Facile green synthesis of nitrogen-doped carbon dots

Sensors and Actuators B 246 (2017) 497–509

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

Facile green synthesis of nitrogen-doped carbon dots using Chionanthus retusus fruit extract and investigation of their suitability for metal ion sensing and biological applications夽 Raji Atchudan a , Thomas Nesakumar Jebakumar Immanuel Edison a , Dasagrandhi Chakradhar b , Suguna Perumal c , Jae-Jin Shim a , Yong Rok Lee a,∗ a

School of Chemical Engineering, Yeungnam University, Gyeongsan 38541, Republic of Korea School of Foodscience and Biotechnology, Kyungpook National University, Daegu 41566, Republic of Korea c Department of Applied Chemistry, Kyungpook National University, Daegu 41566, Republic of Korea b

a r t i c l e

i n f o

Article history: Received 28 November 2016 Received in revised form 17 February 2017 Accepted 20 February 2017 Keywords: Chionanthus retusus Hydrothermal-carbonization Nitrogen-doped carbon dot Bioimaging Candida albicans Cryptococcus neoformans

a b s t r a c t Nitrogen-doped carbon dots (N-CDs) were synthesized from Chionanthus retusus (C. retusus) fruit extract using a simple hydrothermal-carbonization method. Their ability to sense metal ions, and their biological activity in terms of cell viability and bioimaging applications were evaluated. The resulting N-CDs were characterized by various physicochemical techniques such as high-resolution transmission electron microscopy (HRTEM), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), and Raman spectroscopy. The optical properties were characterized by ultraviolet visible (UV-vis) fluorescence spectroscopy techniques. The average size of the N-CDs was approximately 5 ± 2 nm with an interlayer distance of 0.21 nm, as calculated from the HRTEM images. The presence of phytoconstituent functionalities and the percentages of components in the N–CDs were confirmed by XPS studies, and a nitrogen content of 5.3% was detected. The N–CDs demonstrated highly durable fluorescence properties and low cytotoxicity with a quantum yield of 9%. The synthesized N–CDs were then used as probes for the detection of metal ions. The N–CDs exhibited high sensitivity and selectivity towards Fe3+ , with a linear relationship between 0 and 2 ␮M and a detection limit of 70 ␮M. The synthesized N–CDs are anticipated to have diverse biomedical applications, particularly for bioimaging, given their high fluorescence, excellent water solubility, good cell permeability, and negligible cytotoxicity. Finally, the potential of N–CDs as biological probes was investigated using fungal (Candida albicans and Cryptococcus neoformans) strains via fluorescent microscopy. We found that N–CDs were suitable candidates for differential staining applications in yeast cells with good cell permeability, localization with negligible cytotoxicity. Hence, N–CDs may find dual utility as probes for the detection of cellular pools of metal ions (Fe3+ ) and also for early detection of opportunistic yeast infections in biological samples. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Carbon dots (CDs), as a new family of nanomaterials, finds wide attention among the scientific community due to their unique properties, which includes high chemical stability, good biocompatibility, low toxicity, catalytic properties, electrical conductivity, excellent optical properties, and minimal photo bleaching. CDs

夽 Electronic Supplementary Information (ESI) available: Photographs of Chionanthus retusus; Confocal fluorescent microscopy images of C. albicans and C. neoformans yeasts after the uptake of N-CDs under bright field, green and red filters. ∗ Corresponding author. E-mail address: [email protected] (Y.R. Lee). http://dx.doi.org/10.1016/j.snb.2017.02.119 0925-4005/© 2017 Elsevier B.V. All rights reserved.

have a number of applications, especially as photoluminescents, sensors, energy sources, and bioimaging agents [1–9]. Among the CDs, nitrogen-doped carbon dots (N–CDs), have received much attention due to their specific properties and applications, one of which is highly enhanced photoluminescence. The most common methods for the synthesis of CDs/N–CDs are chemical oxidation, ultrasonic synthesis, hydrothermal-carbonization, solvothermal synthesis, microwave synthesis, and laser ablation. Of these, hydrothermal-carbonization is one of the most facile methods. Recently, it has proved to be an eco-friendly, simple, affordable, and soft chemical route for the synthesis of CDs/N–CDs in aqueous media, which produces excellent fluorescent probes for bioimaging applications [10–16]. In addition, one of the most attractive applications of N–CDs is in the field of chemical sens-

498

R. Atchudan et al. / Sensors and Actuators B 246 (2017) 497–509

ing is detection of metal ions, due to their easy fabrication, high quantum yield, tunable emission, low cost, low toxicity, good photostability, and environmental friendliness [17–21]. Iron is the most important transition metal in organisms and biological systems, playing important roles in various biological processes including oxygen transport and exchange, and enzymatic reactions at the cellular level. Thus, the detection of ions of this transition metal (particularly Fe3+ ) is highly coveted due to deepening concerns regarding industrial pollution [22]. Fe3+ is also known to play a vital role in health and disease. Iron imbalance has been linked to various disorders such as anemia, Alzheimer’s, and hemochromatosis, and can serve as a biological stimulus for commensal fungal pathogens such as Candida albicans (C. albicans) and Cryptococcus neoformans (C. neoformans) to become more virulent pathogens and cause secondary infections [23]. The application of fluorescent probes, observed using a fluorescence microscope, can provide information on cellular pools of metal ions and their responses to changes in biological stimuli [24]. Several previously described fluorophores for the detection of biological iron have been used for limited applications in living cells, and hence further development of this technology is essential. The present study was conducted to investigate the efficacy of N–CD synthesis using Chionanthus retusus (C. retusus) fruit extract as a carbon precursor and aqueous ammonia as a nitrogen source using a simple hydrothermal-carbonization method. C. retusus is a climacteric fruit that originates from the Eastern Asian continent, mainly Japan, Korea, Taiwan, and central to south China, that can reach a height of 20 m. The leaf blade is elliptical, oblong, or orbicular, sometimes ovate or obovate, and usually leathery. The white flowers are presented as cymose panicle inflorescences on lateral terminal shoots [25]. Photographs of different growth stages of C. retusus: flowers, unripe, and ripe fruits (purple or dark bluish black) are shown in Fig. S1. C. retusus fruit is an inexpensive and easy to get, it is commonly called Chinese fringetree, is a member of the family Oleaceae and is a prominent food source for birds and wildlife. In general, such fruit contains many chemical components such as phenolic compounds and polysaccharides, which serve as abundant sources of carbon for the preparation of N–CDs. The phenolic compounds and polysaccharides are wealthy in C. retusus fruit. So that, the C. retusus fruit was used as a source for the synthesis of durable fluorescence N–CDs with rich nitrogen, carbonyl and hydroxyl groups. The synthesized N–CDs were used as probes for the detection of metal ions. The rich nitrogen and oxygen on the surface of N–CDs was more affinity with Fe3+ which strongly quenched the fluorescence intensity of N–CDs. In addition, the highly durable water soluble N–CDs were evaluated as fluorescent probes for the selective staining of prokaryotic and eukaryotic

cells using bioimaging in yeast (Candida albicans and Cryptococcus neoformans) pathogens using confocal laser scanning microscopy. To the best of our knowledge, this is the first time C. retusus fruit has been used for the synthesis of N–CDs. The resulting N–CDs exhibited good biocompatibility, meaning that they can be used as staining probe for fluorescence bioimaging and metal ion detection.

2. Experimental 2.1. Materials C. retusus fruit was collected from the Yeungnam University campus, Gyeongsan, Republic of Korea. An aqueous ammonia solution (25%) and quinine sulphate were purchased from SigmaAldrich, and were used without further purification. The analytical reagent grade of metal salts used, AlCl3 , CaCl2 , Cd(CH3 OO)2 , Co(OOCH3 )2 , CrCl3 , CuCl2 , FeCl2 , FeCl3 , HgCl2 , NiCl2 , Pb(NO3 )2 , and ZnCl2 were purchased from Ducksan chemicals and were used as received. The yeast strains C. albicans and C. neoformans were procured from the Korean Cell Type Culture Collection (KCTC), Seoul, Republic of Korea, maintained in 10% v/v glycerol stocks, and stored at −80 ◦ C until further use. Overnight cultures in malt extract (ME) broth were used for the bioimaging experiments. Deionized (DI) water was used throughout this study.

2.2. Preparation of N–CDs The collected C. retusus fruits were washed thoroughly, firstly in running tap water, then in DI water, and were ground using a mixer grinder. The ground fruits were filtered through cotton, followed by Whatman filter paper, and washed with a small amount of DI water. The fruit extract was collected and stored in a clean glass bottle at 5 ◦ C until further use. The procedure for the synthesis of N–CDs was as follows: initially, 25 mL of C. retusus fruit extract was transferred to a Teflon equipped stainless steel autoclave with 50 mL inner volume. Then 1 mL of aqueous ammonia solution was added for the functionalization of the carbon materials with nitrogen. The autoclave was sealed tightly, and heated at 180 ◦ C for 6 h in a hot air oven. The resultant solution was cooled down to room temperature in air. After completion of the hydrothermal process, the solution changed from pale yellowish brown to dark brown due to the formation of N–CDs. The resultant solution was filtered with membrane filter paper to remove the larger or agglomerated carbon particles. The filtrate was collected and stored in a glass vial at 5 ◦ C until further use. Fig. 1 shows a schematic illustration of the preparation and formation mechanism of N–CDs.

Fig. 1. Schematic illustration for the preparation and formation mechanism of N–CDs.


2.3. Characterization methods The synthesized N–CDs were characterized by various analytical techniques including UV–vis spectroscopy, fluorescence spectroscopy, X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), high resolution transmission electron microscopy (HRTEM), and Raman spectroscopy. The optical properties of the synthesized N–CDs were analyzed using an OPTIZEN 3220UV and Hitachi–7000 F spectrometer. The excitation wavelength was varied to determine the maximum luminescence intensity for the fluorescence measurements. The slit width was fixed at 5 nm and the scan speed was set to 240 nm/min. XRD patterns were obtained on a PANalytical X’Pert3 MRD diffractometer using Cu K␣ radiation ( = 1.54 Å, 40 kV, and 30 mA) at an angle of 2. The XRD patterns of N–CDs were recorded in the range 10–90◦ (2). The chemical composition of synthesized materials were examined through XPS analysis and XPS was performed using a K-Alpha (Thermo Scientific). Casa XPS instrument software was used for the deconvolution of the XPS expanded spectrum. HRTEM images of N–CDs were obtained using a FEI-Tecnai TF-20 operated at an acceleration voltage of 200 kV. Samples for HRTEM were prepared by placing droplets of N–CDs suspended in DI water onto a carboncoated polymer micro grid, supported on a Cu grid, and dried at ambient temperature under static conditions. The elemental composition of the synthesized N–CDs was subsequently analyzed using an energy-dispersive X-ray (EDX) spectrometer attached to the HRTEM. Raman spectra of the N–CDs were recorded on a Thermo Scientific DXR SmartRaman spectrometer with a laser excitation of 532 nm. 2.4. Quantum yield measurement The quantum yield of the synthesized N-CDs (QYN-CDs ) was calculated by measuring the fluorescence intensity in an aqueous dispersion by using the following Eq. (1) [26,27]: QYN−CDs = QYR

IN−CDs AR (nN−CDs )2 IR AN−CDs (nR )2

(1)

where, QYN-CDs and QYR are the fluorescence quantum yield of N-CDs and reference, respectively. (Reference: Quinine sulphate, QYR = 0.54), ICDs and IR are the integrated fluorescence intensity of N-CDs and reference, respectively. ACDs and AR are the UV–vis absorption intensity of N-CDs (0.835) and reference (0.821), respectively. n is the refractive index (1) of the solvent for both N-CDs and the reference.

499

28 ◦ C. For staining, yeast (1 × 106 CFU/mL) was washed twice and re-suspended in phosphate buffered saline (PBS, pH 7.4). The cell suspension was mixed with 10 ␮L of N–CDs in distilled water and incubated stationary at 28 ◦ C. At designated time points, the cell suspension (1 mL) was aseptically withdrawn and centrifuged at 6000 rpm for 10 min. The pellet was washed twice with PBS and re-suspended in 0.1 mL of PBS. About 10 ␮L volume of the culture suspension was spotted onto an agarose pad mounted on a microscopic glass slide. Fluorescence imaging was performed using a Nikon eclipse 80 I laser scanning confocal microscope. The fluorescence was observed in deep UV, blue, green and red regions with excitation at 248, 408, 488 and 561 nm respectively. 2.7. Growth inhibition determination The growth inhibiting activity of N–CDs was determined against C. albicans using a broth macrodilution method. Briefly, yeast cells (6 × 106 CFU/mL) were seeded in 10 mL ME medium in glass tubes containing various concentrations of the test agent (range, 5–100 ␮L). The tubes were incubated stationary at 28 ◦ C for 24 h. At regular intervals, the optical density of the medium was measured using a UV–vis spectrophotometer (JASCO, UV530, Japan). Mean% cell growth ± SD of triplicate values was calculated. 2.8. Determination of cytotoxicity of N–CDs in C. albicans The cytotoxicity of N–CDs against the yeast strain, C. albicans (KCTC-11282), was evaluated by determining the redox activity of C. albicans cells using iodonitrotetrazolium salt (INT). Briefly, C. albicans (5 × 106 CFU/mL), prepared in 10 mL of pre-filtered PBS, was added with a concentration gradient of the test agent prepared in water. 50 ␮L of 10 ␮M INT solution was added to each solution, and they were incubated in dark at 28 ◦ C for 6 h. At every 60 min interval, a 1 mL cell suspension was aseptically withdrawn from the tube and centrifuged to pellet the cells. The cells were extracted with 0.5 mL of DMSO and the extracted red iodonitrotetrazolium formazan product was spectrophotometrically measured at 545 nm. Sodium azide (50 ␮M) was used as a positive control. Each experiment was repeated twice and the mean ± SD value of respiratory activity was calculated. The percentage redox activity was determined as per the following Eq. (2): Cell redox activity/Cell viability (%) =

Abs(S) − Abs(B) Abs(C) − Abs(B)

× 100 (2)

where, Abs(S) , Abs(B) , and Abs(C) are the absorbance of the sample, blank, and control, respectively.

2.5. Detection of Fe3+ ions The procedure for the detection of Fe3+ ions was as follows: the fluorescence spectrum of a 1 mL suspension of synthesized N–CDs (0.5 mL of suspension + 0.5 mL of DI water) was recorded and the maximum fluorescence intensity was set as F0 (blank/control). Subsequently, 0.5 mL (500 ␮M) of different metal ions such as Al3+ , Ca2+ , Cd2+ , Co2+ , Cr3+ , Cu2+ , Fe2+ , Fe3+ , Hg2+ , Ni2+ , Pb2+ , and Zn2+ were mixed with the above suspension of synthesized N–CDs (0.5 mL) by gentle shaking. All the experiments were carried out under the same conditions at room temperature, and the corresponding fluorescence spectra were recorded. The highly sensed metal ions were identified and the detection experiments were repeated for these at lower concentrations (0–500 ␮M). 2.6. Fluorescence microscopy Yeast strains C. albicans (KCTC-11282) and C. neoformans (KCTC50554) were grown in ME broth (BD science, CA) overnight at

3. Results and discussion 3.1. Structural characterization of synthesized N–CDs The morphological characteristics of the synthesized N–CDs were observed using HRTEM and the corresponding images (Fig. 2a and b) shows that the synthesized N–CDs were nearly spherical and well dispersed without apparent aggregation. The N–CDs are marked by dotted circles and had well-ordered lattice fringes (Fig. 2b). The lattice fringes of the N–CDs, with an interlayer distance of 0.21 nm, may correspond to the facet of graphitic carbon (100) [28–31]. Thus, the N–CDs consisted of a nanocrystalline sp2 carbon core. The resulting N–CDs appeared relatively uniform in size. Their diameter ranged from 3 to 7 nm, with an average size of 5 ± 2 nm, as shown in the HRTEM images. The particle size distribution is displayed in Fig. 2c, showing a narrow size distribution. The results of the high angle annular dark field scanning transmission electron microscopy (HAADF-STEM) and the corresponding

500


Fig. 2. HRTEM images with different magnification (a and b), particle size distribution graph (c), HAADF image (d), and corresponding elemental mapping images (e) carbon (C), (f) nitrogen (N), (g) oxygen (O) and (h) EDX spectroscopy image of synthesized N–CDs.

elemental mapping (carbon (C), nitrogen (N) and oxygen (O)) of the synthesized N–CDs are shown in Fig. 2d–g. Consistent with the HAADF-STEM elemental mapping, EDX analysis (Fig. 2h) confirmed that the synthesized N–CDs are composed of elements C, N, and O. FT-IR spectroscopy technique has been used extensively for the identification of functional groups of the resulting materials. The C. Retusus fruits extract and synthesized N–CDs in DI water solutions were dried under freeze drying for the analysis. Fig. 3 shows the FTIR spectra of the C. Retusus fruits extract and synthesized N–CDs. The broad absorption band around 3235 cm−1 suggests that the stretching vibrations of −NH and −OH functional groups are interconnected by a hydrogen bond in N–CDs. The absorption band slightly shifted to the lower wavenumber from the OH functional groups (3290 cm−1 ) of the fruits extract which might due to interconnection of NH and OH functional groups. The absorption bands at 2935 and 2875 cm−1 were assigned to the asymmetric and symmetric stretching vibrations of CH bonds, respectively. The band at 1720 cm−1 can be attributed to C O vibration is clearly seen in the FTIR spectrum of our synthesized N–CDs. The strong absorption band of the aromatic C C stretching vibration at 1600 cm−1 indicates the presence of a sp2 hybridized honeycomb lattice. The stretching vibration of the C C intensity increased for the synthesized N–CDs, which may be due to the influence of the formation of N–CDs during hydrothermal-carbonization. The small absorption bands at 1455 cm−1 originates from the stretching vibrations of aromatic C N heterocycles were observed in synthesized N–CDs, which is consistent with the XPS analysis. The absorption band at wavenumber of 1405 cm−1 can be assigned as originating from

out-of-plane bending or wagging vibration of OH (C OH) groups. The absorption band at 1250 cm−1 was assigned to the stretching vibration of C O (acid/C OH) groups from the fruits extract which was absence in the synthesized N CDs. The stretching vibrations of epoxy C O C groups was exhibited by the peaks at 1035 cm−1 for the fruits extract but in case of synthesized N CDs, epoxy C O C (1035 cm−1 ) band merged with C NH C (1080 cm−1 ) band which

Fig. 3. FTIR spectra of C. Retusus fruits extract and the synthesized N–CDs.


confirm the presence of nitrogen moiety functionalized on the CDs [32,33]. The shape and peak positions of the FTIR spectrum of the synthesized N–CDs are quite different from that of C. Retusus fruits extract, implying the corresponding change in the coordination environment of various functional groups in the N–CDs after the hydrothermal-carbonization. Moreover, the FTIR spectra reveals that the presence of amine, acid and hydroxyl moieties on the surface of the synthesized N–CDs without any other impurities and are well in agreement with literature work [34]. The synthesized N–CDs were characterized by XPS to clarify their chemical composition. The XPS scans of synthesized N–CDs show that they contain three elements: a carbon level (C1s), a nitrogen level (N1s), and an oxygen level (O1s) (Fig. 4a). The content of carbon, nitrogen and oxygen at the peak positions of 285, 400, and 532 eV was 77.93, 5.30, and 16.77% respectively. XPS high-

501

resolution spectra of C1s, N1 s and O1 s are shown in Fig. 4b–d, respectively. XPS analysis of the C1 s spectrum revealed the presence of three peaks at 284.4, 285.4, and 287.3 eV, and they are attributed to C C (sp2 )/C C (sp3 ), C N/C N/C O, and C O, respectively [35]. Notably, the energy peak at 284.4 eV clearly indicated that the synthesized N–CDs possessed predominantly sp2 carbons [36,37]. In the high-resolution N1s spectrum, two binding energy peaks at 399.4 and 401 eV could be attributed to C N/C3 N and N H groups, respectively, on the surface of CDs [38]. XPS fitting of O1 s showed two binding energy peaks at 530.4 and 532.1 eV, which were ascribed to C O and C OH/C O C, respectively [39,40]. The XPS results strongly suggested that the synthesized N–CDs contained carbon, nitrogen, and oxygen [41], which was further supported by FTIR spectroscopy analysis and HRTEM with EDX elemental mapping.

Fig. 4. XPS spectra of (a) survey scan, (b) carbon, (c) nitrogen and (d) oxygen for the synthesized NCDs; Wide-angle XRD pattern (e) and Raman spectrum (b) of synthesized N–CDs.

502


The synthesized N–CDs were characterized by powder XRD to investigate the phase and degree of crystallinity. The XRD pattern (Fig. 4e) of the synthesized N–CDs exhibited two diffraction peaks at around 23.2 and 42◦ , which corresponded to graphite lattice spacing (002) and (100), respectively [42–44]. The XRD patterns revealed the moderate graphitic structure of synthesized N–CDs, due to the functionalization of the nitrogen moiety present on the CDs, which was confirmed by XPS analysis. The d-spacing values of 0.38 and 0.21 nm were calculated using the Bragg equation (3) based on the lattice spacing (002) and (100), respectively, and match well with the HRTEM results. The d-spacing value of 0.38 is larger than that of bulk graphite (∼0.34–0.35 nm), possibly due to the oxygen- and nitrogen-containing groups introduced in the synthesis process, which increase the interlayer distance. Additionally, the increase in d-spacing value indicates that the phase tends to be partially amorphous [45]. n␭ = 2dsin

(or)

d=

n␭ 2sin

increased from 300 to 340 nm, and then decreased as the excitation wavelength increased from 350 to 420 nm [56]. The excitationdependent emission wavelengths are shown in Fig. 5d. While the excitation wavelength increased from 300 to 420 nm, the emission wavelength noticeably increased from 407 to 497 nm with variable fluorescence intensity, which may be attributed to the different particle sizes or various surface energy traps of the N–CDs [57]. The florescence intensity of synthesized N–CDs was examined with respect to temperature. Fig. 5e shows the florescence emission spectra of the synthesized N–CDs at different temperatures. The fluorescence intensity decreased when the temperature increased from 30 to 85 ◦ C, but the emission wavelength did not shift with varied temperature (Fig. 5f). Therefore, the stability of photoluminescence of the synthesized N–CDs was excellent under various temperatures, vivid they can be applied in different environments [58]. Thus, the synthesized N–CDs possessed a high florescence intensity with excellent applicability.

(3)

where, n is a positive integer (1), ␭ is the wavelength of the incident X-ray (␭ = 1.54 Å) and is the angle between the incident rays and the surface of the materials. The Raman spectrum of the synthesized N–CDs is shown in Fig. 4f. The spectrum shows two distinct Raman signals at ca. 1350 and 1595 cm−1 , dubbed as D and G-bands, respectively, under our measurement conditions. The D and G-bands originate from disordered carbon and the sp2 -hybridized carbon cluster, respectively [46–49]. The peak intensities of the D-band and G-band are denoted as ID and IG , respectively. Graphitization of carbon nanostructured materials was determined using the ID /IG values [50]. The ID /IG value was ca. 0.75 and the intensity ratio of the D to the G-band indicates that the synthesized N–CDs had a moderate graphitic nature because of its strong fluorescence background [51,52]. The Raman result suggests that the N–CDs had high purity with moderate graphitization. 3.2. Optical properties of synthesized N–CDs Fig. 5 shows the UV–vis absorption spectrum and corresponding fluorescence (FL) spectra of synthesized N–CDs in an aqueous solution (water), confirming their remarkable optical properties. The UV–vis spectrum (Fig. 5a) exhibits two characteristic absorption peaks at ca. 269 and 301 nm, which could be attributed to the ␲–␲* and n–␲* transitions of the carbonyl group (C C/C O) and amine moiety present in the N–CDs, respectively [53]. From the photographic image shown in Fig. 5a inset, the diluted N–CDs aqueous solution is pale yellow under ambient light but exhibits a very intense blue color when irradiated with a UV–light (␭ = 365 nm), further indicating the blue fluorescent property of the N–CDs. The strong fluorescence of the N–CDs may result from the emissive traps of the nitrogen-doped surface [54,55]. The excitation and emission fluorescence spectra of the synthesized N–CDs are shown in Fig. 5b. The spectra show that the maximum excitation started at 340 nm and maximum emission began at 425 nm. Highly bright blue fluorescence can be clearly seen at an emission wavelength of 425 nm in the fluorescence emission spectrum. The QYN-CDs was measured at an excitation wavelength of 340 nm, and the yield was around 9% based on Eq. (1). These fluorescence spectral results strongly suggests that the synthesized N–CDs exhibits a high quantum yield with durable fluorescence. Fig. 5c shows the excitation-dependent emission spectra of the synthesized N–CDs. When the excitation wavelength changes from 300 nm to 420 nm, the maximum fluorescence intensity are observed individually at 340 nm and 425 nm for excitation wavelength and emission wavelength, respectively with a Stokes shift of 85 nm. The fluorescence intensity gradually increased while the excitation wavelength

3.3. Analytical sensor activity of synthesized N–CDs The ability of synthesized N–CDs to sense different metal ions was evaluated, and we found that Fe3+ can quench the fluorescence of N–CDs. Fig. 6a shows the FL spectra of N–CDs in the presence of various metal ions including Al3+ , Ca2+ , Cd2+ , Co2+ , Cr3+ , Cu2+ , Fe2+ , Fe3+ , Hg2+ , Ni2+ , Pb2+ , and Zn2+ all at a concentration of 500 ␮M to evaluate the selectivity of the sensing system. Remarkably, the different metal ions had varying effects on FL intensity, however, only Fe3+ noticeably quenched the FL intensity, as compared with the other metal ions that demonstrated negligible quenching ability. Fig. 6b shows the (F0 -F)/F0 values of synthesized N–CDs in the presence of various metal ions, of which Fe3+ exhibits the highest value. This result suggested that the synthesized N–CDs had outstanding selectivity (outstanding association) for Fe3+ , and the other metal ions had little or no influence on the sensing system. As shown in Fig. 6c and d, the fluorescence intensity of N–CDs was notably quenched in the presence of Fe3+ at concentrations between 0 and 500 ␮M. The selectivity investigation (F0 -F)/F0 versus concentration of N–CDs shows the quenching of fluorescence intensity with types of static and dynamic [59]. If the plot is not straight line/or straight at low concentration and curved at high concentration of analyte (Fe3+ ) while lifetime of the free probe and its complex are same, then it is static quenching i.e. complexation takes place at ground state. In case, life-time of the free probe and its complex are different, then energy transfer occurs through collision and/or close interaction of ␲–␲ overlap in excited state via resonance energy transfer and called dynamic quenching [60]. The synthesized N–CDs exhibits a wide range of quenching/detection ability which might useful for the selective quenching of Fe3+ from the industrial waste (pollution). A strong linear relationship was observed between the fluorescence response of N–CDs and the Fe3+ concentration in the concentration range of 0–2 ␮M (R2 = 0.994) clear from Fig. 6e and f. The limit of detection (LOD) was 70 ␮M calculated based on Eq. (4). The high sensitivity together with the high selectivity for Fe3+ make the N–CDs an ideal fluorescent probe for highly efficient detection of Fe3+ [61]. LOD =

3␴ Slope

(4)

where, ␴ is the standard deviation of the blank signals (n = 10). Apart from the fluorescence spectroscopy study of quenching/detection, the effects of Fe3+ on the UV–vis absorption spectrum of N–CDs was also studied. Fig. S2a shows the UV–vis spectra of synthesized N–CDs suspension before and after addition of Fe3+ . The UV–vis absorption spectrum of N–CDs changed significantly by the addition of Fe3+ . The absorption peaks at 269 and 301 nm were nearly disappeared which demonstrate the presence of Fe3+


503

Fig. 5. (a) UV–vis spectrum, (b) excitation and emission fluorescence spectra, (c) excitation dependent fluorescence (emission) spectra, (d) temperature dependent fluorescence spectra, (e) excitation dependent maximum emission wavelength and (f) temperature dependent maximum emission wavelength at constant excitation of resulting N–CDs.

can influence the surface states of the N–CDs. This results strongly demonstrate that the present sensor has outstanding selectivity towards Fe3+ sensing. 3.4. Mechanism for synthesis and fluorescence quenching of N–CDs The mechanism of fluorescence of CDs/N–CDs is still debated, although it is often credited to surface defects, recombinant, and quantum effects. The proposed formation mechanism of N–CDs is illustrated in Fig. 1. The probable sequence is dehydration, followed by polymerization, and finally carbonization/aromatization. Initially, the nitrogen dopant (ammonia) might react with the

carbonyl groups in the C. retusus fruit extract, which forms stable ammonium salts. Beyond 100 ◦ C, the hydroxyl groups undergo dehydration, subsequently, polymerization yields water soluble polymers. Eventually, the polymers undergo aromatization/carbonization via condensation and cycloaddition due to the high catenation ability of carbon. The resulting material is durable fluorescent N–CDs [62]. Fig. 7 shows a schematic illustration of the fluorescence quenching mechanism of N–CDs in the presence of Fe3+ . There are several reasons behind the strong quenching of fluorescence intensity of N–CDs by Fe3+ , which exhibits a higher affinity for nitrogen and oxygen on the surface of N–CDs. Accordingly, the fluorescence quenching could be believed to be related to the non-radiative electron transfer process between the N–CDs

504


Fig. 6. Fluorescence quenching efficiency of synthesized N–CDs in the presence of (a) and (b) different metal ions (ions concentration: 500 ␮M) and (c) different concentration of Fe3+ from 0 to 500 ␮M, (d) selectivity investigation (F0 -F)/F0 of N–CDs, (e) concentration of Fe3+ from 0 to 2 ␮M and (f) selectivity investigation (F0 -F)/F0 of N–CDs.

and Fe3+ [63]. The oxygen-containing functional groups such as hydroxyl and carboxyl contribute to the water solubility and strong interaction with metal ions, which enable the N–CDs to serve as a fluorescent probe for the highly sensitive and selective detection of Fe3+ , with a detection limit of 70 ␮M [64]. The high sensitivity of synthesized N–CDs could be attributed to the formation of complexes between Fe3+ and the phenolic hydroxyl and/or amine groups. Therefore, the abundant phenolic hydroxyl and/or amine groups on the surface of the synthesized N–CDs may coordinate with Fe3+ which is clear from the Eq. (5) [65]. 6CD–OH/CD–NH2 + Fe3+ → [Fe(OCD)6 ]3− /[Fe(NHCD)6 ]3− + 6H+ (5)

After quenching the fluorescent N–CDs was aggregated by complex formation between the Fe3+ and N–CDs. Fig. S2b shows the HRTEM image of resulting N–CDs after quenching of fluorescent N–CDs. HRTEM images show that the resulting N–CDs were dispersed with apparent aggregation. The monodisperse N–CDs turned to aggregated N–CDs by the addition of Fe3+ which is due strong affinity between the Fe3+ and functional groups (hydroxyl and/or amine) of N–CDs. The results suggest that the Fe with N–CDs complex formation occurred by the addition of Fe3+ . 3.5. Biological activity of the synthesized N–CDs 3.5.1. Fluorescence microscopy study The fluorescence microscopy studies using yeast strains (C. albicans and C. neoformans) suggested that N–CDs at 10 ␮L differ-


505

Fig. 7. Schematic illustration for the plausible fluorescence quenching mechanism of synthesized N–CDs in presence of Fe3+ ions.

Fig. 8. Confocal fluorescent microscopy images of C. albicans (KCTC-11282) yeast after the uptake of N–CDs for 2 and 6 h. The fluorescent images were obtained confocal laser scanning microscopy in bright field (BF), deep UV (248 nm) and UV (408 nm, blue) lasers filters. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

entially stain the yeast cells. C. albicans cells treated with 10 ␮L of N–CDs for 2 h exhibited intense fluorescence at deep UV (286 nm) and blue (408 nm) excitation wavelengths, and this fluorescence remained stable over a period of 6 h as the majority of the cell population retained fluorescence (Fig. 8). However, in the case of C. neoformans, the maximum fluorescence intensity was visualized only up to 2 h, and the fluorescence intensity was slightly

reduced towards the end of the experiment (Fig. 9). The yeast cells failed to exhibit any fluorescence in the green and red laser zones [see the Electronic Supplementary Information (ESI), Figs. S3 and S4]. The untreated control cells of both the strains did not exhibit any detectable auto-fluorescence. Further observations clearly indicated strong fluorescence emitted from N–CDs in the cytosol, showing a blue color upon excitation at 488 nm, suggest-

506


Fig. 9. Confocal fluorescent microscopy images of C. neoformans (KCTC-50554) yeast after the uptake of N–CDs for 2 and 6 h. The fluorescent images were obtained confocal laser scanning microscopy in bright field (BF), deep UV (248 nm) and UV (408 nm, blue) lasers filters. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

ing that N–CDs were internalized in the cytoplasm via endocytosis. As the nucleus in yeast strains remains towards the polar ends of the cell, slightly increased fluorescence towards one end of the cell in both C. albicans and C. neoformans suggested that N–CDs may have interacted with the nuclear material. The N–CDs were hydrophilic in nature and possessed various functional groups (nitrogen, carbonyl and carboxylic acid) that aid in effective engulfment into cellular interiors, and irreversible interaction with the nuclear material. However, N–CDs did not exhibit any fluorescence in the case of Gram-positive and Gram-negative bacteria (Data not shown). The selective fungal staining ability of N–CDs can be explained in terms of the differences in the processes related to cellular uptake of small molecules between bacteria and fungi. Various additional efflux pumps are thought to operate in Gram positive and Gram negative bacteria against various small molecules. However, a further detailed study of bioimaging multiple bacterial strains is warranted.

3.5.2. Growth inhibition study In the growth inhibition assay, the test yeast strains (C. albicans and C. neoformans) grown in the presence of varying concentrations of N–CDs did not exhibit considerable growth inhibition during a 24 h incubation. The growth curves of the test yeast strain were not significantly different from the control, as the growth curves appeared to be similar to untreated growth controls (Fig. 10a). However, N–CDs at a 20-fold increased concentration (100 ␮L) slightly reduced the stationary phase of growth culture and resulted in an overall reduced cell growth rate. However, within a 6 h incubation period there was no profound change in the growth characteristics of the tested strain. Furthermore, only 10 ␮L of the N–CDs was used for bioimaging purposes, suggesting that N–CDs can be utilized as potent in vitro fluorescent probes for detection of fungal infections in biological samples. 3.5.3. Cell viability The respiratory activity of C. albicans in the presence of varying concentrations of the test agent did not differ from the untreated

Fig. 10. (a) Time growth kinetics of C. albicans in the presence of N–CDS. The growth of the C. albicans was determined by tube macro dilution method. Mean ± SD growth of 2 independent experiments with duplicates was represented and (b) respiratory activity of C. albicans in the presence of varying concentrations of N–CDS. The respiratory activity of cells was determined using idonitrotetrazolium chloride. The produced iodonitrotetrazolium formazan (INF) was quantified at 545 nm. Sodium azide (NaN3 ) was used as positive control. Each experiment was repeated twice and mean ± SD of four replicate value was represented.


controls. Only at higher concentrations (>80 ␮L) of the test agent was there a 5.5 ± 3.3% reduction in viability within 2 h, which reached a 14.8 ± 4.1% decrease after an incubation period of 6 h compared to untreated controls. However, volumes lower than 80 ␮L of N–CDs caused no considerable difference in redox activity in comparison with untreated controls after both 2 h and 6 h incubation periods (Fig. 10b). The positive control (sodium azide) at 50 ␮M significantly inhibited redox activity, resulting in a 71.7 ± 0.8% reduction in viability after 6 h of incubation. From these studies, it was evident that the concentration of N–CDs used for bioimaging purposes did not exhibit any considerable cytotoxicity in C. albicans. Similar profiles were also obtained for other test strains (data not shown).

4. Conclusions N–CDs were successfully synthesized in a green and facile manner by a one pot hydrothermal-carbonization process using C. retusus fruit extract and aqueous ammonia as the carbon precursor and nitrogen moiety, respectively. The resulting N–CDs had a narrow size distribution without apparent aggregation and were nearly spherical. The average size was ca. 5 ± 2 nm with an interlayer distance of 0.22 nm. The chemical content of N–CDs with carbon, nitrogen and oxygen was revealed by XPS analysis, and vital nitrogen functional groups with 5.3%. Based on the excellent water solubility and durable high fluorescence intensity, the synthesized N–CDs were used as a promising fluorescent probe for highly sensitive and selective direct detection of Fe3+ , with a detection limit of 70 ␮M, without any further chemical modification. The UV–vis absorption peaks were disappeared for N–CDs with Fe3+ complex which strongly demonstrate that the present sensor has outstanding selectivity towards Fe3+ . The synthesized N–CDs exhibits a wide range of quenching/detection ability which might be useful for the selective quenching of Fe3+ from the industrial waste. Furthermore, the synthesized N–CDs exhibited good cell permeability and acted as a successful probe for the bioimaging of yeast cells such as C. albicans and C. neoformans. The N–CDs were effectively internalized into the fungal cells with strong fluorescent signal from N–CDs. The results of MIC and redox activity tests (cytotoxicity) on yeast in the presence of N–CDs demonstrated the highly biocompatible nature of N–CDs. Furthermore, only 10 ␮L of the N–CDs was used for bioimaging purposes, suggesting that N–CDs can be utilized as potent in vitro fluorescent probes for the detection of fungal infections in biological samples. Overall, the present work describes a new and green method to produce fluorescent N–CDs, for the sensing of Fe3+ and various biological and clinical applications in the near future.

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 (grant number 2012M3A7B4049675). This work was also supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (grant number NRF2014R1A2A1A11052391) and Priority Research Centers Program (grant number 2014R1A6A1031189).

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2017.02.119.

507

References [1] S. Sarkar, D. Banerjee, U.K. Ghorai, N.S. Das, K.K. Chattopadhyay, Size dependent photoluminescence property of hydrothermally synthesized crystalline carbon quantum dots, J. Lumin. 178 (2016) 314–323. [2] S. Niino, S. Takeshita, Y. Iso, T. Isobe, Influence of chemical states of doped nitrogen on photoluminescence intensity of hydrothermally synthesized carbon dots, J. Lumin. 180 (2016) 123–131. [3] L. Wang, Y. Bi, J. Hou, H. Li, Y. Xu, B. Wang, H. Ding, L. Ding, Facile, green and clean one-step synthesis of carbon dots from wool: application as a sensor for glyphosate detection based on the inner filter effect, Talanta 160 (2016) 268–275. [4] L. Song, Y. Cui, C. Zhang, Z. Hu, X. Liu, Microwave-assisted facile synthesis of yellow fluorescent carbon dots from o-phenylenediamine for cell imaging and highly sensitive detection of Fe3+ and H2 O2 , RSC Adv. 6 (2016) 17704–17712. [5] Y. Iqbal, X. Tian, D. Wang, Y. Gong, K. Guoa, Z. Iqbal, W. Wang, W. Qin Liu, Carbon dots prepared by solid state method via citric acid and 1, 10-phenanthroline for selective and sensing detection of Fe2+ and Fe3+ , Sens. Actuators B-Chem. 237 (2016) 408–415. [6] R. Atchudan, T.N.J.I. Edison, S. Perumal, Y.R. Lee, Green synthesis of nitrogen-doped graphitic carbon sheets with use of Prunus persica for supercapacitor applications, Appl. Surf. Sci. 393 (2017) 276–286. [7] W. Li, Z. Yue, C. Wang, W. Zhang, G. Liu, An absolutely green approach to fabricate carbon nanodots from soya bean grounds, RSC Adv. 3 (2013) 20662–20665. [8] J. Jana, M. Ganguly, B. Das, S. Dhara, Y. Negishi, T. Pal, One pot synthesis of intriguing fluorescent carbon dots for sensing and live cell imaging, Talanta 150 (2016) 253–264. [9] J. Yu, N. Song, Y.K. Zhang, S.X. Zhong, A.J. Wang, J. Chen, Green preparation of carbon dots by Jinhua bergamot for sensitive and selective fluorescent detection of Hg2+ and Fe3+ , Sens. Actuators B 214 (2015) 29–35. [10] R. Atchudan, T.N.J.I. Edison, M.G. Sethuraman, Y.R. Lee, Efficient synthesis of highly fluorescent nitrogen-doped carbon dots for cell imaging using unripe fruit extract of Prunus mume, Appl. Surf. Sci. 384 (2016) 432–441. [11] W. Liu, H. Diao, H. Chang, H. Wang, T. Li, W. Wei, Green synthesis of carbon dots from rose-heart radish and application for Fe3+ detection and cell imaging, Sens. Actuators B-Chem. 241 (2017) 190–198. [12] T.N.J.I. Edison, R. Atchudan, J.J. Shim, M.G. Sethuraman, Y.R. Lee, Microwave assisted green synthesis of fluorescent N-doped carbon dots: cytotoxicity and bio-imaging applications, J. Photochem. Photobiol. B 161 (2016) 154–161. [13] R. Atchudan, T.N.J.I. Edison, Y.R. Lee, Nitrogen-doped carbon dots originating from unripe peach for fluorescent bioimaging and electrocatalytic oxygen reduction reaction, J. Colloid Interface Sci. 482 (2016) 8–18. [14] J. Ju, R. Zhang, S. He, W. Chen, Nitrogen-doped graphene quantum dots-based fluorescent probe for the sensitive turn-on detection of glutathione and its cellular imaging, RSC Adv. 4 (2014) 52583–52589. [15] W. Lu, X. Qin, S. Liu, G. Chang, Y. Zhang, Y. Luo, A.M. Asiri, A.O. Al-Youbi, X. Sun, Economical, green synthesis of fluorescent carbon nanoparticles and their use as probes for sensitive and selective detection of mercury(II) ions, Anal. Chem. 84 (2012) 5351–5357. [16] C. Wang, D. Sun, K. Zhuo, H. Zhang, J. Wang, Simple and green synthesis of nitrogen-, sulfur-, and phosphorus-co-doped carbon dots with tunable luminescence properties and sensing application, RSC Adv. 4 (2014) 54060–54065. [17] M.R. Hormozi-Nezhad, M. Taghipour, Quick speciation of iron (II) and iron (III) in natural samples using a selective fluorescent carbon dot-based probe, Anal. Methods 8 (2016) 4064–4068. [18] N. Wang, Y. Wang, T. Guo, T. Yang, M. Chen, J. Wang, Green preparation of carbon dots with papaya as carbon source for effective fluorescent sensing of iron (III) and Escherichia coli, Biosens. Bioelectron. 85 (2016) 68–75. [19] X. Gao, Y. Lu, R. Zhang, S. He, J. Ju, M. Liu, L. Li, W. Chen, One-pot synthesis of carbon nanodots for fluorescence turn-on detection of Ag+ based on the Ag+ -induced enhancement of fluorescence, J. Mater. Chem. C 3 (2015) 2302–2309. [20] X. Gao, C. Du, Z. Zhuang, W. Chen, Carbon quantum dot-based nanoprobes for metal ion detection, J. Mater. Chem. C 4 (2016) 6927–6945. [21] S. Liu, J. Tian, L. Wang, Y. Zhang, X. Qin, Y. Luo, A.M. Asiri, A.O. Al-Youbi, X. Sun, Hydrothermal treatment of grass: a low-cost green route to nitrogen-doped, carbon-rich, photoluminescent polymer nanodots as an effective fluorescent sensing platform for label-free detection of Cu(II) ions, Adv. Mater. 24 (2012) 2037–2041. [22] R. Wang, X. Wang, Y. Sun, One-step synthesis of self-doped carbon dots with highlyphotoluminescence as multifunctional biosensors for detection ofiron ions and pH, Sens. Actuators B-Chem. 241 (2017) 73–79. [23] J.E. Cassat, E.P. Skaar, Iron in infection and immunity, Cell Host Microbe 13 (2013) 509–519. [24] K.P. Carter, A.M. Young, A.E. Palmer, Fluorescent sensors for measuring metal ions in living systems, Chem. Rev. 11 (2014) 4564–4601. [25] J.H. Song, M.K. Oak, S.P. Hong, Morphological traits in an androdioecious species, Chionanthus retusus (Oleaceae), Flora 223 (2016) 129–137. [26] L. Wang, Y. Yin, A. Jain, H.S. Zhou, Aqueous phase synthesis of highly luminescent, nitrogen-doped carbon dots and their application as bioimaging agents, Langmuir 30 (2014) 14270–14275. [27] V.N. Mehta, S. Jha, S.K. Kailasa, One-pot green synthesis of carbon dots by using Saccharum officinarum juice for fluorescent imaging of bacteria

508

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

[44]

[45]

[46]

[47]

[48]

[49]

[50]

[51]

R. Atchudan et al. / Sensors and Actuators B 246 (2017) 497–509 (Escherichia coli) and yeast (Saccharomyces cerevisiae) cells, Mater. Sci. Eng. C 38 (2014) 20–27. R. Atchudan, S. Perumal, T.N.J.I. Edison, Y.R. Lee, Highly graphitic carbon nanosheets synthesized over tailored mesoporous molecular sieves using acetylene by chemical vapor deposition method, RSC Adv. 5 (2015) 93364–93373. Y.Z. Fan, Y. Zhang, N. Li, S.G. Liu, T. Liu, N.B. Li, H.Q. Luo, A facile synthesis of water-soluble carbon dots as a label-free fluorescent probe for rapid, selective and sensitive detection of picric acid, Sens. Actuators B-Chem. 240 (2017) 949–955. J. Ju, R. Zhang, W. Chen, Photochemical deposition of surface-clean silver nanoparticles on nitrogen-doped graphene quantum dots for sensitive colorimetric detection of glutathione, Sens. Actuators B 228 (2016) 66–73. H. Huang, J.J. Lv, D.L. Zhou, N. Bao, Y. Xu, A.J. Wang, J.J. Feng, One-pot green synthesis of nitrogen-doped carbon nanoparticles as fluorescent probes for mercury ions, RSC Adv. 3 (2013) 21691–21696. R. Atchudan, T.N.J.I. Edison, S. Perumal, D. Karthikeyan, Y.R. Lee, Effective photocatalytic degradation of anthropogenic dyes using graphene oxide grafting titanium dioxide nanoparticles under UV-light irradiation, J. Photochem. Photobiol. A 333 (2017) 92–104. Q.Q. Shi, Y.H. Li, Y. Xu, Y. Wang, X.B. Yin, X.W. He, Y.K. Zhang, High-yield and high-solubility nitrogen-doped carbon dots: formation, fluorescence mechanism and imaging application, RSC Adv. 4 (2014) 1563–1566. J. Niu, H. Gao, L. Wang, S. Xin, G.Y. Zhang, Q. Wang, L. Guo, W. Liu, X. Gao, Y. Wang, Facile synthesis and optical properties of nitrogen-doped carbon dots, New J. Chem. 38 (2014) 1522–1527. S. Kesavan, D. Ranjith Kumar, Y.R. Lee, J.J. Shim, Determination of tetracycline in the presence of major interference inhuman urine samples using polymelamine/electrochemically reducedgraphene oxide modified electrode, Sens. Actuators B-Chem. 241 (2017) 455–465. C.W. Lai, Y.H. Hsiao, Y.K. Peng, P.T. Chou, Facile synthesis of highly emissive carbon dots from pyrolysis of glycerol; gram scale production of carbon dots/mSiO2 for cell imaging and drug release, J. Mater. Chem. 22 (2012) 14403–14409. R. Atchudan, T.N.J.I. Edison, S. Perumal, D. Karthikeyan, Y.R. Lee, Facile synthesis of zinc oxide nanoparticles decorated graphene oxide composite via simple solvothermal route and their photocatalytic activity on methylene blue degradation, J. Photochem. Photobiol. B 162 (2016) 500–510. D. Ranjith Kumar, S. Kesavan, T.T. Nguyen, J. Hwang, C. Lamiel, J.J. Shim, Polydopamine@electrochemically reduced graphene oxide-modifiedelectrode for electrochemical detection of free-chlorine, Sens. Actuators B-Chem. 240 (2017) 818–828. T. Liu, J.X. Dong, S.G. Liu, N. Li, S.M. Lin, Y.Z. Fan, J.L. Lei, H.Q. Luo, N.B. Li, Carbon quantum dots prepared with polyethyleneimine as both reducing agent and stabilizer for synthesis of Ag/CQDs composite for Hg2+ ions detection, J. Hazard. Mater. 322 (2017) 430–436. J. Ju, W. Chen, In situ growth of surfactant-free gold nanoparticles on nitrogen-doped graphene quantum dots for electrochemical detection of hydrogen peroxide in biological environments, Anal. Chem. 87 (2015) 1903–1910. Y. Zhang, Y. Wang, X. Feng, F. Zhang, Y. Yang, X. Liu, Effect of reaction temperature on structure and fluorescenceproperties of nitrogen-doped carbon dots, Appl. Surf. Sci. 387 (2016) 1236–1246. R. Atchudan, S. Perumal, T.N.J.I. Edison, Y.R. Lee, Facile synthesis of monodisperse hollow carbon nanospheres using sucrose by carbonization route, Mater. Lett. 166 (2016) 145–149. T. Tian, Y. He, Y. Ge, G. Song, One-pot synthesis of boron and nitrogen co-doped carbon dots as thefluorescence probe for dopamine based on the redox reactionbetween Cr(VI) and dopamine, Sens. Actuators B-Chem. 240 (2017) 1265–1271. R. Atchudan, S. Perumal, T.N.J.I. Edison, A. Pandurangan, Y.R. Lee, Synthesis and characterization of graphenated carbon nanotubes on IONPs using acetylene by chemical vapor deposition method, Physica E 74 (2015) 355–362. C. Yu, T. Xuan, Y. Chen, Z. Zhao, X. Liu, G. Lian, H. Li, Gadolinium-doped carbon dots with high quantum yield as an effective fluorescence and magnetic resonance bimodal imaging probe, J. Alloy Compd. 688 (2016) 611–619. R. Atchudan, A. Pandurangan, Growth of ordered multi-walled carbon nanotubes over mesoporous 3D cubic Zn/Fe-KIT-6 molecular sieves and its use in the fabrication of epoxy nanocomposites, Microporous Mesoporous Mater. 167 (2013) 162–175. S. Thambiraj, D.R. Shankaran, Green synthesis of highly fluorescent carbon quantum dots from sugarcane bagasse pulp, Appl. Surf. Sci. 390 (2016) 435–443. R. Atchudan, A. Pandurangan, K. Subramanian, Synthesis of MWCNTs by the decomposition of acetylene over mesoporous Ni/Cr-MCM-41 catalyst and its functionalization, J. Porous Mater. 19 (2012) 797–805. S. Kesavan, S.A. John, Stable determination of paracetamol in the presence of uric acid in human urine sample using melamine grafted graphene modified electrode, J. Electroanal. Chem. 760 (2016) 6–14. R. Atchudan, S. Perumal, D. Karthikeyan, A. Pandurangan, Y.R. Lee, Synthesis and characterization of graphitic mesoporous carbon using metal-metal oxide by chemical vapor deposition method, Microporous Mesoporous Mater. 215 (2015) 123–132. P. Luo, C. Li, G. Shi, Synthesis of gold@carbon dots composite nanoparticles for surface enhanced Raman scattering, Phys. Chem. Chem. Phys. 14 (2012) 7360–7366.

[52] R. Atchudan, A. Pandurangan, J. Joo, Synthesis of multilayer graphene balls on mesoporous Co-MCM-41 molecular sieves by chemical vapour deposition method, Microporous Mesoporous Mater. 175 (2013) 161–169. [53] D. Gu, S. Shang, Q. Yu, J. Shen, Green synthesis of nitrogen-doped carbon dots from lotus root for Hg(II) ions detection and cell imaging, Appl. Surf. Sci. 390 (2016) 38–42. [54] R. Zhang, W. Chen, Nitrogen-doped carbon quantum dots: facile synthesis and application as a turn-off fluorescent probe for detection of Hg2+ ions, Biosens. Bioelectron. 55 (2014) 83–90. [55] H. Huang, Y. Xu, C.J. Tang, J.R. Chen, A.J. Wang, J.J. Feng, Facile and green synthesis of photoluminescent carbon nanoparticles for cellular imaging, New J. Chem. 38 (2014) 784–789. [56] C. Zhu, J. Zhai, S. Dong, Bifunctional fluorescent carbon nanodots: green synthesis via soy milk and application as metal-free electrocatalysts for oxygen reduction, Chem. Commun. 48 (2012) 9367–9369. [57] Y. Liu, Q. Zhou, J. Li, M. Lei, X. Yan, Selective and sensitive chemosensor for lead ions using fluorescent carbon dots prepared from chocolate by one-step hydrothermal method, Sens. Actuators B-Chem. 237 (2016) 597–604. [58] J. Chen, J. Liu, J. Li, L. Xu, Y. Qiao, One-pot synthesis of nitrogen and sulfur co-doped carbon dots and its application for sensor and multicolor cellular imaging, J. Colloid Interface Sci. 485 (2017) 167–174. [59] J. Xu, Y. Zhou, S. Liu, M. Dong, C. Huang, Low-cost synthesis of carbon nanodots from natural products used as a fluorescent probe for the detection of ferrum(III) ions in lake water, Anal. Methods 6 (2014) 2086–2090. [60] A. Kumar, A. Pandith, H.S. Kim, Pyrene-appended imidazolium probe for 2,4,6-trinitrophenol in water, Sens. Actuators B-Chem. 231 (2016) 293–301. [61] C. Wang, K. Jiang, Z. Xu, H. Lin, C. Zhang, Glutathione modified carbon-dots: from aggregation-induced emission enhancement properties to a turn-on sensing of temperature/Fe3+ ions in cells, Inorg. Chem. Front. 3 (2016) 514–522. [62] T.N.J.I. Edison, R. Atchudan, J.J. Shim, S. Kalimuthu, B.C. Ahn, Y.R. Lee, Turn-off fluorescence sensor for the detection of ferric ion in water using green synthesized N-doped carbon dots and its bio-imaging, J. Photochem. Photobiol. B 158 (2016) 235–242. [63] X. Cui, Y. Wang, J. Liu, Q. Yang, B. Zhang, Y. Gao, Y. Wang, G. Lu, Dual functional N- and S-co-doped carbon dots as the sensor for temperature and Fe3+ ions, Sens. Actuators. B 242 (2017) 1272–1280. [64] J. Ju, W. Chen, Synthesis of highly fluorescent nitrogen-doped graphene quantum dots for sensitive, label-free detection of Fe(III) in aqueous media, Biosens. Bioelectron. 58 (2014) 219–225. [65] Y.L. Zhang, L. Wang, H.C. Zhang, Y. Liu, H.Y. Wang, Z.H. Kang, S.T. Lee, Graphitic carbon quantum dots as a fluorescent sensing platform for highly efficient detection of Fe3+ ions, RSC Adv. 3 (2013) 3733–3738.

Biographies Raji Atchudan was born in Dharmapuri, Tamil Nadu, India, in 1979. He received his B.Sc., (2000) in Chemistry from University of Madras; M.Sc., (2003) in Chemistry from Annamalai University; M.Phil., (2004) in Chemistry from University of Madras; and Ph.D. (2011) in Chemistry (Nanoscience and Nanotechnology) from Anna University, India. He had worked at Kyungpook National University, Republic of Korea as a Postdoctoral Research Fellow with Prof. Jin Joo (2012–2015). At present (2015–till date), he is working as a Foreign Research Professor in the School of Chemical Engineering, Yeungnam University, Republic of Korea. His research interests include facile green synthesis of metal oxide nanoparticles, carbon nanostructured materials-carbon nanotubes, carbon dots, graphene oxide/graphene and carbonrelated nanomaterials, and their application towards the photocatalytic degradation of anthropogenic dyes, electro-catalyst, supercapacitors, sensors, and bioimaging. Thomas Nesakumar Jebakumar Immanuel Edison was born in Madurai, Tamil Nadu, India, in 1984. He received his B.Sc. and M.Sc. degrees in Chemistry from Madurai Kamaraj University, Madurai, in 2004 and 2008, respectively; and Ph.D degree in Chemistry from Gandhigram Rural Institute-DU, Gandhigram, Tamil Nadu, India, in 2014. Since 2015, he has been working as a Foreign Research Professor at the School of Chemical Engineering, Yeungnam University, South Korea. His research interests include green synthesis of metal nanoparticles, semiconductors and carbon nanoparticles and utilizing these materials as catalyst, electro-catalyst, supercapacitors, batteries and sensors. Dasagrandhi Chakradhar received his B.Sc., in microbiology from Sri Venkateswara University in 2002. He obtained a M.Sc., (2004) in applied microbiology specialization from Gandhigram Rural Institute-DU, Gandhigram, Tamil Nadu, India. He joined the Food Science and Biotechnology division of Kyungpook National University, Daegu, South Korea, in 2011 as a Korean Government Scholarship awardee for Ph.D. studies. His graduate research is focused on the development of novel functional materials to detect and curb the drug resistance in multidrug-resistant Staphylococcus aureus. Suguna Perumal has received B.Sc., in Chemistry from Periyar University (Tamil Nadu, India) in 2002, M.Sc., in Chemistry from Bharathidasan University (Tamil Nadu, India) in 2004, and Ph.D. in Chemistry from Freie University Berlin (Germany). She is currently working as a postdoctoral fellow at Kyungpook National University in Applied Chemistry Department. Her main research fields concerned with the synthesis of nanoparticles, block copolymers and graphene-related works.

R. Atchudan et al. / Sensors and Actuators B 246 (2017) 497–509 Jae-Jin Shim received his B.S. in Chemical Engineering from Seoul National University in 1980, his M.S. in Chemical Engineering from Korea Advanced Institute of Science and Technology in 1982, and his Ph.D. in Chemical Engineering from the University of Texas at Austin in 1990. He is a professor in the School of Chemical Engineering at Yeungnam University, Republic of Korea. His current research focuses on graphene-based nano materials for energy storage (supercapacitor), sensor (gas and chemical), and catalysis, mostly using clean solvents such as supercritical fluids, ionic liquids, or water. Yong Rok Lee is a Chunma distinguished professor of the School of Chemical Engineering at Yeungnam University, South Korea. He received his bachelor’s degree

509

(1982) in chemistry from Chonbuk National University (South Korea), and his M.S. (1984) and Ph.D. (1992) in organic chemistry from Seoul National University (South Korea) under the supervision of Prof. Eun Lee. He has worked at Duke University as a postdoctoral fellow with Prof. M. C. Pirrung (1993–1994), Ohio State University as a postdoctoral fellow with Prof. L. Paquette (1995), and Michigan State University as a visiting professor with Prof. W. Wulff (2000). His current research focuses on the synthesis and applications of organic materials, nanomaterials, natural products, and pharmaceuticals.