Efficient synthesis of highly fluorescent nitrogen-doped carbon dots for cell imaging using unripe fruit extract of Prunus mume
Graphical abstract
The green synthesis of highly fluorescent N-CDs was achieved using the extract of unripe P. mume fruit as a carbon precursor by a one-pot simple hydrothermal-carbonization method. The resulting N-CDs were used as a staining agent for the fluorescence imaging of MDA-MB-231 cells.
Introduction
Carbon dots (CDs) with sizes below 10 nm are a new class of carbon nanomaterials [1]. These CDs have attracted considerable attention because of their excellent properties, such as fluorescence properties, which make its applicability in fluorescence cell imaging, energy, catalysis, optics, and sensors [2], [3], [4], [5], [6], [7], [8], [9]. CDs have low cytotoxicity, are eco-friendly, and have good biocompatibility, aqueous solubility and high photostability compared to fluorescent dyes and semiconductor quantum dots. Therefore, CDs are a good alternative for fluorescent dyes and semiconductor quantum dots for cell imaging and biosensing applications [10], [11]. The fluorescence properties of CDs depend on their size, pH, passivating agent, defects, and solvent [12], [13]. Currently, the synthesis of CDs with high fluorescence intensity and different emission properties are a challenging task for nanomaterial researchers. In general, CDs are synthesized using either top-down or bottom-up approaches. The top-down approaches containing arc-discharge, laser ablation and electrochemical methods have been used for the synthesis of CDs. Bottom-up approaches include microwave-assisted carbonization, hydrothermal-carbonization and aqueous based methods. In the top-down methods, graphite or multi-walled carbon nanotubes are used as carbon precursors, whereas in the bottom-up method, carbohydrates or simple organic acids are used as the carbon precursor [14], [15], [16], [17]. Among them, bottom-up methods are quite simple, facile, cost effective, and eco-friendly. In particular, the hydrothermal-carbonization method is simple and large scale with a narrow size distribution for the preparation of CDs. The biomass of fruits, vegetables, milk, hair, caramel, and honey has been utilized as green carbon precursors for the synthesis of fluorescent CDs and N-CDs [18], [19], [20], [21], [22], [23], [24], [25], [26].
In general, the plant extracts containing various bioactive molecules such as acidic constituents (tannin, polyphenols, flavonoids, citric acid, tartaric acid, and ascorbic acid), base constituents (alkaloids), and neutral constituents (carbohydrates) are intriguing when applied in a nanoparticle synthesis. When applied for the nanoparticle synthesis (like carbon dots), the synthetic kinetics sometimes depends on the phytoconstituents present in the particular extract. The extract phytoconstituents present does not only determine the surface functional groups on the carbon dots but also the reactivity of the material. Biswal et al., have investigated the supercapacitor behaviour of carbon particles derived from neem leaf and ashoka leaf. They showed that particles derived from neem leaf have higher supercapacity than those from the ashoka due to the different phytoconstituents present in the neem leaf [27]. Likewise, in CDs, the fluorescence quantum yield (fluorescence intensity) also depends on the type of phytoconstituents, size of particles, solvents, and dopants. This behaviour opens the way for the application of CDs as fluorescent probes for the selective and sensitive detection of metal cations [28], thus making the use of plant extracts an amusing aspect in the CD synthesis. Exploiting the idea, CD synthesis from the P. mume fruit is reported and discussed in this work. P. mume is a deciduous tree belonging to the Rosaceae family and is cultivated widely in China, Japan and South Korea. P. mume fruits are easily available and affordable, and were consumed as food and medicinal materials. The major phytoconstituents present in the fruits are epicatechin, isoquercitrin, rutin, quercetin 3-O-neohesperidoside, and (−)-epicatechin. The immature unripe fruits of P. mume have been used in antitussive, expectoration, antiemetic, antidiarrheal, anthelmintic, and antipyretic treatments [29], [30], [31]. The sucrose and glucose were dominant sugars, and citric acid was dominant organic acids in P. mume fruits, this might be helpful to formation of carbon materials. In addition, the fermented P. mume fruits can meet the standard to be further processed into prune or sauces, and the fermentation broth of P. mume fruits with Lactobacillus fermentium have a good prospect in the development of probiotic beverage [32]. The composition of P. mume is quite different from that of extracts of other fruits including orange and lemon, which was reported previously for the synthesis of CDs and N-CDs. Hence, the P. mume fruits will be chosen as an ideal source for the preparation of N-CDs in this work.
This paper reports the green synthesis of highly fluorescent N-CDs using the unripe fruit extract of P. mume as a carbon precursor via a simple hydrothermal-carbonization method. The N-CDs were synthesized at different pH, such as 2.3 (fruit extract as it is), 5, 7, and 9 (pH was adjusted by aqueous ammonia). The optical properties of the obtained N-CDs were studied by UV–vis and fluorescence spectroscopy. The highly fluorescent N-CDs were characterized by high resolution transmission electron microscopy (HR-TEM), X-ray diffraction (XRD), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), and Fourier transform-infra red (FT-IR) spectroscopy. The highly fluorescent N-CDs were used as a staining agent for the fluorescence imaging of MDA-MB-231 cells. To the best of our knowledge, P. mume is first time used for the synthesis of N-CDs and as a staining probe for fluorescence cell imaging.
Section snippets
Materials
The unripe P. mume fruits were procured from the Yeungnam University campus, Gyeongsan, Republic of Korea. An aqueous ammonia solution (25%), acetonitrile, ethanol and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich and the aqueous ammonia solution was used to adjust the pH of the extract of P. mume. The MDA-MB-231 cells were purchased from the Korean Cell Line Bank. The entire chemicals were used as received. The deionized (DI) water was used in this work.
Preparation of P. mume fruit extract
The unripe fruits of P.
Results and discussion
UV–vis spectroscopy is a useful tool for studying the optical properties of CDs and it is complementary to fluorescence spectroscopy [33]. Fig. 2 shows the UV–vis and fluorescence spectra of the carbon precursor (extract of unripe P. mume fruit) in water with different pH. The UV–vis spectra showed that an extract of unripe P. mume fruit absorbed at 285 and 317 nm at pH of 2.3, 5, and 7. The absorbance intensities of P. mume fruit extract at pH 5 and 7 were lower than that of pH 2.3. On the
Conclusions
This paper reported a simple, cost effective and eco-friendly route for the synthesis of highly fluorescent N-CDs using the extract of P. mume unripe fruit in water by a simple one pot hydrothermal-carbonization method. The N-CDs were synthesized using P. mume fruit extract at various pH at 180 °C. All the synthesized N-CDs showed fluorescence nature. In addition, the fluorescence intensity increased with increasing pH of the P. mume extract from acidic to basic. The intensity difference was
Acknowledgements
This research was supported by the Nano Material Technology Development Program of the Korean National Research Foundation (NRF) funded by the Korean Ministry of Education, Science, and Technology (2012M3A7B4049675). This work was also supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (NRF-2014R1A2A1A11052391) and Priority Research Centers Program (2014R1A6A1031189).
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