Green Synthesis of Luminescent Nitrogen-Doped Carbon Dots from

Letter pubs.acs.org/ac

Green Synthesis of Luminescent Nitrogen-Doped Carbon Dots from Milk and Its Imaging Application Li Wang and H. Susan Zhou* Life Science and Bioengineering Center, Department of Chemical Engineering, Worcester Polytechnic Institute, Gateway Park 4001, Worcester, Massachusetts 01609, United States S Supporting Information *

ABSTRACT: In the present work, a completely green synthetic method for producing fluorescent nitrogen-doped carbon dots by using milk is introduced. The process is environmentally friendly, simple, and efficient. By hydrothermal heating of milk, we produced monodispersed, highly fluorescent carbon dots with a size of about 3 nm. Imaging of U87 cells, a human brain glioma cancer cell line, can be easily achieved with high resolution using the prepared carbon dots as probes and validates their use in imaging applications.

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inexpensive, renewable material. In this work, we develop a green, simple, and low-cost preparative strategy toward highly fluorescent, nitrogen-doped carbon dots by hydrothermal treatment of milk. The synthetic process is illustrated in Scheme 1. A significant advantage of this method is green

anomaterials should be produced economically and cleanly. Over the past decades, green chemistry has captured the imagination of many chemists due to its clean and sustainable feature. Preparation of nanomaterials using nontoxic chemicals, environmentally friendly solvents, and renewable materials is the key issue that should be taken into consideration in a green synthetic strategy.1 In the present work, we present a totally green approach toward the synthesis of fluorescent carbon dots. Because of their unique optical and electronic properties and benign and inexpensive features, carbon dots are very promising in many applications such as optoelectronic devices, catalysts, biological labeling, and biosensors.2−11 Photoluminescent carbon dots are more superior to traditional organic dyes and semiconductor quantum dots in terms of aqueous solubility, functionalizability, toxicity, and biocompatibility.12 Lately, more effort has been put into the preparation of fluorescent nitrogen-doped carbon dots because of their extraordinary photophysical and photochemical properties, good biocompatibility, and the related broad applications in the areas of bioimaging, electrocatalysis, solar cells, and sensors.13−19 However, many of the conventional synthesis methods for these materials are expensive, sophisticated, and tedious.20 The preparation of carbon dots should be simple and environmentally benign as far as possible, and finding new methods that eliminate the use of rigid reaction conditions or toxic materials is highly desired. To this end, a green method using precursors directly from nature is a very promising solution and would be of great benefit to large scale synthesis and widespread applications. Nature provides a limitless source, which gives material scientists the freedom to have inspiration for new ideas, new ways of developing nanomaterials with novel structures and properties, and less environmental impact. Recently, major attention has been focused on developing green methods for preparing carbon dots using natural precursors.21−25 Milk is an © 2014 American Chemical Society

Scheme 1. Illustration of the Formation Process of Carbon Dots from Milk by Hydrothermal Treatment

synthesis, and it requires no severe synthetic conditions (such as strong solvents, surface passivation reagents, and a complicated post-treatment process, which are generally used in conventional methods). Most importantly, we further demonstrate that these carbon dots can serve as very effective fluorescent probes for U87 cell imaging.

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RESULTS AND DISCUSSION Milk, containing carbohydrates, proteins, and a few minerals for nutrition, is widely consumed all over the world. In this work, the hydrothermal treatment of milk at 180 °C can generate a yellow solution (see the experimental section for more details, Supporting Information). Figure 1a shows the transmission electron microscope (TEM) image of the prepared carbon dots, Received: July 9, 2014 Accepted: September 2, 2014 Published: September 2, 2014 8902

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Figure 1. (a) TEM image of the prepared carbon dots. (b) Fluorescence excitation (red line) and emission (black line) spectra of the carbon dots (λ ex = 360 nm, λem = 454 nm). Inset shows the photographs of the carbon dots in aqueous solutions under the irradiation of a 365 nm UV light lamp. (c) Emission spectra of carbon dots at different excitation wavelengths from 330 to 475 nm.

carbon dots are negatively charged due to carboxylic and hydroxy groups. At the same time, these functional groups on the surface may bring in a series of emissive traps, which are very important for the strong photoluminescent property exhibited by the carbon dots.24 The time-resolved fluorescence decay curve measured by the time-correlated single photon counting method is shown in Figure S3 (Supporting Information). The decay curve is very well fitted to a triple-exponential function, and the mean lifetime is calculated to be 1.61 ns; such a short lifetime is comparable to the most available carbon dots reported,6,26−30 and this indicates that the luminescent mechanism is possibly the radioactive recombination of excitations.30 We also noticed that the quantum yield of carbon dots is 12% against the reference of quinine sulfate, and this value is significantly higher than some of the previously reported carbon dots which are surface passivated.22,23,31 The high quantum yield is possibly due to the existence of nitrogencontaining functional groups, which are generally excellent auxochromes. The doped nitrogen can effectively passivate the surface active sites by stabilization of the excitons in the carbon dots, thus greatly enhancing the photluminescent properties.24 In addition, our studies on the photostability showed that the fluorescence of the present carbon dots remained very stable under continuous irradiation. The photoluminescent intensity changed little under continuous excitation (365 nm) with a Xe lamp for 3 h. After 6 months of storage at 4 °C, the fluorescence intensity of carbon dots still remains 90%. The fluorescent stability is comparable to the most available carbon dots reported,6,26,27 and the excellent resistance to photobleaching exhibited by the present carbon dots is much better than the conventional organic fluorophores that suffer from poor photostability, making our carbon dots a good option for use in bioimaging applications. Cell cytotoxicity experiments of carbon dots were evaluated using human brain glioma tumor (U87) cell lines through the CKK-8 assay. As shown in Figure 2a, the obtained carbon dots show no apparent toxicity to the cells even though the carbon dots concentration was increased to 1 mg mL−1. As state above, the as-obtained carbon dots exhibit bright photoluminescent property, low toxicity, and high stability against photobleaching; these features make them promising probes for imaging applications. To examine the capacity of carbon dots for cell imaging, in vitro cellular uptake experiments in U87 cells were then performed and the images were taken under a laser scanning confocal microscope. U87 cells were labeled with the prepared carbon dots after they were cultured and maintained in MEM medium

revealing that these carbon dots are well separated from each other and have a uniform dispersion without apparent aggregation and a relatively narrow size distribution between 2 and 4 nm. The UV−visible absorption spectrum of the carbon dots is given in the Supporting Information (Figure S1). Due to the electron transition of the nanocarbon, the UV−vis absorption spectrum shows a very broad UV absorption band in the range of 250−600 nm with a strong peak at around 280 nm, which could be ascribed to the presence of aromatic π orbitals of carbon dots.24 We observed that the carbon dots give very high photoluminescence under the short-wavelength laser irradiation, as shown in Figure 1b; when excited at 360 nm, the carbon dots show very strong photoluminescence in the range of 400−600 nm, with the maximum located at around 454 nm. Upon the irradiation of 365 nm UV light, the carbon dots solution can immediately generate a blue-green color (Figure 1b inset). This emission, however, is very sensitive to the excitation wavelength (Figure 1c), and with the increase of excitation wavelength, the emission peaks shift to longer wavelengths with decreased intensity. This excitation-dependent emission property of carbon dots has been also found in previous reports.4,17,21−25 Meanwhile, the milk solution itself is nonemissive in the studied region, confirming the bright fluorescence is stemming from the synthesized carbon dots. In order to understand the components and structures of the as-prepared carbon dots, Fourier transformed infrared (FT-IR) and X-ray photoelectron spectroscopy (XPS) were then performed. The FT-IR spectrum (Figure S2a, Supporting Information) exhibits characteristic absorption bands of O−H and N−H stretching vibrations of amine groups at 3289 cm−1, C−H stretching vibrations at 2900 cm−1, C−O stretching vibrations at 1643 cm−1, C−C stretching vibrations at 1583 cm−1, C−N stretching vibrations at 1392 cm−1, and C−O stretching vibrations at 1243 cm−1. The XPS spectrum provides more convincing evidence for the N-doping surface state of carbon dots; as shown in Figure S2b (Supporting Information), the as-prepared carbon dots are composed of carbon, oxygen, and nitrogen, and an obvious N peak was detected with a binding energy at around 400 eV. As seen in FT-IR and XPS spectra, the synthesized carbon dots have multiple functional groups like −COOH, −OH, and a small amount of Ncontaining groups, which can be ascribed to the degradation of milk during the hydrothermal treatment, and the presence of these functional groups imparts carbon dots excellent solubility in water without the requirement of further chemical modification. The zeta potential of the carbon dots was −20.6 mV (Figure S2c, Supporting Information), suggesting 8903

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Letter

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +1-508-831-5275. Fax: +1508-831-5936. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

This work was supported by National Science Foundation (CMMI-1030289). The authors thank Prof. Thomas McCarthy and Dr. Liming Wang (University of Massachusetts Amherst) for some technical support.

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Figure 2. (a) Cytotoxicity testing results via a CCK-8 assay. The values represent percentage cell viability. (b) Laser scanning confocal microscopy image of U87 cells after the cellular uptake of carbon dots.

containing 10 μg/mL carbon dots. As shown in Figure 2b, after being labeled with carbon dots, all cells are lightened, confirming the high cellular uptake of the carbon dots by U87 cells; therefore, U87 cells can be clearly imaged by the confocal microscope, and no cell damage was observed. All these points demonstrate that the carbon dots prepared from milk can serve as a promising substitute for organic dyes which are susceptible to photobleaching or semiconductor quantum dots that have known biotoxicity for bioimaging. In conclusion, we have developed a green, one-step, low-cost method to synthesize highly luminescent carbon dots from milk. The prepared carbon dots are nanometer-sized, exhibit good dispersibility, have strong and stable photoluminescence, which is excitation dependent, and have outstanding photostability. Meanwhile, the carbon dots have almost no cytotoxicity, can be effectively taken up by the human U87 cells, and have been shown to be excellent optical cell-imaging probes.

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ASSOCIATED CONTENT

S Supporting Information *

Experiment section and other characterization data as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org/. 8904

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