Materials Science and Engineering C 38 (2014) 20–27
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One-pot green synthesis of carbon dots by using Saccharum officinarum juice for fluorescent imaging of bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae) cells Vaibhavkumar N. Mehta a, Sanjay Jha b, Suresh Kumar Kailasa a,⁎ a b
Applied Chemistry Department, S. V. National Institute of Technology, Surat, 395 007, India Gujarat Agricultural Biotechnology Institute, Navsari Agricultural University, Surat, 395007, India
a r t i c l e
i n f o
Article history: Received 12 November 2013 Received in revised form 14 December 2013 Accepted 21 January 2014 Available online 29 January 2014 Keywords: Saccharum officinarum juice Fluorescence microscopy DLS TEM Bacteria and yeast cells
a b s t r a c t We are reporting highly economical plant-based hydrothermal method for one-pot green synthesis of waterdispersible fluorescent carbon dots (CDs) by using Saccharum officinarum juice as precursor. The synthesized CDs were characterized by UV-visible, fluorescence, Fourier transform infrared (FT-IR), dynamic light scattering (DLS), high-resolution transmission electron microscopic (HR-TEM), and laser scanning confocal microscopic techniques. The CDs are well dispersed in water with an average size of ~3 nm and showed bright blue fluorescence under UV-light (λex = 365 nm). These CDs acted as excellent fluorescent probes in cellular imaging of bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae). © 2014 Elsevier B.V. All rights reserved.
1. Introduction The monitoring of the biomolecules of real-time dynamics plays a key role in the translocation of molecules and interacts with partners in cell biology [1]. Recent breakthroughs of nanoscience in cell biology have provided new approaches to address and to solve problems in biological assays and in clinical diagnostics. Furthermore, the evaluation of subcellular structures by light microscopy is important in many fields, including cell biology and pathology diagnostics [2,3]. In this connection, two-photon fluorescence nanomaterials have been integrated in light microscopic methods for the visualization of cells in tissues with high resolutions [4,5]. Among the fluorescent nanomaterials, fluorescent carbon dots have received considerable interest in the field of optoelectronic devices and biocompatible imaging probes [6–8]. CDs are the new class of small oxygenous carbon nanoparticles with sizes b 10 nm, which exhibit excitation wavelength-dependent photoluminescence properties based on their displaying size. Importantly, CDs have several advantages over fluorescent semiconductor nanomaterials, such as excellent optical properties, high chemical stability, and low environmental hazard, making them promising light-emitting materials to visualize the cell in vitro and in vivo. The CDs showed superiority in chemical inertness and lower toxicity than the traditional quantum dots (QDs) [9]. However, the preparations of CDs involve the use of chemicals and several experimental setups to synthesize the CDs with multi-color ⁎ Corresponding author. Tel.: +91 261 2201730; fax: +91 261 2227334. E-mail addresses: [email protected], [email protected] (S.K. Kailasa). 0928-4931/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2014.01.038
emission. Literature survey reveals that a variety of synthetic routes such as arc-discharge [10], electrochemical oxidation [11,12], hydrothermal [13], laser ablation [14], microwave heating [15,16], combustion [9,17], and supported-synthesis [18] have been developed for the preparation of fluorescent CDs and used as imaging probes for metal ions and biomolecules. However, these approaches require expensive precursors and involve specific/sophisticated experimental setup/ condition and complex or post-treatment processes for the synthesis of fluorescent CDs. As cost is an important criterion in the success of fluorescent CDs, new green approaches are essentially needed to produce fluorescent CDs without the use of organic chemicals and tedious experimental setups. In recent years, several researchers have devoted their time on the preparation of fluorescent CD emitters without the use of organic chemicals in a single step, which is called as green chemistry concept [19,20]. To support this assumption, several green synthetic approaches have been developed for the preparation of CDs by using inexpensive renewable resources as the precursor. Briefly, Han's and Zou's groups developed green synthetic approach for the synthesis of water-soluble fluorescent CDs by using watermelon peel as raw resource at lowtemperature carbonization and used as live cell imaging probes [21]. Lu and co-workers [22] reported a simple, economical, and green preparative hydrothermal strategy for synthesis of water-soluble, fluorescent carbon CDs with a quantum yield of ~ 6.9% by using pomelo peel as a carbon source and used as fluorescent probes for detection of Hg2+. The same group described the use of flour as the carbon source for the microwave-assisted rapid green synthesis of photoluminescent
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CDs (1–4 nm) and used as a fluorescent sensor for the detection of metal ions [15]. Pandey's and Sharon's teams described the potential use of Indian water plant Trapa bispinosa peel for the preparation of luminescent water-soluble CDs (5–10 nm) with exceptionally biocompatible against MDCK cells [23]. The photoluminescent CDs with (26% quantum yield) have been prepared in a single step by the hydrothermal treatment of orange juice and used as fluorescent probes for cellular imaging [24]. Recently, luminescent CDs were prepared by using renewable resources such as jaggery, bread, and sugar at room temperature [25]. Apart from these methods, Li's group described a simple approach for the synthesis of fluorescent CDs by using gelatin as carbon source in the hydrothermal treatment and used as biocompatible materials for the visualization of cells [26]. Wang et al. [27] developed a one-pot fabrication approach for the synthesis of fluorescent CDs by using ascorbic acid at 90 °C. Koshizaki and co-workers [28] prepared photoluminescent CDs by using laser rapid passivation technique with ordinary organic solvents as carbon source. Similarly, Chang's group described a simple one-pot hydrothermal method for the preparation of photoluminescent CDs by using low-cost organic compounds as carbon sources and used as fluorescent probes for imaging of MCF-10A and MCF-7 cells [29]. Recently, Wei et al. [30–32] described a series of facile chemical routes for the preparation of highly water-dispersible fluorescent organic nanoparticles and their uses as promising fluorescent bioprobes for the visualization of cells in biological samples. With the inspiration of these achievements, we report a novel green chemistry synthetic approach for the preparation of water-soluble fluorescent CDs (~3 nm) in a single step by using the Saccharum officinarum (sugar cane) juice as a rich carbon source. The synthesized CDs were characterized by UV-visible, fluorescence, FT-IR, 1H- and 13C-NMR, DLS, and HR-TEM techniques. The synthesized CDs are well dispersed in water and exhibited strong visible fluorescence and up-conversion photoluminescence. These CDs acted multicolor-emitting probes for cell imaging of bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae).
2. Materials and methods
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2.3. Cell labeling assay E. coli DH5α cells were grown in Luria–Bertani (LB) medium (10 mg/mL Tryptone, 5 mg/mL Yeast Extract and 10 mg/mL NaCl pH 7.0) at 37 °C, 180 rpm. The diploid strain S. cerevisiae were grown in yeast extract-peptone-dextrose (YPD) medium (10 mg/mL yeast extract, 20 mg/mL Bacto Peptone, 20 mg/mL glucose, pH 6.5 ± 0.2) at 30 °C. The cultures were shaken at 150 rpm, and growth was monitored by measuring the absorbance at 600 nm. E. coli and S. cerevisiae cells were collected from the middle of the exponential growth phase and used for confocal microscopy analysis. For CD internalization studies, cells were fixed and the fixation was performed with 70% (v/v) ethanol at 4 °C for 5 min. The fixed cells were resuspended in 100 mM of phosphate buffer containing CDs (CDs: 40 μg/mL) at room temperature for 10 min. The stained cells were washed twice and subjected to confocal microscopy. Cell imaging was done under a Carl Zeiss 510 LSM laser scanning confocal microscope with a laser excitation of 405, 488, and 561 nm, and fluorescence was collected in blue, green, and red region. 2.4. Cytotoxicity study of CDs The growth curve of E. coli and S. cerevisiae was plotted by growing the cells in modified M9 and YNB (Yeast Nitrogen Base) medium in 150 mL Erlenmeyer flasks at 37 °C and 28 °C for 12–16 h. To these flasks, 0, 10, 20, 40, 100, 200, and 400 mg/mL of CD concentrations were added and incubated by using orbital shaker at 180 rpm. In order to know cell viability, a 2-mL culture broth was collected at different time intervals (0–24 h) and measured for their optical density (at 600 nm) using a spectrophotometer (CARY 50 UV, Australia).
2.5. Quantum yield measurement The quantum yield of the CDs was calculated by measuring the fluorescence intensity in aqueous dispersion by using the following equation [33],
2.1. Chemicals and materials S. officinarum juice was purchased (rupees 20 per 500 mL) from the local market of Surat, India. Ethanol, dichloromethane, acetone, sodium chloride, and glucose were purchased from Merck Ltd., India. All chemicals were of analytical grade and used without further purification. Milli-Q-purified water was used for sample preparations.
2.2. Synthesis of carbon dots from Saccharum officinarum juice The hydrothermal method was used for the preparation of waterdispersible CDs by using S. officinarum juice as carbon source. The water-dispersible fluorescent CDs were synthesized according to the reported method with minor modification [24]. Briefly, 350 mL of S. officinarum juice was mixed with 150 mL ethanol, and then the mixture was transferred into a 700-mL Teflon-lined stainless-steel autoclave. The reaction mixture was heated at constant temperature (120 °C) for 180 min until a dark brown solution formed. The autoclave was allowed to cool down naturally. The resulted dark brown solution was washed with dichloromethane to remove unreacted organic moieties. The aqueous solution was collected and centrifuged at 5000 rpm for 20 min to separate the less-fluorescent deposit. Excess acetone was added to the upper brown solution and centrifuged at 13,000 rpm for 15 min to obtain highly fluorescent CDs with ~2.5–3.0 nm. In order to know the optimum temperature for high efficient fluorescent CDs, we prepared the water-dispersible CDs at different temperatures 100 °C, 120 °C, and 140 °C, and the obtained CDs solutions were confirmed by UV-visible absorption and fluorescence emission spectra.
Q CD ¼ Q R
I CD AR η2CD IR ACD η2R
ð1Þ
where Q is the quantum yield, I is the intensity of luminescent spectra, A is the absorbance at exited wavelength, and η is the refractive index of the solvent used, using quinine sulfate (quantum yield 54%) in 0.1 M H2SO4 solution as the reference. The subscripts CD for carbon dots and R for reference are used in this equation. The quantum yield of CDs was measured at an excitation wavelength of 360 nm, and yield was found to be 5.76%.
2.6. Instrumentation UV-visible spectra were measured by using Maya Pro 2000 spectrophotometer (Ocean Optics, USA) at room temperature. The fluorescence spectra were recorded using an RF-5301 PC Shimadzu spectroflourometer. Fourier transform infrared (FT-IR) spectra were recorded on a Perkin Elmer (FT-IR spectrum BX, Germany). The morphology and the microstructure of the CDs were examined by HR-TEM on a JEOL 3010 with an accelerating voltage of 200 kV. The samples for HR-TEM were made by dropping an aqueous solution onto a 300-mesh copper grid coated with a lacy carbon film. The x-ray diffraction (XRD) profiles of the prepared samples were recorded on a Rigaku diffractometer (Rigaku, Japan) equipped with graphite monochromatized CuKα (λ = 0.15405 nm) radiation in the range from 10° to 70°. DLS measurements were performed by using Zetasizer Nano ZS90 (Malvern, UK).
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13000 rpm
Hydrothermal o
Extraction with DCM
120 C for 180 min Crude CDs
Centrifuge
CDs in aqueous layer
CDs in water CDs under UV light at 365 nm
Fig. 1. Schematic representation for the formation of carbon dots through the hydrothermal treatment of Saccharum officinarum juice.
3. Results and discussion 3.1. Characterization The multicolor emission CDs were prepared by using different carbon source materials, nucleation, and growth conditions. The carbon source material and conditions play a key role for the preparation of CDs with tunable emission and with high quantum yield. The color of S. officinarum juice was changed from green to dark brown after heating the juice at different temperatures 100 °C, 120 °C, and 140 °C for 180 min, confirming the formation of CDs. Fig. 1 shows the schematic representation for the preparation of CDs by using S. officinarum juice as carbon source. The carbonization of S. officinarum juice was
100
120
140
Temperature (oC)
100
120
140
Temperature (oC) at λex 302 nm
performed at different reaction temperatures from 100 °C to 140 °C. To confirm the best optimum temperature, the obtained CDs were placed on UV light and irradiated at excitation wavelengths 254, 302, and 365 nm (Fig. 2). It can be observed that the CDs showed intense bright blue color emission only at UV light excitation wavelength 365 nm, and the CDs did not exhibit any emission at excitation wavelengths 254 and 302 nm. As shown in Fig. 2, at 120 °C, carbonization solution was well dispersed with good brown color and exhibited bright blue color emission under UV light at the excitation wavelength of 365 nm compared with carbonizations at 100 °C and 140 °C, confirming that 120 °C is the best temperature for the preparation of water-dispersible CDs with bright blue emission. Fig. 3 shows the UVvisible absorption and emission spectra of CDs (1.0 mg/mL). The CDs
100
120
140
Temperature (oC) at λex 254 nm
100
120
140
Temperature (oC) at λex 365 nm
Fig. 2. Photographic images of synthesized CDs at different temperatures (100 °C, 120 °C, and 140 °C) and their fluorescence properties under UV light excitation wavelengths 254, 302, and 365 nm.
V.N. Mehta et al. / Materials Science and Engineering C 38 (2014) 20–27 UV-visible FL emission
474 nm
2.0
Absorbance
370 nm
1.5
15
1.0
10
0.5
5
0.0
Fluorescence intensity
20
0 300
400
500
600
700
Wavelength (nm) Fig. 3. UV-visible absorption and fluorescence emission spectra (λex = 390 nm) of CDs (1.0 mg/mL) synthesized at 120 °C. Inset picture shows the carbon dots under daylight (left) and UV light at 365 nm wavelength (right).
show the UV absorption peak at 370 nm, which is the characteristic peak for CDs by carbonization of renewable carbon source. This absorption peak confirms the carbon π system or the n–p* transition of the carbonyl [24,26,27].
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We also measured the UV-visible and fluorescent spectra of CDs at different carbonization temperatures (100 °C, 120 °C, and 140 °C). The well-water-dispersed CDs exhibit bright blue emission under UV light (λex = 365 nm), which could be easily observed with the naked eye (Inset in Fig. 3). The water-dispersed CDs showed a strong fluorescence emission band at 474 nm by exciting the CDs solution at 390 nm, which confirms that the formation of fluorescent CDs by using S. officinarum juice as carbon source in hydrothermal method. Supporting Information of Fig. S1 shows the UV-visible spectra of CDs at different temperatures of 100 °C, 120 °C, and 140 °C, respectively. Supporting Information of Fig. S2 shows the fluorescence emission spectra of CDs at different temperatures (100 °C, 120 °C, and 140 °C) under excitation wavelength 390 nm. It can be observed that the fluorescence emission intensity was gradually increased with increasing temperature from 100 °C to 120 °C, after that temperature the fluorescence emission intensity of CDs was decreased. Supporting Information of Fig. S3 reveals that excitation peaks are located at 390 and 410 nm, which indicates that the emission may be related to two kinds of transitions [26]. The fluorescence behavior of CDs is attributed to the presence of surface energy traps, which results to generate different emission colors through the surface passivation in nanosize CDs [14]. Therefore, we anticipate that this characteristic feature plays a key role to use them as multicoloremitting probes for cell imaging by using fluorescence microscope. Furthermore, the quantum yield of CDs is 5.67%, which is well comparable with the reported methods green synthetic approaches in the literature [29,34,35].
a
COO-
-C-H
C-O C=O C=C
C-O-C
-OH
b Intensity (a.u.)
(002) (101)
2 Theta Fig. 4. (a) FT-IR spectrum and (b) XRD pattern of CDs.
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We also studied FT-IR spectroscopy for the identification of functional groups present in CDs. As shown in Fig. 4a, the CDs exhibit peaks at 3379 and 2978 cm- 1 correspond to –OH and C-H groups stretching and bending vibrations, which is attributed to the presence of carbohydrates (sucrose and glucose) in S. officinarum juice. The peaks at 1045 and 1644 cm−1 belong to the stretching vibration, indicating that the existence of −C–O–C− and –C = C− groups. The characteristic absorption peak at 1235 cm− 1 was attributed due to the presence of –C–O stretching. The absorption peaks at 1693 and 1376 cm−1 correspond to –C = O vibration and symmetric carboxylate stretching. These are confirming that the aldoses/ketoses are oxidized during the carbonization of S. officinarum juice. Furthermore, we also studied XRD for the confirmation of structural properties of CDs. As shown in Fig. 4b, the CDs have exhibited the intense peak at 2θ = 22.72º (002) and a weak peak at 2θ = 42.60º (101), confirming the diffraction patterns of graphitic carbon in sugar cane juice, which is strongly agreed with reported methods [25–27]. Supporting Information of Figs. S4 and S5 show the 1H- and 13C-NMR spectra of CDs. The peaks at 2.0–3.0 ppm corresponded to methylene protons, and the peak at 8.2 ppm represents the protons of OH in CDs solution. Similarly, the 13C-NMR spectrum of CDs shows the peaks at 15–40 ppm correspond to aliphatic sp3 carbon atoms in CDs. The peaks at 97–105 ppm are attributed to O − C* = CH− and O − C = C*H− groups in CDs. In order to investigate the morphology and average size of synthesized CDs, we studied HR-TEM and DLS for the characterization of CDs. Fig. 5a–b shows the typical HR-TEM images of CDs at 20 and 10 nm scale bar, in which most of CDs are well dispersed in aqueous solution with uniform in size and possessed a nearly spherical shape with an average size of ~3 nm. Fig. 5c shows the DLS data of CDs, in which CDs are well monodispersed with an average hydrodynamic diameter of ~2.71 nm. These data are well agreed with the above HR-TEM images.
3.2. CDs as fluorescent imaging probes for bacteria (E. coli) and yeast (S. cerevisiae) Nanobiotechnologists have found ways to visualize cells in microorganisms by using CDs as fluorescent imaging probes. The CDs are fluorescent nanometer-scale crystals that consist of an electron-dense core composed of carbon in order to facilitate bioconjugation. The CDs are becoming increasingly popular as cellular probes for light microscopic imaging because of their unique optical and physical properties. These include high fluorescent quantum yield, resistance to photobleaching, a large absorption cross section, and the ability to precisely tune the fluorescence emission. To extend the application potential of CDs as fluorescent imaging probes, we have investigated the CDs as in vivo cell imaging probes for bacteria (E. coli) and yeast (S. cerevisiae). Fig. 6 shows the laser confocal fluorescence microscopic images of E. coli DH5α under different excitation wavelengths at 405, 488, and 561 nm laser in bright field with fluorescence mode and in only fluorescence mode by using CDs as fluorescent imaging probes. As shown in Fig. 6b–d, the confocal images of bacteria cells show obvious blue, green, and red emissions by using CDs (40 μg/mL) as fluorescent probe under bright field with fluorescence mode. We collected optical images at 10 μm in the z-direction at 408 nm (blue), 488 nm (green), and 561 nm (red) emission and images were shown in Fig. 6e–g. As depicted in Fig. 6, strong blue, green, and red fluorescence of E. coli cells can be seen after incubation with CDs for 1–6 h. Furthermore, we have also taken the confocal images of E. coli DH5α under different excitation wavelengths at 405, 488, and 561 nm laser in the fluorescence mode (without bright field) by using CDs as fluorescent imaging probes (Fig. 6e–g). These images indicate that CDs are widely dispersed and appeared in the membrane and cytoplasmic area of E. coli cells. Importantly, there are no CDs were found in cell membrane at both imaging modes (Fig. 6b–g).
a
b
20 nm
10 nm
c Number (%)
Average hydrodynamic diameter: ~ 2.71 nm
Size (d.nm) Fig. 5. HR-TEM images of carbon dots at different magnification (a) 20 nm and (b) 10 nm. (c) DLS measurement of CDs in aqueous solution.
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a
25
b
10 μm
10 μm
c
d
10 μm
10 μm
e
f
10 μm
10 μm
g
10 μm Fig. 6. Confocal laser microscopic images of Escherichia coli cells after incubation at 37 °C for 1–6 h (a) bright field without CDs and with bright field and fluorescence mode at excitation wavelengths (b) 405 (blue), (c) 488 (green), and (d) 561 (red) nm. Confocal laser microscopic images of CDs with E. coli at excitation wavelengths (e) 405 (blue), (f) 488 (green), and (g) 561 (red) nm, without bright field. Scale bar indicates 10 μm.
Fig. 7 shows the laser confocal fluorescence microscopic images of yeast (S. cerevisiae) by using CDs (40 μg/mL) as fluorescent imaging probes at 405, 488, and 561 nm. To this, we incubated yeast (S. cerevisiae) cells in CDs for 1–6 h and monitored by confocal fluorescence microscopy. As shown in Fig. 7b–d, the strong blue, green, and red fluorescence exhibited from CDs-labeled yeast cells upon the excitation of CDs-conjugated cells at 405, 488, and 561 nm in bright-field
fluorescence emission mode, whereas no fluorescence was observed from the control sample without the treatment of CDs. These results illustrate that the CDs were effectively up taken to the cells, which results a possible endocytosis mechanism through their homogeneous distributions inside the cell [29]. As can be seen in Fig. 7b–g, more than 90% of yeast cells are remained alive and exhibited intense blue, green, and red fluorescence, which demonstrates that the prepared CDs are
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a
b
10 μm
c
10 μm
10 μm
e
f
10 μm
d
10 μm
g
10 μm
10 μm
Fig. 7. Confocal laser microscopic images of Saccharomyces cerevisiae cells after incubation at 37 °C for 1–6 h (a) bright field without CDs and with bright field and fluorescence mode at excitation wavelengths (b) 405 (blue), (c) 488 (green), and (d) 561 (red) nm. Confocal laser microscopic images of CDs with S. cerevisiae at excitation wavelengths (e) 405 (blue), (f) 488 (green), and (g) 561 (red) nm, without bright field. Scale bar indicates 10 μm.
exhibited beautiful fluorescence colors with low cytotoxicity. To further confirm the localization of CDs inside the yeast nucleus, we have measured the confocal fluorescence microscopic images of yeast cells by using CDs as imaging probes at fluorescence mode only (without bright field) (Fig. 7e–g). It can be seen that CDs were preferentially well dispersed and located at the nucleus of yeast. These results successfully illustrate that CDs possess good biocompatibility and can be effectively applied multiple fluorescence emission for in vivo cell imaging and
a 0 mg/mL
OD
10 mg/mL 20 mg/mL 40 mg/mL 100 mg/mL 200 mg/mL 400 mg/mL
Time (h)
4. Conclusions
b 0 mg/mL 10 mg/mL 20 mg/mL
OD
biological labeling. Importantly, it has been observed that these results are quite different from the report of CdSe QDs as fluorescent imaging probes for yeasts [36]. As comparison of CDs with the biosynthesized CdSe QDs as fluorescent probes, the CDs acted as an effective fluorescent probes for in vivo cell imaging of yeast as well as bacteria with intense multiple colors (blue, green, and red). Based on the above observations, we assume that the highly water-dispersible CDs (~3 nm) in the yeast cells possibly involves extracellular growth, and a subsequent endocytosis pathway is responsible for in vivo imaging. Furthermore, we also studied the cytotoxicity of CDs in bacteria and yeast cells. Fig. 8 shows the cytotoxicity study of CDs in E. coli and S. cerevisiae by using CD concentration ranging from 0 to 400 mg/mL. These results indicate that CDs are exhibited non-toxicity up to 400 mg/mL for bacteria and yeast, which confirms that the CDs acted as eco-friendly biological fluorescent-labeling probes for cellular and in vivo imaging applications. It was found that 40 μg/mL of CDs is sufficient for the tagging of multiple cell of E. coli and S. cerevisiae. These results indicate that the multicolor CDs derived from S. officinarum juice have exhibited very good biocompatibility and acted as nontoxic good candidates for fluorescence imaging of wide variety cells (bacteria and yeast), which are alternative to semiconductor nanocrystals.
40 mg/mL 100 mg/mL 200 mg/mL 400 mg/mL
Time (h) Fig. 8. Cytotoxicity of CDs on (a) Escherichia coli and (b) Saccharomyces cerevisiae. The values are plotted as mean ± SD (n = 3).
In this paper, we described a novel, efficient and simple green synthetic approach for the preparation of highly fluorescent CDs by using S. officinarum juice as carbon source. The synthesized CDs exhibited an average size ~3 nm with intense bright blue color emission under UVlight, and these are characterized by UV-visible, fluorescence, FT-IR, and HR-TEM techniques. The synthesized CDs showed high fluorescence ability for in vitro cellular imaging of bacteria and yeast. As a result, CDs were successfully conjugated with cells of bacteria and yeast and showed different fluorescence colors (blue, green, and red) depending on the excitation wavelength. Therefore, the present method allows upward scalability in terms of one-pot green synthesis of fluorescent and biocompatible CDs, which can be used as fluorescent imaging probes for in vitro and in vivo bioimaging applications in cell biology and other sensing applications.
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Acknowledgements Authors thanks Prof. Z. V. P. Murthy, Chemical Engineering Department, S. V. National Institute of Technology, Surat, for providing DLS instrument facility to this work. We also thank the Department of Science and Technology for providing Maya Pro 2000 spectrophotometer (Ocean Optics, USA) under the Fast-Track Young Scientist Scheme (2011–2014). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2014.01.038. References [1] Y.P. Chang, F. Pinaud, J. Antelman, S. Weiss, J. Biophotonics 1 (2008) 287–298. [2] S. Kurabuchi, S. Tanaka, Cell Tissue Res. 288 (1997) 485–496. [3] B. Lontay, Z. Serfozo, P. Gergely, M. Ito, D.J. Hartshorne, F. Erdodi, J. Comp. Neurol. 478 (2004) 72–87. [4] F. Helmchen, W. Denk, Nat. Methods 2 (2005) 932–940. [5] D.R. Larson, W.R. Zipfel, R.M. Williams, S.W. Clark, M.P. Bruchez, F.W. Wise, W.W. Webb, Science 300 (2003) 1434–1436. [6] F. Wang, Y.H. Chen, C.Y. Liu, D.G. Ma, Chem. Commun. 47 (2011) 3502–3504. [7] 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, Angew. Chem. Int. Ed. 49 (2010) 4430–4434. [8] B.R. Selvi, D. Jagadeesan, B.S. Suma, G. Nagashankar, M. Arif, K. Balasubramanyam, M. Eswaramoorthy, T.K. Kundu, Nano Lett. 8 (2008) 3182–3188. [9] S.C. Ray, A. Saha, N.R. Jana, R. Sarkar, J. Phys. Chem. C 113 (2009) 18546–18551. [10] X. Xu, R. Ray, Y. Gu, H.J. Ploehn, L. Gearheart, K. Raker, W.A. Scrivens, J. Am. Chem. Soc. 126 (2004) 12736–12737. [11] J. Zhou, C. Booker, R. Li, X. Zhou, T.K. Sham, X. Sun, Z. Ding, J. Am. Chem. Soc. 129 (2007) 744–745.
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[12] L. Zheng, Y. Chi, Y. Dong, J. Lin, B. Wang, J. Am. Chem. Soc. 131 (2009) 4564–4565. [13] Y. Sha, J. Lou, S. Bai, D. Wu, B. Liu, Y. Ling, Mater. Res. Bull. 48 (2013) 1728–1731. [14] Y.P. Sun, B. Zhou, Y. Lin, W. Wang, K.A.S. Fernando, P. Pathak, M.J. Meziani, B.A. Harruff, X. Wang, H. Wang, P.G. Luo, H. Yang, M.E. Kose, B. Chen, L.M. Veca, S.Y. Xie, J. Am. Chem. Soc. 128 (2006) 7756–7757. [15] X. Qin, W. Lu, A.M. Asiri, A.O. Al-Youbi, X. Sun, Sens. Actuators B 184 (2013) 156–162. [16] H. Zhu, X. Wang, Y. Li, Z. Wang, F. Yang, X. Yang, Chem. Commun. 34 (2009) 5118–5120. [17] H. Liu, T. Ye, C. Mao, Angew. Chem. Int. Ed. 46 (2007) 6473–6475. [18] R. Liu, D. Wu, S. Liu, K. Koynov, W. Knoll, Q. Li, Angew. Chem. 121 (2009) 4668–4671. [19] 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. [20] Y. Fang, S. Guo, D. Li, C. Zhu, W. Ren, S. Dong, E. Wang, ACS Nano 6 (2011) 400–409. [21] J. Zhou, Z. Sheng, H. Han, M. Zou, C. Li, Mater. Lett. 66 (2012) 222–224. [22] W. Lu, X. Qin, S. Liu, G. Chang, Y. Zhang, Y. Luo, A.M. Asiri, A.O. Al-Youbi, X. Sun, Anal. Chem. 84 (2012) 5351–5357. [23] A. Mewada, S. Pandey, S. Shinde, N. Mishra, G. Oza, M. Thakur, M. Sharon, M. Sharon, Mater. Sci. Eng. C 33 (2013) 2914–2917. [24] S. Sahu, B. Behera, T. Maiti, S. Mohapatra, Chem. Commun. 48 (2012) 8835–8837. [25] S. Pandey, A. Mewada, G. Oza, M. Thakur, N. Mishra, M. Sharon, M. Sharon, Nanosci. Nanotechnol. Lett. 5 (2013) 775–779. [26] Q. Liang, W. Ma, Y. Shi, Z. Li, X. Yang, Carbon 60 (2013) 421–428. [27] X. Jia, J. Lia, E. Wang, Nanoscale 4 (2012) 5572–5575. [28] X. Li, H. Wang, Y. Shimizu, A. Pyatenko, K. Kawaguchi, N. Koshizaki, Chem. Commun. 47 (2011) 932–934. [29] P.C. Hsu, H.T. Chang, Chem. Commun. 48 (2012) 3984–3986. [30] X. Zhang, X. Zhang, S. Wang, M. Liu, L. Tao, Y. Wei, Nanoscale 5 (2013) 147–150. [31] X. Zhang, M. Liu, B. Yang, X. Zhang, Y. Wei, Colloids Surf. B: Biointerfaces 112 (2013) 81–86. [32] X. Zhang, M. Liu, B. Yang, X. Zhang, Z. Chi, S. Liu, J. Xu, Y. Wei, Polym. Chem. 4 (2013) 5060–5064. [33] J.R. Lakowicz, Principles of fluorescence spectroscopy, 2nd ed. Kluwer Academic/Plenum Publishers, New York, 1999. [34] H. Huang, J.J. Lv, D.L. Zhou, N. Bao, Y. Xu, A.J. Wang, J.J. Feng, RSC Adv. 3 (2013) 21691–21696. [35] B. De, N. Karak, RSC Adv. 3 (2013) 8286–8290. [36] H. Bao, N. Hao, Y. Yang, D. Zhao, Nano Res. 3 (2010) 481–489.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
One-pot green synthesis of carbon dots by using Saccharum officinarum juice for fluorescent imaging of bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae) cells Vaibhavkumar N. Mehta a, Sanjay Jha b, Suresh Kumar Kailasa a,⁎ a b
Applied Chemistry Department, S. V. National Institute of Technology, Surat, 395 007, India Gujarat Agricultural Biotechnology Institute, Navsari Agricultural University, Surat, 395007, India
a r t i c l e
i n f o
Article history: Received 12 November 2013 Received in revised form 14 December 2013 Accepted 21 January 2014 Available online 29 January 2014 Keywords: Saccharum officinarum juice Fluorescence microscopy DLS TEM Bacteria and yeast cells
a b s t r a c t We are reporting highly economical plant-based hydrothermal method for one-pot green synthesis of waterdispersible fluorescent carbon dots (CDs) by using Saccharum officinarum juice as precursor. The synthesized CDs were characterized by UV-visible, fluorescence, Fourier transform infrared (FT-IR), dynamic light scattering (DLS), high-resolution transmission electron microscopic (HR-TEM), and laser scanning confocal microscopic techniques. The CDs are well dispersed in water with an average size of ~3 nm and showed bright blue fluorescence under UV-light (λex = 365 nm). These CDs acted as excellent fluorescent probes in cellular imaging of bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae). © 2014 Elsevier B.V. All rights reserved.
1. Introduction The monitoring of the biomolecules of real-time dynamics plays a key role in the translocation of molecules and interacts with partners in cell biology [1]. Recent breakthroughs of nanoscience in cell biology have provided new approaches to address and to solve problems in biological assays and in clinical diagnostics. Furthermore, the evaluation of subcellular structures by light microscopy is important in many fields, including cell biology and pathology diagnostics [2,3]. In this connection, two-photon fluorescence nanomaterials have been integrated in light microscopic methods for the visualization of cells in tissues with high resolutions [4,5]. Among the fluorescent nanomaterials, fluorescent carbon dots have received considerable interest in the field of optoelectronic devices and biocompatible imaging probes [6–8]. CDs are the new class of small oxygenous carbon nanoparticles with sizes b 10 nm, which exhibit excitation wavelength-dependent photoluminescence properties based on their displaying size. Importantly, CDs have several advantages over fluorescent semiconductor nanomaterials, such as excellent optical properties, high chemical stability, and low environmental hazard, making them promising light-emitting materials to visualize the cell in vitro and in vivo. The CDs showed superiority in chemical inertness and lower toxicity than the traditional quantum dots (QDs) [9]. However, the preparations of CDs involve the use of chemicals and several experimental setups to synthesize the CDs with multi-color ⁎ Corresponding author. Tel.: +91 261 2201730; fax: +91 261 2227334. E-mail addresses: [email protected], [email protected] (S.K. Kailasa). 0928-4931/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2014.01.038
emission. Literature survey reveals that a variety of synthetic routes such as arc-discharge [10], electrochemical oxidation [11,12], hydrothermal [13], laser ablation [14], microwave heating [15,16], combustion [9,17], and supported-synthesis [18] have been developed for the preparation of fluorescent CDs and used as imaging probes for metal ions and biomolecules. However, these approaches require expensive precursors and involve specific/sophisticated experimental setup/ condition and complex or post-treatment processes for the synthesis of fluorescent CDs. As cost is an important criterion in the success of fluorescent CDs, new green approaches are essentially needed to produce fluorescent CDs without the use of organic chemicals and tedious experimental setups. In recent years, several researchers have devoted their time on the preparation of fluorescent CD emitters without the use of organic chemicals in a single step, which is called as green chemistry concept [19,20]. To support this assumption, several green synthetic approaches have been developed for the preparation of CDs by using inexpensive renewable resources as the precursor. Briefly, Han's and Zou's groups developed green synthetic approach for the synthesis of water-soluble fluorescent CDs by using watermelon peel as raw resource at lowtemperature carbonization and used as live cell imaging probes [21]. Lu and co-workers [22] reported a simple, economical, and green preparative hydrothermal strategy for synthesis of water-soluble, fluorescent carbon CDs with a quantum yield of ~ 6.9% by using pomelo peel as a carbon source and used as fluorescent probes for detection of Hg2+. The same group described the use of flour as the carbon source for the microwave-assisted rapid green synthesis of photoluminescent
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CDs (1–4 nm) and used as a fluorescent sensor for the detection of metal ions [15]. Pandey's and Sharon's teams described the potential use of Indian water plant Trapa bispinosa peel for the preparation of luminescent water-soluble CDs (5–10 nm) with exceptionally biocompatible against MDCK cells [23]. The photoluminescent CDs with (26% quantum yield) have been prepared in a single step by the hydrothermal treatment of orange juice and used as fluorescent probes for cellular imaging [24]. Recently, luminescent CDs were prepared by using renewable resources such as jaggery, bread, and sugar at room temperature [25]. Apart from these methods, Li's group described a simple approach for the synthesis of fluorescent CDs by using gelatin as carbon source in the hydrothermal treatment and used as biocompatible materials for the visualization of cells [26]. Wang et al. [27] developed a one-pot fabrication approach for the synthesis of fluorescent CDs by using ascorbic acid at 90 °C. Koshizaki and co-workers [28] prepared photoluminescent CDs by using laser rapid passivation technique with ordinary organic solvents as carbon source. Similarly, Chang's group described a simple one-pot hydrothermal method for the preparation of photoluminescent CDs by using low-cost organic compounds as carbon sources and used as fluorescent probes for imaging of MCF-10A and MCF-7 cells [29]. Recently, Wei et al. [30–32] described a series of facile chemical routes for the preparation of highly water-dispersible fluorescent organic nanoparticles and their uses as promising fluorescent bioprobes for the visualization of cells in biological samples. With the inspiration of these achievements, we report a novel green chemistry synthetic approach for the preparation of water-soluble fluorescent CDs (~3 nm) in a single step by using the Saccharum officinarum (sugar cane) juice as a rich carbon source. The synthesized CDs were characterized by UV-visible, fluorescence, FT-IR, 1H- and 13C-NMR, DLS, and HR-TEM techniques. The synthesized CDs are well dispersed in water and exhibited strong visible fluorescence and up-conversion photoluminescence. These CDs acted multicolor-emitting probes for cell imaging of bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae).
2. Materials and methods
21
2.3. Cell labeling assay E. coli DH5α cells were grown in Luria–Bertani (LB) medium (10 mg/mL Tryptone, 5 mg/mL Yeast Extract and 10 mg/mL NaCl pH 7.0) at 37 °C, 180 rpm. The diploid strain S. cerevisiae were grown in yeast extract-peptone-dextrose (YPD) medium (10 mg/mL yeast extract, 20 mg/mL Bacto Peptone, 20 mg/mL glucose, pH 6.5 ± 0.2) at 30 °C. The cultures were shaken at 150 rpm, and growth was monitored by measuring the absorbance at 600 nm. E. coli and S. cerevisiae cells were collected from the middle of the exponential growth phase and used for confocal microscopy analysis. For CD internalization studies, cells were fixed and the fixation was performed with 70% (v/v) ethanol at 4 °C for 5 min. The fixed cells were resuspended in 100 mM of phosphate buffer containing CDs (CDs: 40 μg/mL) at room temperature for 10 min. The stained cells were washed twice and subjected to confocal microscopy. Cell imaging was done under a Carl Zeiss 510 LSM laser scanning confocal microscope with a laser excitation of 405, 488, and 561 nm, and fluorescence was collected in blue, green, and red region. 2.4. Cytotoxicity study of CDs The growth curve of E. coli and S. cerevisiae was plotted by growing the cells in modified M9 and YNB (Yeast Nitrogen Base) medium in 150 mL Erlenmeyer flasks at 37 °C and 28 °C for 12–16 h. To these flasks, 0, 10, 20, 40, 100, 200, and 400 mg/mL of CD concentrations were added and incubated by using orbital shaker at 180 rpm. In order to know cell viability, a 2-mL culture broth was collected at different time intervals (0–24 h) and measured for their optical density (at 600 nm) using a spectrophotometer (CARY 50 UV, Australia).
2.5. Quantum yield measurement The quantum yield of the CDs was calculated by measuring the fluorescence intensity in aqueous dispersion by using the following equation [33],
2.1. Chemicals and materials S. officinarum juice was purchased (rupees 20 per 500 mL) from the local market of Surat, India. Ethanol, dichloromethane, acetone, sodium chloride, and glucose were purchased from Merck Ltd., India. All chemicals were of analytical grade and used without further purification. Milli-Q-purified water was used for sample preparations.
2.2. Synthesis of carbon dots from Saccharum officinarum juice The hydrothermal method was used for the preparation of waterdispersible CDs by using S. officinarum juice as carbon source. The water-dispersible fluorescent CDs were synthesized according to the reported method with minor modification [24]. Briefly, 350 mL of S. officinarum juice was mixed with 150 mL ethanol, and then the mixture was transferred into a 700-mL Teflon-lined stainless-steel autoclave. The reaction mixture was heated at constant temperature (120 °C) for 180 min until a dark brown solution formed. The autoclave was allowed to cool down naturally. The resulted dark brown solution was washed with dichloromethane to remove unreacted organic moieties. The aqueous solution was collected and centrifuged at 5000 rpm for 20 min to separate the less-fluorescent deposit. Excess acetone was added to the upper brown solution and centrifuged at 13,000 rpm for 15 min to obtain highly fluorescent CDs with ~2.5–3.0 nm. In order to know the optimum temperature for high efficient fluorescent CDs, we prepared the water-dispersible CDs at different temperatures 100 °C, 120 °C, and 140 °C, and the obtained CDs solutions were confirmed by UV-visible absorption and fluorescence emission spectra.
Q CD ¼ Q R
I CD AR η2CD IR ACD η2R
ð1Þ
where Q is the quantum yield, I is the intensity of luminescent spectra, A is the absorbance at exited wavelength, and η is the refractive index of the solvent used, using quinine sulfate (quantum yield 54%) in 0.1 M H2SO4 solution as the reference. The subscripts CD for carbon dots and R for reference are used in this equation. The quantum yield of CDs was measured at an excitation wavelength of 360 nm, and yield was found to be 5.76%.
2.6. Instrumentation UV-visible spectra were measured by using Maya Pro 2000 spectrophotometer (Ocean Optics, USA) at room temperature. The fluorescence spectra were recorded using an RF-5301 PC Shimadzu spectroflourometer. Fourier transform infrared (FT-IR) spectra were recorded on a Perkin Elmer (FT-IR spectrum BX, Germany). The morphology and the microstructure of the CDs were examined by HR-TEM on a JEOL 3010 with an accelerating voltage of 200 kV. The samples for HR-TEM were made by dropping an aqueous solution onto a 300-mesh copper grid coated with a lacy carbon film. The x-ray diffraction (XRD) profiles of the prepared samples were recorded on a Rigaku diffractometer (Rigaku, Japan) equipped with graphite monochromatized CuKα (λ = 0.15405 nm) radiation in the range from 10° to 70°. DLS measurements were performed by using Zetasizer Nano ZS90 (Malvern, UK).
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13000 rpm
Hydrothermal o
Extraction with DCM
120 C for 180 min Crude CDs
Centrifuge
CDs in aqueous layer
CDs in water CDs under UV light at 365 nm
Fig. 1. Schematic representation for the formation of carbon dots through the hydrothermal treatment of Saccharum officinarum juice.
3. Results and discussion 3.1. Characterization The multicolor emission CDs were prepared by using different carbon source materials, nucleation, and growth conditions. The carbon source material and conditions play a key role for the preparation of CDs with tunable emission and with high quantum yield. The color of S. officinarum juice was changed from green to dark brown after heating the juice at different temperatures 100 °C, 120 °C, and 140 °C for 180 min, confirming the formation of CDs. Fig. 1 shows the schematic representation for the preparation of CDs by using S. officinarum juice as carbon source. The carbonization of S. officinarum juice was
100
120
140
Temperature (oC)
100
120
140
Temperature (oC) at λex 302 nm
performed at different reaction temperatures from 100 °C to 140 °C. To confirm the best optimum temperature, the obtained CDs were placed on UV light and irradiated at excitation wavelengths 254, 302, and 365 nm (Fig. 2). It can be observed that the CDs showed intense bright blue color emission only at UV light excitation wavelength 365 nm, and the CDs did not exhibit any emission at excitation wavelengths 254 and 302 nm. As shown in Fig. 2, at 120 °C, carbonization solution was well dispersed with good brown color and exhibited bright blue color emission under UV light at the excitation wavelength of 365 nm compared with carbonizations at 100 °C and 140 °C, confirming that 120 °C is the best temperature for the preparation of water-dispersible CDs with bright blue emission. Fig. 3 shows the UVvisible absorption and emission spectra of CDs (1.0 mg/mL). The CDs
100
120
140
Temperature (oC) at λex 254 nm
100
120
140
Temperature (oC) at λex 365 nm
Fig. 2. Photographic images of synthesized CDs at different temperatures (100 °C, 120 °C, and 140 °C) and their fluorescence properties under UV light excitation wavelengths 254, 302, and 365 nm.
V.N. Mehta et al. / Materials Science and Engineering C 38 (2014) 20–27 UV-visible FL emission
474 nm
2.0
Absorbance
370 nm
1.5
15
1.0
10
0.5
5
0.0
Fluorescence intensity
20
0 300
400
500
600
700
Wavelength (nm) Fig. 3. UV-visible absorption and fluorescence emission spectra (λex = 390 nm) of CDs (1.0 mg/mL) synthesized at 120 °C. Inset picture shows the carbon dots under daylight (left) and UV light at 365 nm wavelength (right).
show the UV absorption peak at 370 nm, which is the characteristic peak for CDs by carbonization of renewable carbon source. This absorption peak confirms the carbon π system or the n–p* transition of the carbonyl [24,26,27].
23
We also measured the UV-visible and fluorescent spectra of CDs at different carbonization temperatures (100 °C, 120 °C, and 140 °C). The well-water-dispersed CDs exhibit bright blue emission under UV light (λex = 365 nm), which could be easily observed with the naked eye (Inset in Fig. 3). The water-dispersed CDs showed a strong fluorescence emission band at 474 nm by exciting the CDs solution at 390 nm, which confirms that the formation of fluorescent CDs by using S. officinarum juice as carbon source in hydrothermal method. Supporting Information of Fig. S1 shows the UV-visible spectra of CDs at different temperatures of 100 °C, 120 °C, and 140 °C, respectively. Supporting Information of Fig. S2 shows the fluorescence emission spectra of CDs at different temperatures (100 °C, 120 °C, and 140 °C) under excitation wavelength 390 nm. It can be observed that the fluorescence emission intensity was gradually increased with increasing temperature from 100 °C to 120 °C, after that temperature the fluorescence emission intensity of CDs was decreased. Supporting Information of Fig. S3 reveals that excitation peaks are located at 390 and 410 nm, which indicates that the emission may be related to two kinds of transitions [26]. The fluorescence behavior of CDs is attributed to the presence of surface energy traps, which results to generate different emission colors through the surface passivation in nanosize CDs [14]. Therefore, we anticipate that this characteristic feature plays a key role to use them as multicoloremitting probes for cell imaging by using fluorescence microscope. Furthermore, the quantum yield of CDs is 5.67%, which is well comparable with the reported methods green synthetic approaches in the literature [29,34,35].
a
COO-
-C-H
C-O C=O C=C
C-O-C
-OH
b Intensity (a.u.)
(002) (101)
2 Theta Fig. 4. (a) FT-IR spectrum and (b) XRD pattern of CDs.
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We also studied FT-IR spectroscopy for the identification of functional groups present in CDs. As shown in Fig. 4a, the CDs exhibit peaks at 3379 and 2978 cm- 1 correspond to –OH and C-H groups stretching and bending vibrations, which is attributed to the presence of carbohydrates (sucrose and glucose) in S. officinarum juice. The peaks at 1045 and 1644 cm−1 belong to the stretching vibration, indicating that the existence of −C–O–C− and –C = C− groups. The characteristic absorption peak at 1235 cm− 1 was attributed due to the presence of –C–O stretching. The absorption peaks at 1693 and 1376 cm−1 correspond to –C = O vibration and symmetric carboxylate stretching. These are confirming that the aldoses/ketoses are oxidized during the carbonization of S. officinarum juice. Furthermore, we also studied XRD for the confirmation of structural properties of CDs. As shown in Fig. 4b, the CDs have exhibited the intense peak at 2θ = 22.72º (002) and a weak peak at 2θ = 42.60º (101), confirming the diffraction patterns of graphitic carbon in sugar cane juice, which is strongly agreed with reported methods [25–27]. Supporting Information of Figs. S4 and S5 show the 1H- and 13C-NMR spectra of CDs. The peaks at 2.0–3.0 ppm corresponded to methylene protons, and the peak at 8.2 ppm represents the protons of OH in CDs solution. Similarly, the 13C-NMR spectrum of CDs shows the peaks at 15–40 ppm correspond to aliphatic sp3 carbon atoms in CDs. The peaks at 97–105 ppm are attributed to O − C* = CH− and O − C = C*H− groups in CDs. In order to investigate the morphology and average size of synthesized CDs, we studied HR-TEM and DLS for the characterization of CDs. Fig. 5a–b shows the typical HR-TEM images of CDs at 20 and 10 nm scale bar, in which most of CDs are well dispersed in aqueous solution with uniform in size and possessed a nearly spherical shape with an average size of ~3 nm. Fig. 5c shows the DLS data of CDs, in which CDs are well monodispersed with an average hydrodynamic diameter of ~2.71 nm. These data are well agreed with the above HR-TEM images.
3.2. CDs as fluorescent imaging probes for bacteria (E. coli) and yeast (S. cerevisiae) Nanobiotechnologists have found ways to visualize cells in microorganisms by using CDs as fluorescent imaging probes. The CDs are fluorescent nanometer-scale crystals that consist of an electron-dense core composed of carbon in order to facilitate bioconjugation. The CDs are becoming increasingly popular as cellular probes for light microscopic imaging because of their unique optical and physical properties. These include high fluorescent quantum yield, resistance to photobleaching, a large absorption cross section, and the ability to precisely tune the fluorescence emission. To extend the application potential of CDs as fluorescent imaging probes, we have investigated the CDs as in vivo cell imaging probes for bacteria (E. coli) and yeast (S. cerevisiae). Fig. 6 shows the laser confocal fluorescence microscopic images of E. coli DH5α under different excitation wavelengths at 405, 488, and 561 nm laser in bright field with fluorescence mode and in only fluorescence mode by using CDs as fluorescent imaging probes. As shown in Fig. 6b–d, the confocal images of bacteria cells show obvious blue, green, and red emissions by using CDs (40 μg/mL) as fluorescent probe under bright field with fluorescence mode. We collected optical images at 10 μm in the z-direction at 408 nm (blue), 488 nm (green), and 561 nm (red) emission and images were shown in Fig. 6e–g. As depicted in Fig. 6, strong blue, green, and red fluorescence of E. coli cells can be seen after incubation with CDs for 1–6 h. Furthermore, we have also taken the confocal images of E. coli DH5α under different excitation wavelengths at 405, 488, and 561 nm laser in the fluorescence mode (without bright field) by using CDs as fluorescent imaging probes (Fig. 6e–g). These images indicate that CDs are widely dispersed and appeared in the membrane and cytoplasmic area of E. coli cells. Importantly, there are no CDs were found in cell membrane at both imaging modes (Fig. 6b–g).
a
b
20 nm
10 nm
c Number (%)
Average hydrodynamic diameter: ~ 2.71 nm
Size (d.nm) Fig. 5. HR-TEM images of carbon dots at different magnification (a) 20 nm and (b) 10 nm. (c) DLS measurement of CDs in aqueous solution.
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a
25
b
10 μm
10 μm
c
d
10 μm
10 μm
e
f
10 μm
10 μm
g
10 μm Fig. 6. Confocal laser microscopic images of Escherichia coli cells after incubation at 37 °C for 1–6 h (a) bright field without CDs and with bright field and fluorescence mode at excitation wavelengths (b) 405 (blue), (c) 488 (green), and (d) 561 (red) nm. Confocal laser microscopic images of CDs with E. coli at excitation wavelengths (e) 405 (blue), (f) 488 (green), and (g) 561 (red) nm, without bright field. Scale bar indicates 10 μm.
Fig. 7 shows the laser confocal fluorescence microscopic images of yeast (S. cerevisiae) by using CDs (40 μg/mL) as fluorescent imaging probes at 405, 488, and 561 nm. To this, we incubated yeast (S. cerevisiae) cells in CDs for 1–6 h and monitored by confocal fluorescence microscopy. As shown in Fig. 7b–d, the strong blue, green, and red fluorescence exhibited from CDs-labeled yeast cells upon the excitation of CDs-conjugated cells at 405, 488, and 561 nm in bright-field
fluorescence emission mode, whereas no fluorescence was observed from the control sample without the treatment of CDs. These results illustrate that the CDs were effectively up taken to the cells, which results a possible endocytosis mechanism through their homogeneous distributions inside the cell [29]. As can be seen in Fig. 7b–g, more than 90% of yeast cells are remained alive and exhibited intense blue, green, and red fluorescence, which demonstrates that the prepared CDs are
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a
b
10 μm
c
10 μm
10 μm
e
f
10 μm
d
10 μm
g
10 μm
10 μm
Fig. 7. Confocal laser microscopic images of Saccharomyces cerevisiae cells after incubation at 37 °C for 1–6 h (a) bright field without CDs and with bright field and fluorescence mode at excitation wavelengths (b) 405 (blue), (c) 488 (green), and (d) 561 (red) nm. Confocal laser microscopic images of CDs with S. cerevisiae at excitation wavelengths (e) 405 (blue), (f) 488 (green), and (g) 561 (red) nm, without bright field. Scale bar indicates 10 μm.
exhibited beautiful fluorescence colors with low cytotoxicity. To further confirm the localization of CDs inside the yeast nucleus, we have measured the confocal fluorescence microscopic images of yeast cells by using CDs as imaging probes at fluorescence mode only (without bright field) (Fig. 7e–g). It can be seen that CDs were preferentially well dispersed and located at the nucleus of yeast. These results successfully illustrate that CDs possess good biocompatibility and can be effectively applied multiple fluorescence emission for in vivo cell imaging and
a 0 mg/mL
OD
10 mg/mL 20 mg/mL 40 mg/mL 100 mg/mL 200 mg/mL 400 mg/mL
Time (h)
4. Conclusions
b 0 mg/mL 10 mg/mL 20 mg/mL
OD
biological labeling. Importantly, it has been observed that these results are quite different from the report of CdSe QDs as fluorescent imaging probes for yeasts [36]. As comparison of CDs with the biosynthesized CdSe QDs as fluorescent probes, the CDs acted as an effective fluorescent probes for in vivo cell imaging of yeast as well as bacteria with intense multiple colors (blue, green, and red). Based on the above observations, we assume that the highly water-dispersible CDs (~3 nm) in the yeast cells possibly involves extracellular growth, and a subsequent endocytosis pathway is responsible for in vivo imaging. Furthermore, we also studied the cytotoxicity of CDs in bacteria and yeast cells. Fig. 8 shows the cytotoxicity study of CDs in E. coli and S. cerevisiae by using CD concentration ranging from 0 to 400 mg/mL. These results indicate that CDs are exhibited non-toxicity up to 400 mg/mL for bacteria and yeast, which confirms that the CDs acted as eco-friendly biological fluorescent-labeling probes for cellular and in vivo imaging applications. It was found that 40 μg/mL of CDs is sufficient for the tagging of multiple cell of E. coli and S. cerevisiae. These results indicate that the multicolor CDs derived from S. officinarum juice have exhibited very good biocompatibility and acted as nontoxic good candidates for fluorescence imaging of wide variety cells (bacteria and yeast), which are alternative to semiconductor nanocrystals.
40 mg/mL 100 mg/mL 200 mg/mL 400 mg/mL
Time (h) Fig. 8. Cytotoxicity of CDs on (a) Escherichia coli and (b) Saccharomyces cerevisiae. The values are plotted as mean ± SD (n = 3).
In this paper, we described a novel, efficient and simple green synthetic approach for the preparation of highly fluorescent CDs by using S. officinarum juice as carbon source. The synthesized CDs exhibited an average size ~3 nm with intense bright blue color emission under UVlight, and these are characterized by UV-visible, fluorescence, FT-IR, and HR-TEM techniques. The synthesized CDs showed high fluorescence ability for in vitro cellular imaging of bacteria and yeast. As a result, CDs were successfully conjugated with cells of bacteria and yeast and showed different fluorescence colors (blue, green, and red) depending on the excitation wavelength. Therefore, the present method allows upward scalability in terms of one-pot green synthesis of fluorescent and biocompatible CDs, which can be used as fluorescent imaging probes for in vitro and in vivo bioimaging applications in cell biology and other sensing applications.
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Acknowledgements Authors thanks Prof. Z. V. P. Murthy, Chemical Engineering Department, S. V. National Institute of Technology, Surat, for providing DLS instrument facility to this work. We also thank the Department of Science and Technology for providing Maya Pro 2000 spectrophotometer (Ocean Optics, USA) under the Fast-Track Young Scientist Scheme (2011–2014). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2014.01.038. References [1] Y.P. Chang, F. Pinaud, J. Antelman, S. Weiss, J. Biophotonics 1 (2008) 287–298. [2] S. Kurabuchi, S. Tanaka, Cell Tissue Res. 288 (1997) 485–496. [3] B. Lontay, Z. Serfozo, P. Gergely, M. Ito, D.J. Hartshorne, F. Erdodi, J. Comp. Neurol. 478 (2004) 72–87. [4] F. Helmchen, W. Denk, Nat. Methods 2 (2005) 932–940. [5] D.R. Larson, W.R. Zipfel, R.M. Williams, S.W. Clark, M.P. Bruchez, F.W. Wise, W.W. Webb, Science 300 (2003) 1434–1436. [6] F. Wang, Y.H. Chen, C.Y. Liu, D.G. Ma, Chem. Commun. 47 (2011) 3502–3504. [7] 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, Angew. Chem. Int. Ed. 49 (2010) 4430–4434. [8] B.R. Selvi, D. Jagadeesan, B.S. Suma, G. Nagashankar, M. Arif, K. Balasubramanyam, M. Eswaramoorthy, T.K. Kundu, Nano Lett. 8 (2008) 3182–3188. [9] S.C. Ray, A. Saha, N.R. Jana, R. Sarkar, J. Phys. Chem. C 113 (2009) 18546–18551. [10] X. Xu, R. Ray, Y. Gu, H.J. Ploehn, L. Gearheart, K. Raker, W.A. Scrivens, J. Am. Chem. Soc. 126 (2004) 12736–12737. [11] J. Zhou, C. Booker, R. Li, X. Zhou, T.K. Sham, X. Sun, Z. Ding, J. Am. Chem. Soc. 129 (2007) 744–745.
27
[12] L. Zheng, Y. Chi, Y. Dong, J. Lin, B. Wang, J. Am. Chem. Soc. 131 (2009) 4564–4565. [13] Y. Sha, J. Lou, S. Bai, D. Wu, B. Liu, Y. Ling, Mater. Res. Bull. 48 (2013) 1728–1731. [14] Y.P. Sun, B. Zhou, Y. Lin, W. Wang, K.A.S. Fernando, P. Pathak, M.J. Meziani, B.A. Harruff, X. Wang, H. Wang, P.G. Luo, H. Yang, M.E. Kose, B. Chen, L.M. Veca, S.Y. Xie, J. Am. Chem. Soc. 128 (2006) 7756–7757. [15] X. Qin, W. Lu, A.M. Asiri, A.O. Al-Youbi, X. Sun, Sens. Actuators B 184 (2013) 156–162. [16] H. Zhu, X. Wang, Y. Li, Z. Wang, F. Yang, X. Yang, Chem. Commun. 34 (2009) 5118–5120. [17] H. Liu, T. Ye, C. Mao, Angew. Chem. Int. Ed. 46 (2007) 6473–6475. [18] R. Liu, D. Wu, S. Liu, K. Koynov, W. Knoll, Q. Li, Angew. Chem. 121 (2009) 4668–4671. [19] 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. [20] Y. Fang, S. Guo, D. Li, C. Zhu, W. Ren, S. Dong, E. Wang, ACS Nano 6 (2011) 400–409. [21] J. Zhou, Z. Sheng, H. Han, M. Zou, C. Li, Mater. Lett. 66 (2012) 222–224. [22] W. Lu, X. Qin, S. Liu, G. Chang, Y. Zhang, Y. Luo, A.M. Asiri, A.O. Al-Youbi, X. Sun, Anal. Chem. 84 (2012) 5351–5357. [23] A. Mewada, S. Pandey, S. Shinde, N. Mishra, G. Oza, M. Thakur, M. Sharon, M. Sharon, Mater. Sci. Eng. C 33 (2013) 2914–2917. [24] S. Sahu, B. Behera, T. Maiti, S. Mohapatra, Chem. Commun. 48 (2012) 8835–8837. [25] S. Pandey, A. Mewada, G. Oza, M. Thakur, N. Mishra, M. Sharon, M. Sharon, Nanosci. Nanotechnol. Lett. 5 (2013) 775–779. [26] Q. Liang, W. Ma, Y. Shi, Z. Li, X. Yang, Carbon 60 (2013) 421–428. [27] X. Jia, J. Lia, E. Wang, Nanoscale 4 (2012) 5572–5575. [28] X. Li, H. Wang, Y. Shimizu, A. Pyatenko, K. Kawaguchi, N. Koshizaki, Chem. Commun. 47 (2011) 932–934. [29] P.C. Hsu, H.T. Chang, Chem. Commun. 48 (2012) 3984–3986. [30] X. Zhang, X. Zhang, S. Wang, M. Liu, L. Tao, Y. Wei, Nanoscale 5 (2013) 147–150. [31] X. Zhang, M. Liu, B. Yang, X. Zhang, Y. Wei, Colloids Surf. B: Biointerfaces 112 (2013) 81–86. [32] X. Zhang, M. Liu, B. Yang, X. Zhang, Z. Chi, S. Liu, J. Xu, Y. Wei, Polym. Chem. 4 (2013) 5060–5064. [33] J.R. Lakowicz, Principles of fluorescence spectroscopy, 2nd ed. Kluwer Academic/Plenum Publishers, New York, 1999. [34] H. Huang, J.J. Lv, D.L. Zhou, N. Bao, Y. Xu, A.J. Wang, J.J. Feng, RSC Adv. 3 (2013) 21691–21696. [35] B. De, N. Karak, RSC Adv. 3 (2013) 8286–8290. [36] H. Bao, N. Hao, Y. Yang, D. Zhao, Nano Res. 3 (2010) 481–489.
