Outright Green Synthesis of Fluorescent Carbon Dots from Eutrophic

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An Outright Green Synthesis of Fluorescent Carbon Dots from Eutrophic Algal Blooms for In Vitro Imaging Ramanan Vadivel, Senthil Kumar Thiyagarajan, Kaviyarasan Raji, Raghupathy Suresh, Rajkumar Sekar, and Perumal Ramamurthy ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/acssuschemeng.6b00935 • Publication Date (Web): 26 Jul 2016 Downloaded from http://pubs.acs.org on July 28, 2016

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ACS Sustainable Chemistry & Engineering

An Outright Green Synthesis of Fluorescent Carbon Dots from Eutrophic Algal Blooms for In Vitro Imaging Vadivel Ramanan,a Thiyagarajan Senthil Kumar,a Raji Kaviyarasan,a Ragupathy Suresh,a Sekar Rajkumar,b Perumal Ramamurthy*a,b a

National Centre for Ultrafast Processes, University of Madras, Taramani Campus, Chennai – 600113.

b

Department of Inorganic Chemistry, University of Madras, Guindy Campus, Chennai – 600025.

ABSTRACT:

Carbon dots (CDs) synthesized from biological sources has attracted much interest in bioimaging and biomedical applications due to their excellent biocompatibility and thus a facile synthesis of CDs with high fluorescence quantum yield (QY) is requisite for practical applications. In this work, we report a simple, rapid and green approach to synthesize photoluminescent CDs using eutrophic algal blooms as the carbon source. This method offers a possibility for large scale production of highly luminescent CDs (QY = 13%) with the average particle size ~8 nm. These CDs are highly water soluble and exhibit nanosecond fluorescence

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lifetime with high photostability, luminescence stability in different environments, low cytotoxicity and excellent cell permeability. Laser Scanning Confocal Microscopy shows the uptake of CDs by MCF-7 cells and the destined application of these CDs as a potential biomarker is demonstrated.

Keywords: Eutrophication, Microwave synthesis, Stability of CDs, Zeta potential, Photophysics of CDs, Cytotoxicity, MCF-7 cell imaging.

INTRODUCTION Eutrophication, the over enrichment of waters by nutrients, threatens and degrades many ecosystems around the world. The two most acute symptoms of eutrophication are hypoxia (or oxygen depletion) and harmful algal blooms, which among other things can destroy aquatic life in affected areas.1 Reduction of oxygen in water, release and accumulation of toxic substances in the water, pollution of aquatic environment, suffocation and death of aquatic organisms, drinking water treatment problems due to foul taste, color and unpleasant odor are the direct consequences of eutrophication while, shellfish poisoning, swimmer’s itch, decreasing bio-diversity, changes in species composition and dominance, global warming, climate change and loss in economy of a country are the indirect impacts of it.2 Hypoxia, considered to be the most severe symptom of eutrophication, has escalated over the past 60 years, increasing from about 10 documented cases in 1960 to at least 169 in 2007.3 Hypoxia occurs when algae and other organisms die, sink to the bottom, and are decomposed by bacteria, using the available dissolved oxygen lead to the formation of a hypoxic or “dead” zone.4 To mitigate hypoxia, elimination of algal blooms in water surface is necessary. So, what can we do with the tonnes and tonnes of these eutrophic


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algal biomass, a natural water pollutant? Conversion of these copious, ubiquitous, green, biowater pollutants into a useful material would be a suitable alternative. Some valuable contributions has made by M.Sevilla and M.M.Titirici research groups in converting the microalgae into hydrothermal microporous carbons.5,6 In this connection, we report on a rapid, one-step, green synthesis of highly luminescent carbon dots (CDs) from algal blooms, collected from a eutrophic stagnant water, as the carbon precursor. CDs, spherical nanoparticles with sizes below 10 nm, as a new member of the carbonaceous materials family, have attracted tremendous attention since their accidental discovery,7 owing to their advantageous properties such as stable photoluminescence (PL), broad excitation spectra, multicolored fluorescence, surface tunable functionalities, and water solubility. Although semiconductor quantum dots (QDs) such as CdS and CdSe provide several advantages, the use of toxic precursors for the preparation of QDs and the leaching of toxic metal ions from QDs into biological systems have raised safety issues.8,9 The alternative metal photoluminescent nanaomaterials such as gold nanodots and silver nanoclusters are suffered from the requirement of expensive precursors, lower QY values, and poor photostability. In contrast with them, CDs comprising nontoxic elements, in addition, they exhibit low cytotoxicity, excellent biocompatibility and lower environmental hazards which signify that CDs are potential substitute for conventional toxic metal-based semiconductor quantum dots especially in biomedical applications.8,10,11 For commercial production of CDs, one should be concerned with the cost and availability of the precursor and also with the ease, energy consumption, time consumption and cost of sophisticated instruments, materials and methods. Generally, CDs are mainly synthesized using top-down or bottom-up strategies.12,13 In the top-down approaches, C-dots are prepared through


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electro-oxidation,14 acid-assisted chemical oxidation,15 or laser ablation16 using graphite, lampblack or candle soot as the precursor. However, these approaches require complex and extreme synthetic conditions. For example, laser ablation and ion beam radiation suffer a main drawback as the use of expensive energy-consuming devices. With the use of strong acid, chemical oxidation of proper carbon source is away from the desirable green method. Thus, bottom-up strategy has received growing attention because the CDs, can be easily prepared via carbonization of precursors (such as citric acid,17 amino acid,18 and polyethylene glycol19) through hydrothermal treatment,20 thermal decomposition,21 and microwave-assisted method.22 Although, hydrothermal pyrolysis is an excellent route to produce CDs with high quantum yield, it requires the maintenance of high temperature and pressure for several hours (usually from 5 hr to 12 hr). Hence, it is obvious that the route is highly energy consuming which eliminates it from the consideration of green synthesis. That’s why the complicated multi-step procedure23 in the production of CDs from algae residue involving calcination at 600°C precludes the credit of being a green approach. On the contrary, microwave irradiation offers energy efficient, rapid, and uniform heating of the reaction medium, thus dramatically shortening the reaction time (usually few minutes) and improving the product yields and quality.24 Green synthesis of CDs is a highly attractive research topic, which exploits the use of natural, renewable precursors. Nevertheless, it is always exciting to explore the green sources of CDs because these are inexpensive, clean, nontoxic, and easily accessible. However, their fluorescence quantum yields were regretfully low (most of them lying below QY 6 times) to remove the unbound CDs from the surface of the cell membranes. Fluorescent images of the MCF-7 cells treated with CDs were acquired using a laser scanning confocal microscope (LEICA – TCS SP2 SE) with an excitation of 458 nm. The fluorescence images of CDs were examined to evaluate the cellular internalization of CDs. RESULTS AND DISCUSSION Characterization The size distribution and morphology of CDs were characterized by TEM (Fig. 1a). As shown in Fig. 1a, the as prepared CDs are quasispherical, monodispersed and are well separated from each other. The histogram in Fig. 1b shows the corresponding size distribution of CDs. The statistical diameter of the CDs is 8.5 ± 5.6 nm on analyzing about 100 particles as shown in Fig. 1b. An Xray diffraction (XRD) pattern of CDs (Fig. 1c) showed a single broad peak centred at 2θ = 24.1°, which is consistent with the (002) lattice spacing of carbon-based materials with abundant sp3 disorder. The interlayer spacing is 3.7 A˚, larger than that of bulk graphite (3.3 A˚), indicating


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poor crystallization. The selected area electron diffraction (SAED) pattern (inset of Fig. 1a) of CDs is in agreement with these results. Hence, the obtained XRD and SAED patterns confirm the amorphous nature of CDs. In Fig. 1d, the Raman spectrum of the carbon dots exhibits two peaks at 1352 and 1564 cm-1, corresponding to the D and G bands of carbon, respectively. The D band is associated with the vibrations of carbon atoms with dangling bonds in the termination plane of disordered graphite or glassy carbon. The G band corresponds to the E2g mode of the graphite and is related to the vibration of sp2-bonded carbon atoms in a two-dimensional (2D) hexagonal lattice. The ratio of ID/IG is 1.91, which is characteristic of the disorder extent and the ratio of sp3/sp2 carbon, implying that there is a plenty of structural defects in the CDs, during the oxidative microwave treatment. Further, the obtained ratio clearly depicts that the nanoparticles formed are amorphous carbon quantum dots and not graphene quantum dots. Hence, it is rational to name it as CDs. The XPS survey spectrum in Fig. 2a, reveals that the CDs are composed of carbon (59%) and oxygen (41%). A sign of Na was also noticed because of the residues of NaOH in the neutralization procedure of phosphoric acid treated CDs. The C 1s (Fig. 2b) and O 1s (Fig. 2c) spectra were deconvoluted into four and two constituent peaks respectively. The peaks at 286.2 eV in C 1s spectrum and at 531.0 eV in O 1s spectrum were attributed to the presence of C-O bonds on the surface of CDs. Similarly, the peaks at 288.7 eV in C 1s spectrum and at 533.6 eV in O 1s spectrum were assigned to the carboxyl C=O bonds on the surface. The presence of sp3 C-C/sp2 C=C at 284.8 eV in addition with a broad, π-π* shake-up satellite feature of sp2 carbon domain at 291.4 eV were observed in the C 1s spectrum of our CDs. We have recorded FTIR spectrum (Fig. 2d) for the CDs to identify its surface functionalities. A broad absorption from 3360 to 2550 cm-1, centered at 3050 cm-1; a sharp and strong absorption at 1763 cm-1; and a weak absorption at 1279 cm-1 are attributed to the O-H, C=O and C-O stretching


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vibrations respectively of carboxylic acid functionality. Absorption at 1763 cm-1 reveals that the surface carboxylic groups are unconjugated. Two weak absorptions at 2670 cm-1 and 2630 cm-1 are the fine structure bands of intramolecular hydrogen bonded O-H of carboxyl functional group which illustrate that the surface carboxyl groups are very close to each other, meaning that the CDs possess greater density of surface carboxyl groups. A strong absorption at 1590 cm-1, infers that the CDs are composed of well conjugated and/or aromatic C=C moieties (carbon core of CDs). Peaks at 2960 cm-1 and at 1495 cm-1 are assigned to the sp3 C-H stretching and in-plane bending vibrations respectively of surface methyl/methylene groups. Finally, the carbonization peak has appeared at about 570 cm-1. A highly negative apparent zeta potential value (Fig. S2, Supporting information) of -22.3 ± 8.39 mV, further substantiates that the CDs are negatively charged which is obvious for a carboxyl-rich nanoparticle on its periphery. From Raman spectrum, XPS, FTIR spectrum, and zeta potential value it is very clear that the degree of carboxylic acid functionalization is quite high in our CDs. Photophysical Studies The UV-Vis absorption and excitation spectrum of the CDs are shown in the Fig. 3a. The absorption spectrum exhibits two poorly resolved bands around 250 and 330 nm and extends to 580 nm without noticeable structures. According to previous studies,40 the absorption at 250 nm is ascribed to π−π* transition of aromatic C=C bonds, while the absorption at 320 nm attributes to n−π* transition of C=O bonds. C=O groups may originate from the surface of CDs, while C=C from the core. The absence of background absorbance in the visible region indicates the absence of other carbonaceous materials resulting from partial carbonization which usually absorb at higher wavelengths. As shown in the inset of Fig. 3a, the CDs display relatively strong blue luminescence under 365 nm UV irradiation (also see Fig. S1, Supporting information). The


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corresponding excitation spectrum shows two peaks at 258 nm and at 345 nm which matches well with the core and surface absorption respectively. Like most luminescent carbon nanoparticles, these CDs also exhibit an excitation-dependent photoluminescence (PL) behavior (Fig. 3b). The prominent emission is observed at 438 nm when excited at 360 nm. The corresponding normalized emission spectrum (Fig. S4, Supporting inormation) at increasing excitation wavelengths shows the red shift clearly. The quantum yield of the CDs is about 13% in comparison with quinine sulfate (QY 0.54 in 0.1 N H2SO4).

The 2D fluorescence

topographical map (Fig. 3c) of the CDs show two distinct contours (corresponding to core and surface) appears at the same emission maximum, at 433 nm i.e., irrespective of the excitation from the core absorption band or surface absorption band the PL maximum is observed at the same position. It portrays clearly that, even when the excitons are generated from the carbogenic core, they get trapped and relaxes at the surface defects before recombination.41 The PL decay curve measured at room temperature was performed to analyze the lifetime of the CDs at 440 nm (blue emission) with 375 nm excitation (Fig. 3d). The lifetime data of CDs were very well fitted to a tri-exponential function as shown in Fig. 3d and Fig. S6 (Supporting information). The parameters generated from iterative reconvolution of the decay with the instrument response function (IRF) are listed in the supportive information (table S5). The observed lifetimes of the CDs are τ1 = 1.01 ± 0.06 ns, τ2 = 2.53 ± 0.02 ns, and τ3 = 9.60 ± 0.37 ns, and the calculated average lifetime, τavg is 1.92 ns. The observed nanosecond lifetime of CDs, suggests that the synthesized CDs are most suitable for optoelectronic and biological applications.42 Stability of as-prepared CDs The CDs solution exhibits a long-term homogeneous phase without any noticeable precipitation at room temperature. In addition, the fluorescence intensity has no obvious change during long-


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term storage for 120 days (Fig. 4a). The observed zeta potential value (-22.3 ± 8.39 mV) is very high for a hydrophilic colloid which further supports the long-term storage stability of the CDs without significant aggregation or flocculation. The influence of pH change on the luminescence of CDs is analyzed by monitoring the luminescence intensity at various pH (Fig. 4b). The luminescence of the CDs is unaffected on a broad range of pH (from pH 3 to pH 11) which is a constructive property for a fluorescent probe to be employed on biological systems. In addition, the luminescence is almost unaffected over a broad range of ionic strength, from 0 M to 2 M as shown in the Fig. 4c. The CDs are subjected to UV irradiation for 100 min using a 365 nm UV lamp to investigate its photostability. It is found from Fig. 4d that the photostability of the CDs is superior when compared with that of rhodamine 6G (Rh6G), a commercial dye for cell imaging. All these results representing that our CDs are appropriate for bioimaging applications. Cell cytotoxicity by introducing CDs in MCF-7 cells We used MCF-7 cell line as a sample to test the inherent cytotoxicity of our CDs so that to test the practicality of the as-prepared CDs for cell imaging. The results show that the obtained CDs are scarcely toxic towards the cells (Fig. 5) even at doses (0.5 mg/mL, 12 h incubation time) substantially higher than the usual concentrations (

Outright Green Synthesis of Fluorescent Carbon Dots from Eutrophic

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