36 (1985) 893–907. [45] M.M. Titirici, M. Antonietti, Chemistry and materials options of sustainable carbon materials made by hydrothermal carbonization, Chem. Soc. Rev. 39 (2010) 103–116. [46] B. Hu, K. Wang, L. Wu, S.H. Yu, M. Antonietti, M.M. Titirici, Engineering carbon materials from the hydrothermal carbonization process of biomass, Adv. Mater. 22 (2010) 813–828.
