Research article Received: 8 April 2014,
Revised: 8 October 2014,
Accepted: 19 November 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.2834
The preparation of ethylenediamine-modified fluorescent carbon dots and their use in imaging of cells Wei Dong,a* Siqi Zhou,b Yan Dong,c Jingwen Wang,a Xin Gea and Lili Suia ABSTRACT: In this work, fluorescent carbon dots (CDs) were synthesized using a hydrothermal method with glucose as the carbon source and were surface-modified with ethylenediamine. The properties of as-prepared CDs were analyzed by transmission electron microscopy (TEM), Fourier transform infrared (FTIR), ultraviolet–visible light (UV/vis) absorption and fluorescent spectra. Furthermore, CDs conjugated with mouse anti-(human carcinoembryonic antigen) (CEA) monoclonal antibody were successful employed in the biolabeling and fluorescent imaging of human gastric carcinoma cells. In addition, the cytotoxicity of CDs was also tested using human gastric carcinoma cells. There was no apparent cytotoxicity on human gastric carcinoma cells. These results suggest the potential application of the as-prepared CDs in bioimaging and related fields. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: carbon dots; ethylenediamine; cell imaging; cytotoxicity
Introduction Fluorescence semiconductor quantum dots (QDs), such as CdTe, CdS and CdSe, have attracted much attention due to their particular optical properties as fluorescence probes. However, the known toxicity of heavy metals, for example cadmium, the main ingredient of QDs, and severe synthetic and environmental hazards have limited their bio-application (1,2). Recently, carbon dots (CDs) have attracted intense attention due to their unique advantages. CDs are usually carbon nanocrystals of 2–10 nm in diameter, which not only exhibit many excellent optical properties, such as photoinduced electron transfer, photoluminescence (PL), electrochemiluminescence (ECL) and chemiluminescence (CL), but also present some additional advantages over luminescent semiconductor QDs, such as good biocompatibility and low toxicity (3–6). In recent years, various methods have been developed to obtain CDs including: oxidation by a strong oxidant, electrochemical methods, laser ablation and microwave heating (7–11). Although all these CDs prepared as above showed great PL, many drawbacks, such as long and complex processes, strict experimental conditions and producing CDs with low quantum yields (QYs) limit their application in the life sciences. Thus, a rapid, convenient and efficient method for the synthesis of CDs remains a challenge. To date, passivation has been a popular approach to enhance the fluorescence of CDs (12). Several organic polymers and diamine compounds, such as polyethylene glycol) (PEG) (13–16), polyethyleneimine (PEI) (17), citrate and 4-aminoantipyrine (18,19) have been used to passivate CDs. All these CDs showed high photostability, tunable excitation and emission wavelength, good biocompatibility and low toxicity, and could be applied in the imaging of normal and cancer cells. In this work, we report a facile, economic and green synthesis route towards photoluminescent CDs, resulting in a quantum yield of 3.0%. The CDs were synthesized by a hydrothermal
Luminescence 2015
method with glucose as the carbon source and modified with ethylenediamine (EDA). The as-prepared dots were linked with mouse anti-(human CEA) antibody to form an immunoprobe. The CDs–antibody probe was successfully used to label human gastric carcinoma cells (MGC-803) via the specific reaction between antibody and antigen. Compared with other routes, the direct labeling procedure was relatively simpler. The cytotoxicity of CDs was also tested using gastric carcinoma cells. It was observed that none of the cell lines show any statistically significant differences in cell survival within 48 h at a CD concentration of 0–160 μg/ml. All these exciting results indicated that CD-based probes are ideal fluorescent markers with excellent spectral properties, photostability and biocompatibility.
Experimental Materials Glucose, PEG 400 and EDA (99%) were obtained from Sinopharm Chemical Reagent Co. Ltd (Shenyang, China). Mouse anti-(human CEA) was purchased from Beijing Biosynthesis Biotechnology Co. (Beijing, China). N-Hydroxysuccinimide (NHS) was obtained from Fluka (New York, USA). Dulbecco’s modified Eagle’s medium/Ham′s * Correspondence to: W. Dong, Department of Chemistry, Shenyang Medical college, Shenyang 110034, people’s Republic of China. E-mail: [email protected] a
Department of Chemistry, Shenyang Medical College, Shenyang, People’s Republic of China
b
ICU, Fengtian Hospital Affiliated to Shenyang Medical College, Shenyang, People’s Republic of China
c
Experiment Center of Traditional Chinese Medicine Department, Shenyang Pharmaceutical University, People’s Republic of China
Copyright © 2015 John Wiley & Sons, Ltd.
W. Dong et al. F12 media (DMEM/F12) was obtained from Gibco (New York, USA). Cell counting kit-8 (CCK-8) was obtained from Shenyang Tongren Institute of Chemistry (Shenyang, China). All other reagents were of analytical reagent grade. The water used in all experiments was prepared using a quartzose double-distilling system. Gastric carcinoma cells (MGC-803) used in the study were obtained from the Department of Pharmacology of Shenyang Medical College. Preparation of fluorescent CDs.
CDs were synthesized according to a procedure described previously (20). Briefly, 540 mg of glucose was thoroughly mixed with 25 ml of PEG/distilled water (1: 1, v/v/) in a high-pressure nitrifying pot and heated at 180 °C for 3 h. After cooling to room temperature naturally, 100 μl EDA was added to the system. The mixture was then removed to the high-pressure nitrifying pot for passivation at 120 °C for 12 h. Finally, the dark yellow solution of luminescent CDs was passed through a 0.22 μm filter membrane to remove the larger product, acetone was added to the solution and the CDs were collected by centrifugation at 3500 r.p.m. for 15 min. The precipitation was dried a under nitrogen flow and dispersed in distilled water to obtain a clear solution for further experiments. The absorption and emission spectra, TEM images and FTIR spectra of CDs were recorded. The water-soluble CDs were conjugated with CEA using NHS as a cross-linker. Typically, 100 μl of mouse anti-(human CEA) antibody (1 mg/ml) in 100 μl of PBS (10 mmol/L) was mixed with 50 μl of as-prepared CDs (0.4 mg/ml) and incubated in an incubator shaker for 1.5 h at 37 °C after being activated by 50 μl of a mixture of 500 mmol/l EDC and 50 mmol/l NHS for 10 min at room temperature to form CDs–CEA. Gastric carcinoma cells were then incubated with the CDs–CEA at 37 °C for 2 h in an electro-heating constant temperature incubator. Preparation of CDs–antibody conjugates.
The quantum yield of the CDs was measured using the following equation: Quantum yield measurements.
Yu ¼ Ys
F u As nu 2 F s Au ns 2
Where Y is the quantum yield, F is integrated area under fluorescence spectra, A is absorbance and n is refractive index of the solvent. The subscript ‘s’ refers to a reference fluorophore of known quantum yield, for an example, quinine sulfate as used in present work. The quinine sulfate (literature quantum yield 0.54) was dissolved in 0.1 M H2SO4 (n = 1.33) and the CDs were dissolved in distilled water (n = 1.33). Cell culture, fixation and fluorescence microscopic imaging. Gastric carcinoma cells were cultured on a glass slides in DMEM/F12 media containing 10% fetal bovine serum in a culture box (heraeus BB 16UV). All cells were incubated at 37 °C under an atmosphere containing 5% CO2. To label fixed gastric carcinoma cells, the cells cultured on slides were gently washed three times with PBS and fixed with 4% formaldehyde solution for 10 min at room temperature. For the immunofluorescence experiment, slides containing fixed gastric carcinoma cells were blocked with 5% skim milk powder at 37 °C for 30 min. Unbound CDs were removed by washing thoroughly with cool PBS. The cancer marker detections of the gastric carcinoma cells were performed after direct labeling by using a Leica, TCS inverted fluorescence microscope (with a × 40/10 objective), and the fluorescence images were captured.
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Cell viability assay. Gastric carcinoma cells were also used to study the cytotoxicity of as-prepared CDs. Cell viability assay was carried out using CCK-8, determining the absorbance at 450 nm to explain the cell viability (21). Gastric carcinoma cells (80 μl) were seeded on a 96-well microtiter plate and maintained at 37 °C in a 5% CO2 incubator. After 24 h incubation, 10 μl aliquots of different concentrations of CDs were loaded into each well in triplicate for each CDs concentration. The CDs solutions were prepared by reconstitution of 16 mg of CDs with 5 ml of PBS, and twofold serial dilutions were carried out to give the desired concentrations of 1600, 800, 400, 200, 100 and 50 μg/ml. No CDs were added to the control cells. After 24 and 48 h of incubation, 10 μl of CCK-8 reagent was added to each well and the mixture was incubated at 37 °C for another 4 h to evaluate cell viability. The final concentration of CDs in the wells was 5, 10, 20, 40, 80 and 160 μg/ml in a total volume of 100 μl per well, respectively. The optical absorbance of each well was measured at 450 nm on a microplate reader.
Fluorescence emission and excitation spectra were acquired using a LS-55 luminescence spectrometer (Norwalk, USA). Absorption spectra were recorded on a UV2100 UV/vis spectrometer (Ruili Analytical Instrument Company, Beijing, China). The excitation or emission slit width was 10 nm and Cuvettes of 1 cm path length were used to measure the fluorescence spectra and absorption spectra separately. FTIR were recorded by using a Bruker Tensor 27 FTIR spectrometer (Berlin, Germany) in KBr media. All optical measurements were carried out at room temperature under ambient conditions. The sizes of the CDs were determined using a HITACHIH-7650 (Tokyo, Japan), for which the CDs solution were dropped onto a copper grid and dried at room temperature. During the conjugation process, CDs and antibody were mixed and then shaken using the CHA-S Reciprocating Oscillator (Jintan, Jiangshu, China). The gastric carcinoma cells were incubated with the CD–antibody conjugates in a electro-heating constant temperature incubator (DHP-9082, Yi heng, Shanghai, China). The aggregation and fluorescence emission of CDs in gastric carcinoma cells were supported by images taken with a fluorescence microscope (Munich, Germany).
Characterization.
Results and discussion Optical properties of the prepared CDs The absorption and emission spectra of the as-prepared CDs emitting blue fluorescence are shown in Fig. 1(A), which shows the desirable fluorescence properties. The characteristic absorption bank peaks of the CDs centered around 250–300 nm, and were attributed to the π–π* transition of the conjugated C = C band. The most intense PL of the CDs appeared at 451 nm upon excitation at 350 nm and the aqueous solution of CDs emitted strong blue luminescence when excited by a 365 nm UV light (Fig. 1A, insert). The EDA-modified CDs have a broad range of emission wavelengths which depend on the excitation wavelengths. When the CDs were excited from 360 to 440 nm in 20 nm increments, the fluorescence spectra ranged from 458 to 508 nm, as clearly shown in Fig. 1(B). Typical TEM images of CDs are shown in Fig. 1(C). The particle sizes of the water-soluble CDs were ~ 20 nm, with desirable dispersibility and uniformity. Furthermore, because EDA was chosen as the capped molecule, the surfaces of the CDs were bound in free amino groups,
Copyright © 2015 John Wiley & Sons, Ltd.
Luminescence 2015
Preparation of EDA-modified CDs and their use in the imaging of cells 350
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Figure 1. (A) Absorption (a) and emission (b) spectra and fluorescent image (insert) (4 × 10 mg/ml). (B) Emission spectra at an excitation wavelength of 360–440 nm with 20 nm increments. (C) TEM image of the prepared CDs.
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Figure 2. FTIR of EDA (A) and as prepared CDs (B).
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Direct labeling of gastric carcinoma cells In the study, the direct approach for cell labeling is the use of asprepared CDs by direct linking with CEA antibody towards gastric carcinoma cells. The CDs were first conjugated with mouse anti-(human CEA) antibody to give a specific probe. As shown
Luminescence 2015
in Fig. 3, the fluorescence peak position of CD–CEA was obviously blue-shifted (Δλ = 7 nm) compared with the bare CDs. This blue-shift in the maximum emission peak was probably due to
Fluorescence Intensity /a.u.
making it water soluble, biocompatible and perfect candidates for biomarkers. FTIR spectra were recorded to identify the organic functional groups on EDA-modified CDs. As shown in Fig. 2(B), the EDAfunctionalized CDs have many characteristic absorption bands of EDA (NH of 3400 and 1473 cm–1,CH2 of 2924 and 720 cm–1). Furthermore, a new sharp peak associated with amide linkage (-CONH-) found at 1651 cm–1 indicates that abundant EDA should be coated at the surface of bare CDs by the amide linkages. The quantum yield (QY) of the fluorescent CDs was calculated using quinine sulfate (excited at 310 nm) as a reference. The QY of the EDA-modified CDs was 3.0%, which was much higher than the value for bare CDs (1.3%). The high QY of the prepared EDAmodified CDs meant that the surfaces of the bare CDs are well passivated attributed to the strong linkage (-CONH-).
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Wavelength /nm Figure 3. Fluorescence spectra of CDs (a), and CDs–mouse anti-human CEA (b), –3 the CD concentration was 4 × 10 mg/ml.
Copyright © 2015 John Wiley & Sons, Ltd.
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W. Dong et al.
Figure 4. Fixed cells alone (a) at 405 nm excitation wavelength, and incubated with bare CDs for 2 h (b) and CDs–CEA for 2 h (c).
the conjugation of antibody molecules with CDs via a covalent bond. Conjugation via linking of the amino groups on CDs and the carboxylic groups of the antibody might weaken the dipole–dipole interaction and shorten the Stokes shifts, leading to the blue-shift in the emission peaks (22). It was also observed that the fluorescence intensity was enhanced due to surface passivation during conjugate formation. The CD–antibody conjugates were used as probes to label gastric carcinoma cells as described in the Experimental section. As shown in Fig. 4(a), the shape of the gastric carcinoma cells was hardly visible in the dark-field at 405 nm excitation wavelength, showing that gastric carcinoma cells themselves did not emit fluorescence. When cells were incubated with CDs without CEA antibody for 2 h, no fluorescence could be seen (Fig. 4b), whereas, when they were incubated with CD–CEA conjugates for 2 h, a strong blue fluorescence was detected Fig. 4c), indicating successful labeling. Compared with other routes, this type of labeling procedure was relatively simple. It may be expected that the CDs prepared here could be used as probes with further applications in immunolabeling and other biological areas.
Cytotoxicity For bioprobes, biocompatibility is highly important for practical applications. As far as live cell imaging is concerned, the effect of bioprobes on the natural physiological behaviors of cells should be emphasized (23). Accordingly, the cytotoxicity of CDs for human gastric carcinoma cells was studied using the CKK-8 method. All data shown in Fig. 5 are an average of three experiments. It can be seen that 20 μg/ml of CDs were almost non-toxic to the gastric carcinoma cells over 48 h. When the CD 100
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C (ug/mL) Figure 5. The viability of human gastric carcinoma cells with CDs of different concentrations for 24 and 48 h.
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concentration increased to 160 μg/ml, the cell viability of CDs was still high at 90%. Therefore, CDs can be thought of as biocompatible and suitable for live cell imaging. Such satisfactory biocompatibility can be attributed mainly to the use of the new materials, which are chemically inert, non-toxic, and do not release any toxic species even in harsh environments (24).
Conclusions Water-soluble CDs were synthesized by the hydrothermal treatment of glucose and then modified using EDA to enhance the luminescent intensity. The structure of the as-prepared CDs was proved by a series of characterizations, such as TEM, FTIR, UV/vis absorption and fluorescence spectra. The as-prepared CDs were successfully used to label fixed human gastric carcinoma cells through linking with mouse anti-(human CEA). With 48 h incubation, the as-prepared CDs scarcely showed any detectable cytotoxicity. In addition, the concentration and incubation time needed for cell labeling and imaging in vivo are much lower than the conditions used for cytotoxicity testing. As the synthesized CDs are biologically compatible and show excellent optical properties, they are promising imaging agents in biomedical and related applications.
References 1. Derfus AM, Chen WC, Bhatia SN. Probing the cytotoxicity of semiconductor quantum dots[J]. Nano Letters 2004;4(1):11–8. 2. Li JJ, Zou L, Hartono D, Ong CN, Bay BH, Yung LL. Gold nanoparticles induce oxidative damage in lung fibroblasts in vitro[J]. Advanced Materials 2008;20:138–42. 3. Fu CC, Lee HY, Chen K, Lim TS, Wu HY, Lin PK, et al. Characterization and application of single fluorescent nanodiamonds as cellular biomarkers[J]. Proceedings of the National Academy of Sciences of the United States of America 2007;104:727–32. 4. Dou XN, Lin Z, Chen H, Zheng YZ, Lu C, Lin JM. Production of superoxide anion radicals as evidence for carbon nanodots acting as electron donors by the chemiluminescence method[J]. Chemical Communications 2013;49:5871–73. 5. Lin Z, Xue W, Chen H, Lin JM. Peroxynitrous-Acid-Induced Chemiluminescence of Fluorescent Carbon Dots for Nitrite Sensing[J]. Analytical Chemistry 2011;83:8245–51. 6. Xue W, Lin Z, Chen H, Lin JM. Enhancement of Ultraweak Chemiluminescence from Reaction of Hydrogen Peroxide and Bisulfite by Water-Soluble Carbon Nanodots[J]. Journal of Physical Chemistry C 2011;115:21707–14. 7. Bottini M, Blasubramanian C, Dawson MI, Bergamaschi A, Bellucci S, Mustelin T. Isolation and characterization of fluorescent nanoparticles
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Preparation of EDA-modified CDs and their use in the imaging of cells
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from pristine and oxidized electric arc-produced single-walled carbon nanotubes[J]. Journal of Physical Chemistry B 2006;110:831–36. Zhou JG, Booker C, Li RY, Zhou XT, Sham TK, Sun XL, et al. An electrochemical avenue to blue luminescent nanocrystals from multiwalled carbon nanotubes (MWCNTs)[J]. Journal of the American Chemical Society 2007;129:744–45. Cao L, Wang X, Meziani MJ, Lu F, Wang H, Luo PG, et al. Carbon dots for multiphoton bioimaging[J]. Journal of the American Chemical Society 2007;129:11318–19. Sun YP, Zhou B, Lin Y, Wang W, Fernando KAS, Pathak P, et al. Quantum-sized carbon dots for bright and colorful photoluminescence[J]. Journal of the American Chemical Society 2006;128:7756–57. Zhu H, Wang XL, Li YL, Wang ZJ, Yang F, Yang XR. Microwave synthesis of fluorescent carbon nanoparticles with electrochemiluminescence properties[J]. Chemical Communications 2009; 34:5118–20. Zhang HZ, Wang QL, Long YJ, Zhang HJ, Huang XX, Zhu R. Enhancing the luminescence of carbon dots with a reduction pathway[J]. Chemical Communications 2011;47:10650–52. Hu SL, Niu KY, Sun J, Yang J, Zhao NQ, Du XW. One-step synthesis of fluorescent carbon nanoparticles by laser irradiation[J]. Journal of Materials Chemistry 2009;19:484–8. Mao XJ, Zheng HZ, Long YJ, Du J, Hao JY, Wang LL, et al. Study on the fluorescence characteristics of carbon dots[J]. Journal of Spectrochimica Acta Part A 2010;75:553–7. Qiao ZA, Wang YF, Gao Y, Li HW, Dai TY, Liu YL, et al. Commercially activated carbon as the source for producing multicolor photoluminescent carbon dots by chemical oxidation[J]. Journal of Chemical Communications 2010;46:8812–14.
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16. Zhang JC, Shen WQ, Pan DY, Zhang ZW, Fang YG, Wu MH. Controlled synthesis of green and blue luminescent carbon nanoparticles with high yields by the carbonization of sucrose[J]. New Journal of Chemistry 2010;34:591–3. 17. Han BF, Wang WX, Wu HY, Fang F, Wang NZ, Zhang XJ, et al. Polyethyleneimine modified fluorescent carbon dots and their application in cell labeling[J]. Colloids and Surfaces B 2012;100:209–14. 18. Liu CJ, Zhang P, Tian F, Li WC, Li F, Liu WG. One-step synthesis of surface passivated carbon nanodots by microwave assisted pyrolysis for enhanced multicolor photoluminescence and bioimaging[J]. Journal of Materials Chemistry 2011;21:13163–67. 19. Bourlinos AB, Stassinopoulos A, Anglos D, Zboril R, Karakassides M, Giannelis EP. Surface functionalized carbogenic quantum dots[J]. Small 2008;4:455–8. 20. Peng H, Travas-Sejdic J. Simple aqueous solution route to luminescent carbogenic dots from carbohydrates[J]. Chemistry of Materials 2009;21:5563–65. 21. Hashimoto D, Ohmuraya M, Hirota M, Yamamoto A, Suyama K, Ida S, et al. Involvement of autophagy in trypsinogen avtivation within the parcreatic acinar cells[J]. Journal of Cell Biology 2008;181:1065–72. 22. Zhu CQ, Zhao DH, Chen JL, Li YX, Wang LY, Wang L. Application of Lcysteine-capped nano-ZnS as a fluorescence probe for the determination of proteins[J]. Analytical and Bioanalytical Chemistry 2004; 378:811–15. 23. Zhang J, Jia X, Lv XJ, Deng YL, Xie XY. Fluorescent quantum dot-labeled aptamer bioprobes specifically targeting mouse liver cancer cells[J]. Talanta 2010;81:505–9. 24. Wang F, Xie Z, Zhang H, Liu CY, Zhang YG. Highly luminescent organosilane-functionalized carbon dots[J]. Advanced Functional Materials 2011;21:1027–31.
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/luminescence
Revised: 8 October 2014,
Accepted: 19 November 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.2834
The preparation of ethylenediamine-modified fluorescent carbon dots and their use in imaging of cells Wei Dong,a* Siqi Zhou,b Yan Dong,c Jingwen Wang,a Xin Gea and Lili Suia ABSTRACT: In this work, fluorescent carbon dots (CDs) were synthesized using a hydrothermal method with glucose as the carbon source and were surface-modified with ethylenediamine. The properties of as-prepared CDs were analyzed by transmission electron microscopy (TEM), Fourier transform infrared (FTIR), ultraviolet–visible light (UV/vis) absorption and fluorescent spectra. Furthermore, CDs conjugated with mouse anti-(human carcinoembryonic antigen) (CEA) monoclonal antibody were successful employed in the biolabeling and fluorescent imaging of human gastric carcinoma cells. In addition, the cytotoxicity of CDs was also tested using human gastric carcinoma cells. There was no apparent cytotoxicity on human gastric carcinoma cells. These results suggest the potential application of the as-prepared CDs in bioimaging and related fields. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: carbon dots; ethylenediamine; cell imaging; cytotoxicity
Introduction Fluorescence semiconductor quantum dots (QDs), such as CdTe, CdS and CdSe, have attracted much attention due to their particular optical properties as fluorescence probes. However, the known toxicity of heavy metals, for example cadmium, the main ingredient of QDs, and severe synthetic and environmental hazards have limited their bio-application (1,2). Recently, carbon dots (CDs) have attracted intense attention due to their unique advantages. CDs are usually carbon nanocrystals of 2–10 nm in diameter, which not only exhibit many excellent optical properties, such as photoinduced electron transfer, photoluminescence (PL), electrochemiluminescence (ECL) and chemiluminescence (CL), but also present some additional advantages over luminescent semiconductor QDs, such as good biocompatibility and low toxicity (3–6). In recent years, various methods have been developed to obtain CDs including: oxidation by a strong oxidant, electrochemical methods, laser ablation and microwave heating (7–11). Although all these CDs prepared as above showed great PL, many drawbacks, such as long and complex processes, strict experimental conditions and producing CDs with low quantum yields (QYs) limit their application in the life sciences. Thus, a rapid, convenient and efficient method for the synthesis of CDs remains a challenge. To date, passivation has been a popular approach to enhance the fluorescence of CDs (12). Several organic polymers and diamine compounds, such as polyethylene glycol) (PEG) (13–16), polyethyleneimine (PEI) (17), citrate and 4-aminoantipyrine (18,19) have been used to passivate CDs. All these CDs showed high photostability, tunable excitation and emission wavelength, good biocompatibility and low toxicity, and could be applied in the imaging of normal and cancer cells. In this work, we report a facile, economic and green synthesis route towards photoluminescent CDs, resulting in a quantum yield of 3.0%. The CDs were synthesized by a hydrothermal
Luminescence 2015
method with glucose as the carbon source and modified with ethylenediamine (EDA). The as-prepared dots were linked with mouse anti-(human CEA) antibody to form an immunoprobe. The CDs–antibody probe was successfully used to label human gastric carcinoma cells (MGC-803) via the specific reaction between antibody and antigen. Compared with other routes, the direct labeling procedure was relatively simpler. The cytotoxicity of CDs was also tested using gastric carcinoma cells. It was observed that none of the cell lines show any statistically significant differences in cell survival within 48 h at a CD concentration of 0–160 μg/ml. All these exciting results indicated that CD-based probes are ideal fluorescent markers with excellent spectral properties, photostability and biocompatibility.
Experimental Materials Glucose, PEG 400 and EDA (99%) were obtained from Sinopharm Chemical Reagent Co. Ltd (Shenyang, China). Mouse anti-(human CEA) was purchased from Beijing Biosynthesis Biotechnology Co. (Beijing, China). N-Hydroxysuccinimide (NHS) was obtained from Fluka (New York, USA). Dulbecco’s modified Eagle’s medium/Ham′s * Correspondence to: W. Dong, Department of Chemistry, Shenyang Medical college, Shenyang 110034, people’s Republic of China. E-mail: [email protected] a
Department of Chemistry, Shenyang Medical College, Shenyang, People’s Republic of China
b
ICU, Fengtian Hospital Affiliated to Shenyang Medical College, Shenyang, People’s Republic of China
c
Experiment Center of Traditional Chinese Medicine Department, Shenyang Pharmaceutical University, People’s Republic of China
Copyright © 2015 John Wiley & Sons, Ltd.
W. Dong et al. F12 media (DMEM/F12) was obtained from Gibco (New York, USA). Cell counting kit-8 (CCK-8) was obtained from Shenyang Tongren Institute of Chemistry (Shenyang, China). All other reagents were of analytical reagent grade. The water used in all experiments was prepared using a quartzose double-distilling system. Gastric carcinoma cells (MGC-803) used in the study were obtained from the Department of Pharmacology of Shenyang Medical College. Preparation of fluorescent CDs.
CDs were synthesized according to a procedure described previously (20). Briefly, 540 mg of glucose was thoroughly mixed with 25 ml of PEG/distilled water (1: 1, v/v/) in a high-pressure nitrifying pot and heated at 180 °C for 3 h. After cooling to room temperature naturally, 100 μl EDA was added to the system. The mixture was then removed to the high-pressure nitrifying pot for passivation at 120 °C for 12 h. Finally, the dark yellow solution of luminescent CDs was passed through a 0.22 μm filter membrane to remove the larger product, acetone was added to the solution and the CDs were collected by centrifugation at 3500 r.p.m. for 15 min. The precipitation was dried a under nitrogen flow and dispersed in distilled water to obtain a clear solution for further experiments. The absorption and emission spectra, TEM images and FTIR spectra of CDs were recorded. The water-soluble CDs were conjugated with CEA using NHS as a cross-linker. Typically, 100 μl of mouse anti-(human CEA) antibody (1 mg/ml) in 100 μl of PBS (10 mmol/L) was mixed with 50 μl of as-prepared CDs (0.4 mg/ml) and incubated in an incubator shaker for 1.5 h at 37 °C after being activated by 50 μl of a mixture of 500 mmol/l EDC and 50 mmol/l NHS for 10 min at room temperature to form CDs–CEA. Gastric carcinoma cells were then incubated with the CDs–CEA at 37 °C for 2 h in an electro-heating constant temperature incubator. Preparation of CDs–antibody conjugates.
The quantum yield of the CDs was measured using the following equation: Quantum yield measurements.
Yu ¼ Ys
F u As nu 2 F s Au ns 2
Where Y is the quantum yield, F is integrated area under fluorescence spectra, A is absorbance and n is refractive index of the solvent. The subscript ‘s’ refers to a reference fluorophore of known quantum yield, for an example, quinine sulfate as used in present work. The quinine sulfate (literature quantum yield 0.54) was dissolved in 0.1 M H2SO4 (n = 1.33) and the CDs were dissolved in distilled water (n = 1.33). Cell culture, fixation and fluorescence microscopic imaging. Gastric carcinoma cells were cultured on a glass slides in DMEM/F12 media containing 10% fetal bovine serum in a culture box (heraeus BB 16UV). All cells were incubated at 37 °C under an atmosphere containing 5% CO2. To label fixed gastric carcinoma cells, the cells cultured on slides were gently washed three times with PBS and fixed with 4% formaldehyde solution for 10 min at room temperature. For the immunofluorescence experiment, slides containing fixed gastric carcinoma cells were blocked with 5% skim milk powder at 37 °C for 30 min. Unbound CDs were removed by washing thoroughly with cool PBS. The cancer marker detections of the gastric carcinoma cells were performed after direct labeling by using a Leica, TCS inverted fluorescence microscope (with a × 40/10 objective), and the fluorescence images were captured.
wileyonlinelibrary.com/journal/luminescence
Cell viability assay. Gastric carcinoma cells were also used to study the cytotoxicity of as-prepared CDs. Cell viability assay was carried out using CCK-8, determining the absorbance at 450 nm to explain the cell viability (21). Gastric carcinoma cells (80 μl) were seeded on a 96-well microtiter plate and maintained at 37 °C in a 5% CO2 incubator. After 24 h incubation, 10 μl aliquots of different concentrations of CDs were loaded into each well in triplicate for each CDs concentration. The CDs solutions were prepared by reconstitution of 16 mg of CDs with 5 ml of PBS, and twofold serial dilutions were carried out to give the desired concentrations of 1600, 800, 400, 200, 100 and 50 μg/ml. No CDs were added to the control cells. After 24 and 48 h of incubation, 10 μl of CCK-8 reagent was added to each well and the mixture was incubated at 37 °C for another 4 h to evaluate cell viability. The final concentration of CDs in the wells was 5, 10, 20, 40, 80 and 160 μg/ml in a total volume of 100 μl per well, respectively. The optical absorbance of each well was measured at 450 nm on a microplate reader.
Fluorescence emission and excitation spectra were acquired using a LS-55 luminescence spectrometer (Norwalk, USA). Absorption spectra were recorded on a UV2100 UV/vis spectrometer (Ruili Analytical Instrument Company, Beijing, China). The excitation or emission slit width was 10 nm and Cuvettes of 1 cm path length were used to measure the fluorescence spectra and absorption spectra separately. FTIR were recorded by using a Bruker Tensor 27 FTIR spectrometer (Berlin, Germany) in KBr media. All optical measurements were carried out at room temperature under ambient conditions. The sizes of the CDs were determined using a HITACHIH-7650 (Tokyo, Japan), for which the CDs solution were dropped onto a copper grid and dried at room temperature. During the conjugation process, CDs and antibody were mixed and then shaken using the CHA-S Reciprocating Oscillator (Jintan, Jiangshu, China). The gastric carcinoma cells were incubated with the CD–antibody conjugates in a electro-heating constant temperature incubator (DHP-9082, Yi heng, Shanghai, China). The aggregation and fluorescence emission of CDs in gastric carcinoma cells were supported by images taken with a fluorescence microscope (Munich, Germany).
Characterization.
Results and discussion Optical properties of the prepared CDs The absorption and emission spectra of the as-prepared CDs emitting blue fluorescence are shown in Fig. 1(A), which shows the desirable fluorescence properties. The characteristic absorption bank peaks of the CDs centered around 250–300 nm, and were attributed to the π–π* transition of the conjugated C = C band. The most intense PL of the CDs appeared at 451 nm upon excitation at 350 nm and the aqueous solution of CDs emitted strong blue luminescence when excited by a 365 nm UV light (Fig. 1A, insert). The EDA-modified CDs have a broad range of emission wavelengths which depend on the excitation wavelengths. When the CDs were excited from 360 to 440 nm in 20 nm increments, the fluorescence spectra ranged from 458 to 508 nm, as clearly shown in Fig. 1(B). Typical TEM images of CDs are shown in Fig. 1(C). The particle sizes of the water-soluble CDs were ~ 20 nm, with desirable dispersibility and uniformity. Furthermore, because EDA was chosen as the capped molecule, the surfaces of the CDs were bound in free amino groups,
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Preparation of EDA-modified CDs and their use in the imaging of cells 350
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Figure 1. (A) Absorption (a) and emission (b) spectra and fluorescent image (insert) (4 × 10 mg/ml). (B) Emission spectra at an excitation wavelength of 360–440 nm with 20 nm increments. (C) TEM image of the prepared CDs.
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Direct labeling of gastric carcinoma cells In the study, the direct approach for cell labeling is the use of asprepared CDs by direct linking with CEA antibody towards gastric carcinoma cells. The CDs were first conjugated with mouse anti-(human CEA) antibody to give a specific probe. As shown
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in Fig. 3, the fluorescence peak position of CD–CEA was obviously blue-shifted (Δλ = 7 nm) compared with the bare CDs. This blue-shift in the maximum emission peak was probably due to
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making it water soluble, biocompatible and perfect candidates for biomarkers. FTIR spectra were recorded to identify the organic functional groups on EDA-modified CDs. As shown in Fig. 2(B), the EDAfunctionalized CDs have many characteristic absorption bands of EDA (NH of 3400 and 1473 cm–1,CH2 of 2924 and 720 cm–1). Furthermore, a new sharp peak associated with amide linkage (-CONH-) found at 1651 cm–1 indicates that abundant EDA should be coated at the surface of bare CDs by the amide linkages. The quantum yield (QY) of the fluorescent CDs was calculated using quinine sulfate (excited at 310 nm) as a reference. The QY of the EDA-modified CDs was 3.0%, which was much higher than the value for bare CDs (1.3%). The high QY of the prepared EDAmodified CDs meant that the surfaces of the bare CDs are well passivated attributed to the strong linkage (-CONH-).
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W. Dong et al.
Figure 4. Fixed cells alone (a) at 405 nm excitation wavelength, and incubated with bare CDs for 2 h (b) and CDs–CEA for 2 h (c).
the conjugation of antibody molecules with CDs via a covalent bond. Conjugation via linking of the amino groups on CDs and the carboxylic groups of the antibody might weaken the dipole–dipole interaction and shorten the Stokes shifts, leading to the blue-shift in the emission peaks (22). It was also observed that the fluorescence intensity was enhanced due to surface passivation during conjugate formation. The CD–antibody conjugates were used as probes to label gastric carcinoma cells as described in the Experimental section. As shown in Fig. 4(a), the shape of the gastric carcinoma cells was hardly visible in the dark-field at 405 nm excitation wavelength, showing that gastric carcinoma cells themselves did not emit fluorescence. When cells were incubated with CDs without CEA antibody for 2 h, no fluorescence could be seen (Fig. 4b), whereas, when they were incubated with CD–CEA conjugates for 2 h, a strong blue fluorescence was detected Fig. 4c), indicating successful labeling. Compared with other routes, this type of labeling procedure was relatively simple. It may be expected that the CDs prepared here could be used as probes with further applications in immunolabeling and other biological areas.
Cytotoxicity For bioprobes, biocompatibility is highly important for practical applications. As far as live cell imaging is concerned, the effect of bioprobes on the natural physiological behaviors of cells should be emphasized (23). Accordingly, the cytotoxicity of CDs for human gastric carcinoma cells was studied using the CKK-8 method. All data shown in Fig. 5 are an average of three experiments. It can be seen that 20 μg/ml of CDs were almost non-toxic to the gastric carcinoma cells over 48 h. When the CD 100
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concentration increased to 160 μg/ml, the cell viability of CDs was still high at 90%. Therefore, CDs can be thought of as biocompatible and suitable for live cell imaging. Such satisfactory biocompatibility can be attributed mainly to the use of the new materials, which are chemically inert, non-toxic, and do not release any toxic species even in harsh environments (24).
Conclusions Water-soluble CDs were synthesized by the hydrothermal treatment of glucose and then modified using EDA to enhance the luminescent intensity. The structure of the as-prepared CDs was proved by a series of characterizations, such as TEM, FTIR, UV/vis absorption and fluorescence spectra. The as-prepared CDs were successfully used to label fixed human gastric carcinoma cells through linking with mouse anti-(human CEA). With 48 h incubation, the as-prepared CDs scarcely showed any detectable cytotoxicity. In addition, the concentration and incubation time needed for cell labeling and imaging in vivo are much lower than the conditions used for cytotoxicity testing. As the synthesized CDs are biologically compatible and show excellent optical properties, they are promising imaging agents in biomedical and related applications.
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