Hydrothermal conversion of Magnolia liliiflora into nitrogen-doped carbon dots as an effective turn-off fluorescence sensing, multi-colour cell imaging and fluorescent ink

Accepted Manuscript Title: Hydrothermal conversion of Magnolia liliiflora into nitrogen-doped carbon dots as an effective turn-off fluorescence sensing, multi-colour cell imaging and fluorescent ink Authors: Raji Atchudan, Thomas Nesakumar Jebakumar Immanuel Edison, Kanikkai Raja Aseer, Suguna Perumal, Yong Rok Lee PII: DOI: Reference:

S0927-7765(18)30316-3 https://doi.org/10.1016/j.colsurfb.2018.05.032 COLSUB 9351

To appear in:

Colloids and Surfaces B: Biointerfaces

Received date: Revised date: Accepted date:

27-3-2018 11-5-2018 14-5-2018

Please cite this article as: Raji Atchudan, Thomas Nesakumar Jebakumar Immanuel Edison, Kanikkai Raja Aseer, Suguna Perumal, Yong Rok Lee, Hydrothermal conversion of Magnolia liliiflora into nitrogen-doped carbon dots as an effective turnoff fluorescence sensing, multi-colour cell imaging and fluorescent ink, Colloids and Surfaces B: Biointerfaces https://doi.org/10.1016/j.colsurfb.2018.05.032 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Hydrothermal conversion of Magnolia liliiflora into nitrogen-doped carbon dots as an effective turn-off fluorescence sensing, multi-colour cell imaging

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and fluorescent ink† Raji Atchudana*, Thomas Nesakumar Jebakumar Immanuel Edisona, Kanikkai Raja Aseerb, Suguna Perumalc, Yong Rok Leea* a

School of Chemical Engineering, Yeungnam University, Gyeongsan 38541, Republic of Korea

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Department of Biotechnology, Daegu University, Kyungsan, Kyungbuk 38453, Republic of

Department of Applied Chemistry, Kyungpook National University, Daegu 41566, Republic of

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c

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Korea

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Korea *

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Corresponding authors.

E-mail addresses: [email protected] (R. Atchudan); [email protected] (Y.R. Lee) Electronic Supplementary Information (ESI) available: XRD pattern, FTIR spectrum, Raman

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†

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spectrum, UV-vis spectra, fluorescence spectra, long-term stability analysis, zeta potential,

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biocompatibility measurement and plausible formation mechanism of the synthesized N-CDs;

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FTIR spectrum and zeta potential of the N-CDs-Fe3+ complex.

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Graphical Abstract N-CDs were synthesized using M. liliiflora flower and were potentially utilized for the detection

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of Fe3+ ions, multi-colour cellular imaging and fluorescent ink.

Research Highlights

Hydrothermal conversion of M. liliiflora into N-CDs by one-pot hydrothermal method

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N-CDs was applied as a fluorescent probe for the effective detection of Fe3+ ions

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N-CDs could offer a multi-colour cell imaging without any chemical modification

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N-CDs was used as a fluorescent ink without any pretreatment of the sample

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Abstract The present work illustrates the potential uses of nitrogen-doped multi-fluorescent carbon dots 2

(N-CDs) for Fe3+ sensing, cellular multi-colour imaging, and fluorescent ink. N-CDs were synthesized using Magnolia liliiflora flower by the simple hydrothermal method. The resulted NCDs was found to be nearly spherical in shape with the size of about 41 nm and showed

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competitive quantum yield around 11%. The synthesized N-CDs with uniform size distribution and high content of nitrogen and oxygen-bearing functional groups exhibit excellent dispersibility in aqueous media. The N-CDs were able to detect a high concentration of Fe3+ ions (1–1000 μM) with a limit of detection is about 1.2 M by forming N-CDs-Fe3+ complex due to

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the functional groups such as nitrogen, carbonyl and carboxyl on the surface of N-CDs. Thus they could be used to remove pollutants from industrial wastewater. The electronic charge on the

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surface of the N-CDs and N-CDs-Fe3+ complex (zeta potential) is around –36 and 18 mV,

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respectively. In addition, these N-CDs show excitation-dependent fluorescence that was utilized for multi-colour in vitro cellular imaging in rat liver cells (Clone 9 hepatocytes). The N-CDs are

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rapidly uptake in the cell cytoplasm and showed high cytocompatibility on cellular morphology.

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Moreover, as the N-CDs possess strong fluorescence and anti-coagulation they could be utilized

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in fluorescent ink pens.

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Keywords: Magnolia liliiflora; Hydrothermal method; Carbon dot; Metal ion sensing; Clone 9

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hepatocytes; Multi-colour imaging; Fluorescent ink

1.

Introduction

In recent years, carbon dots (CDs) have great attention in the scientific community owing to their wide applications in various field includes fluorescence probe for detection of metal ions, 3

fluorescent labelling, bioimaging, fluorescent ink, electrocatalyst for oxygen reduction reaction and oxygen evaluation reaction, electrode material for supercapacitors and batteries [1–8]. The CDs exhibits excellent monodispersibility and bright fluorescence with high quantum yield (QY).

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Their optical and electrical properties including photostability and bright fluorescence with high QY are enhanced by the doping with heteroatoms such as nitrogen (N), sulphur (S) and etc. [9– 12]. These advantages lead to the synthesis of many new N-doped CDs (N-CDs), S-doped CDs (S-CDs) and N, S-co-doped CDs (N, S-CDs). In general, several techniques such as hydrothermal treatment, solvothermal treatment, microwave pyrolysis, plasma treatment, laser

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ablation, electrochemical process have been adopted for the synthesis of CDs including N-CDs,

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S-CDs and N, S-CDs. Among them, the hydrothermal method is preferably used for the

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synthesis of CDs owing to their distinct advantages such as simple, eco-friendly, low-cost and

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scalable [13–17]. Based on their merits, the hydrothermally derived CDs are of great interest in

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various applications. Natural/Bio-waste converted CDs are facile, cost-less and environmental

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friendly when compared to citrate based compounds derived CDs. Thus, many researchers are focused on synthesizing CDs from natural bio-sources [18–20]. In general, the natural bio-

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sources including flowers, fruits, seeds and stems containing numerous bioactive molecules such

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as acidic constituents, base constituents, and neutral constituents are fascinating when applied in the synthesis of the nanoparticle. During the synthesis of nanoparticles such as CDs/N-CDs, the

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phytoconstituents present in particular bio-source, plays crucial role in the reaction kinetics and the phytoconstituents determine the surface functional groups on the resulting nanoparticles (CDs) as well as the reactivity of the resulting nanoparticles [21]. Similarly, the QY of fluorescent CDs/N-CDs depends on the type of phytoconstituents, size of CDs/N-CDs, solvent, and dopants. This behaviour opens the way for the application of CDs/N-CDs as fluorescent 4

probes for the selective and sensitive detection of metal ions in aqueous medium [22], thus the use of natural bio-extracts is an amusing aspect in the synthesis of CDs/N-CDs. Inspired by the earlier reported work [18–20]. Here an attempt has been made to synthesize the N-CDs from

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Magnolia liliiflora (M. liliiflora) in a cheap and easy manner. M. liliiflora is native to southwest China but cultivated for many centuries elsewhere in China, Japan and also in Korea. It is a deciduous shrub, which grow up to 4 meters tall and belongs to the Magnoliaceae family (Genus: Magnolia). M. liliiflora is an affordable flower and consumed for many medicinal uses [23]. The extracts of the M. liliiflora flower consist of many phytoconstituents, such as essential

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oils (trans-α-Farnesene, -Cadinene and etc) [24, 25] and volatile components (1,8-cineole,

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farnesol, sabinene, -pinene, α-pinene, camphor and etc) [26]. These essential oils and volatile

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components might be helpful to the formation of carbon nanostructured materials. Therefore, in the present work, the extracts of the M. liliiflora flowers was chosen as a precursor for the

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preparation of N-CDs.

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Fe3+ is one of the most common and essential transition metal ions in the biological system and plays a crucial role in many physiological and pathological processes, such as oxygen uptake and

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transport, cellular metabolism, enzyme catalysis and electron transfer process. However,

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abnormal variations of Fe3+ ion in the human body can induce serious disorders such as anemia, intelligence decline, heart failure, diabetes and so on [27, 28]. Also, Fe3+ ion plays an important

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role in natural aquatic systems and seawater. Thus, the considerable importance has been given to find the selective techniques for the determination of Fe3+ ion. As a result, various analytical techniques have been developed but during analysis, several other metal ions interfere with the analysis. To avoid this complicated pretreatment procedures and sophisticated instrumentations 5

are needed. Recently, fluorescent techniques have been widely employed for the selective and sensitive detection of Fe3+ ions in biological as well as environmental systems owing to their simplicity and using the sample without any pretreatment [29]. However, fluorescence probes

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such as semiconducting quantum dots and organic dyes intrinsically suffer from poor solubility, potential cytotoxicity, and poor photostability which hinders their potential in biological and environmental systems. Hence, an ideal fluorescence probe is needed to overcome the above drawbacks.

In this study, the N-CDs have been synthesized from M. liliiflora flower to investigate the

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optical properties and were applied towards the sensing of metal ions, living cell imaging, and

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fluorescent ink. The M. liliiflora itself served as both nitrogen as well as carbon precursor for

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synthesizing N-CDs. The morphology, surface functional groups, chemical state and optical

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properties of resulted N-CDs were investigated extensively in the aqueous medium. The N-CDs

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exhibit narrow size distribution with durable fluorescence and it showed commendable potential

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on selective and sensitive detection of the Fe3+ ion. In addition, multi-colour cellular imaging, as well as biocompatibility studies have been carried out in Clone 9 hepatocytes, which indicated

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the utility of the N-CDs for multi-colour cell imaging and various biomedical applications. On

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the other hand, the synthesized N-CDs was directly utilized as a fluorescent ink without any chemical modification. To the best of our knowledge, this is the first report on the synthesis of

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N-CDs from M. liliiflora and their use in fluorescence probe for highly selective detection of Fe3+ ion, cell imaging with good biocompatibility and fluorescent ink without any pretreatment of the sample. 2. Experimental 2.1. Materials 6

M. liliiflora flowers were procured from Yeungnam University Campus, Gyeongsan, Republic of Korea. Phosphate buffered saline (PBS), p-formaldehyde, quinine sulphate, sulphuric acid (H2SO4) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich,

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Republic of Korea. 30-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) purchased from Generay Biotech, Shanghai, China. The analytical reagent grade of metal salts such as AlCl3, CaCl2, Cd(CH3OO)2, Co(OOCH3)2, CrCl3, CuCl2, FeCl3, HgCl2, NiCl2, Pb(NO3)2 and ZnCl2 was purchased from Ducksan chemicals, Republic of Korea. Clone 9 hepatocytes, which belongs to Rat liver cells were obtained from ATCC (CRL-1439, Manassas, VA, USA).

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2.2. Synthesis of N-CDs

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the preparation of samples throughout this study.

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All the chemicals were used as received. Double-distilled (DD) water was used as a solution for

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The hydrothermal method was used for the synthesis of N-CDs from M. liliiflora flower.

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Briefly, M. liliiflora flower was cut into small pieces, 3 g of flower and 30 mL of DD water were

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taken into a Teflon-lined stainless steel autoclave with a 50 mL capacity of inner volume. Then, the mixture was sealed and placed in a hot air oven followed by hydrothermal treatment at 240

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C for 12 h. During this process, the colourless solution turned to dark brown which indicated

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the formation of N-CDs. After completion of the reaction, the mixture was cooled down to room temperature and the resulting N-CDs were purified by filtration through a membrane filter with

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pore size of 0.22 m to remove residual carbon nanoparticles (large or aggregated nanoparticles). Fluorescence of the obtained yellow-brown coloured solution was observed under UV light with an excitation wavelength of 365 nm. Finally, a dark yellow-brown solid N-CDs were obtained after freeze drying. The hydrothermal temperature was varied from 160 to 240 C to determine 7

the high fluorescence N-CDs with similar reaction condition. The resulted N-CDs with excellent photoluminescence properties were applied for various applications which are clearly

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demonstrated in Fig. 1.

2.3. Characterization

The morphology and selected area electron diffraction (SAED) patterns of the N-CDs were captured by high-resolution transmission electron microscopy (HRTEM) with accelerating voltage of 200 kV (FEI-Tecnai TF-20). A drop of diluted N-CDs solution was placed on the

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carbon-coated copper grid and was air dried before HRTEM analysis. The topography image was

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obtained by an atomic force microscopy (AFM), Tapping mode-NanoscopeIIIa, Digital

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Instruments, Inc. X-ray diffraction (XRD) analysis was performed using powder diffractometer

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PANalytical X’Pert3 MRD employing monochromatized Cu Kα radiation (λ = 1.54 Å) at 40 kV

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and 30 mA and was recorded in the range from 10 to 90° (2θ) to determine the

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crystallinity/graphitization of the N-CDs. The powder sample was packed on top of a glass substrate and was measured in reflection geometry. Attenuated total reflectance Fourier

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transform infrared (ATR-FTIR) spectroscopy was performed to analyze the functional groups present on the surface of N-CDs using Perkin Elmer Spectrum Two by the addition of 16 scans at

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a resolution of 16 cm−1. Raman spectrum of the N-CDs was recorded on a Thermo Scientific

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DXR SmartRaman spectrometer with laser excitation at 532 nm. X-ray photoelectron spectroscopy (XPS) analysis was performed to find the chemical composition of N-CDs by Thermo Scientific K-Alpha. The non-monochromatic Al Kα line at 180–200 W was used as the primary excitation. Casa XPS instrument software was used for the deconvolution of the highresolution XPS spectrum. The ultraviolet-visible (UV–vis) absorption spectrum of the N-CDs 8

was recorded in DD water using OPTIZEN 3220UV spectrophotometer. The clear dispersion of the sample in DD water was prepared by sonication process. Fluorescence excitation and emission spectra of the N-CDs were recorded using a Hitachi F-7000 fluorescence

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spectrophotometer using a 1 cm path length quartz cell. The slit width and scan speed were fixed at 5 nm and 240 nm/min, respectively. The zeta potential was measured for an aqueous solution of N-CDs and Fe3+-N-CDs complex at 25 C by a Zetasizer Nano ZS ZEN3600, Malvern. Cellular imaging was performed using confocal laser scanning microscope (X400; LSM700, Carl

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Zeiss, Oberkochen, Germany).

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2.4. Quantum yield measurements

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The QY of the synthesized N-CDs was measured based on the established procedure [30]. In

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brief, quinine sulphate in 0.1 M H2SO4 (QY is 0.54 at 350 nm) was used as the reference

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standard. The QY was calculated according to the following equation (1):

(1)

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ISA R (n S ) 2 QY (%) = QYR I R AS (n R ) 2

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where, “I” is the measured integrated fluorescent emission intensity, “n” is the refractive index of

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the solvent, and “A” is the absorbance. The subscript “R” and “S” refers to the corresponding parameter of known fluorescent standard and for the synthesized sample, respectively.

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2.5. Metal ion sensing The detection of Fe3+ was performed at room temperature with a fluorescence excitation

wavelength of 340 nm. The fluorescence maximum emission intensity was fixed as a blank which is an equal amount of DD water and N-CDs solution (0.5 mL). The selectivity for the Fe3+ 9

ion of N-CDs was confirmed by adding 0.5 mL of eleven kinds of metal ion solutions such as Al3+, Ca2+, Cd2+, Co2+, Cr3+, Cu2+, Fe3+, Hg2+, Ni2+, Pb2+ and Zn2+ with a concentration of 1 mM to 0.5 mL of the N-CDs solution. The fluorescence emission spectra were measured after 2 min

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of reaction at ambient condition for all the experiments. To determine the detection range of Fe3+ in the presence of synthesized N-CDs, 0.5 mL aqueous solution of N-CDs was taken in a quartz cell, followed by the addition of 0.5 mL of different concentrations of Fe3+ ion (1–250 M) and the fluorescence emission spectra were recorded.

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2.6. Cells imaging and MTT assay

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Cells were cultured on coverslips in 6-well plates for the analysis of fluorescent confocal

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imaging. The staining was achieved by incubating the cells in the presence and absence of the

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synthesized N-CDs in a humidified chamber at 37 °C for 24 h. After staining, the cells were fixed with 4% p-formaldehyde, washed thrice with PBS, and mounted in fluorescence mounting

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medium (Dako North America Inc., Carpinteria, CA, USA). The fluorescence of the stained cells

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was viewed and imaged using different filters such as blue, green, red and also bright field (BF) by a confocal microscopy. Rat liver cell (Clone 9 hepatocytes) was chosen to determine the

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cytotoxicity of the synthesized N-CDs by MTT viability assay. Cell viability was evaluated

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based on the reduction of yellowish MTT into an insoluble purple formazan product. Typically, Clone 9 hepatocytes were cultured in F-12K medium containing 10 % FBS in a 96-well plate at

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37 °C under 5 % CO2 atmosphere until confluence, followed by treatment with different concentrations of N-CDs (0 – 100 μg mL-1) for 24 h. The MTT solution (5 mg MTT/mL in PBS) was added to each well of the microtiter plate and incubated for 4 h at 37 °C in a dark humidified chamber. Finally, purple-Coloured formazan crystals formed only by living cells and were then 10

dissolved in DMSO and absorbance was recorded at 540 nm using a microtiter plate reader. 3. Results and discussion 3.1. Characterization of the N-CDs

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The existence of the graphite-like structure in the N-CDs was confirmed by HRTEM, XRD and Raman spectroscopy. The HRTEM images of synthesized N-CDs are shown in Fig. 2a and b, indicating a nearly spherical shape with an uniform dispersion and a narrow size distribution with an average size of 4  1 nm. Comparative lattice fringes are found in the HRTEM image

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(Fig. 2b), indicating the graphitic nature of N-CDs. The lattice space is 0.21 nm, which is in good

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agreement with the lattice space of (100) plane of graphitic carbon [31–33]. In addition, the

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HRTEM clearly demonstrate the high and poor graphitization in the inner and outer surface of

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the N-CDs, respectively. In agreement with HRTEM results, the AFM topography image of the resulted N-CDs shows the monodisperse CDs with an average size of about 5 nm which is clear

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from Fig. 2c. The XRD pattern (Fig. S1a) exhibits a broad peak centered at 2θ = 23.2 with a

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lattice space of 0.38 nm and a small peak at 2θ = 43.1 with a lattice space of 0.21 nm corresponding to the graphite lattice spacing of (002) and (100) planes, respectively. The lattice

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space of 0.38 nm, which belongs to (002) plane is larger than that of normal graphite (0.34 nm),

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possibly owing to the existence of oxygen-containing functional groups [34]. The broad peak and lower 2θ in the XRD pattern confirm their smaller size and moderate crystallization, respectively.

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The presence of the moderate crystallization/ graphitization in the N-CDs might be due to functional groups on the surface of N-CDs. These XRD results agree well with HRTEM analysis [35–38]. Raman spectroscopy is a powerful tool used in observing the graphitization of the NCDs. Fig. S1b shows the Raman spectrum of the synthesized N-CDs. The spectrum shows two 11

distinct peaks at 1365 and 1590 cm-1 corresponding to the D-band and G-band from the in-plane of sp3 hybridized carbons and sp2 carbons, respectively. The ratio of the D- and G-band intensities (ID/IG) of the N-CDs is calculated to be 0.71 which is little higher than that of the pristine graphene. The

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high value might be due to the presence of surface defects and surface functional groups in the NCDs nanostructure [39–41].

ATR-FTIR spectroscopy and XPS were carried out to evaluate the surface functionalization and chemical composition of N-CDs. The ATR-FTIR spectrum (Fig. S2) showed a broad band

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around 3230 cm-1 corresponding to the stretching vibrations of hydroxyl (O–H) functional

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groups and physically adsorbed water molecules (H–O–H) on the surface of the N-CDs [42; 43].

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The bands at 2862 and 2922 cm-1 corresponds to symmetric and asymmetric C–H stretching

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vibrations, respectively. The obvious bands at 1680, 1590, 1400, 1290 and 1072 cm-1 are

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associated with C=O, C=C, C=N, C–OH, C–O–C/C–N stretching vibrations, respectively [44–

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46]. The presence of functional groups in N-CDs enhances its solubility in an aqueous media even without further chemical modification. Furthermore, the absence of prominent N–H in-

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plane stretching of amine group in the FTIR spectrum, suggests the successful incorporation of

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nitrogen atoms into the CDs. The XPS survey spectrum (Fig. 3a) displays three distinguish signals at 284, 400 and 532 eV attributed to the C1s, N1s, and O1s with the atomic percentage of

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70, 6 and 24 %, respectively. Deconvolution of the C1s spectrum (Fig. 3b) shows four peaks at 284.08, 284.78, 285.68 and 288.98 eV corresponding to C=C, C–C, C–N/C–OH and C–O–C/O– C=O groups, respectively [47–49]. As shown in Fig. 3c, deconvolution of the N1s spectrum exhibit two broad peaks at 399.38 and 401.88 eV attributed to N associated with pyridinic (C–N– C) and graphitic carbons (C)3–N), respectively [50–54]. The O1s spectrum (Fig. 3d) can be 12

resolved into three peaks at 530.28, 531.98 and 535.98 eV, corresponding to the C–OH/C–O–C, C=O, and H–O–H chemical environments, respectively [55, 56]. The existence of C, N, and O in the synthesized N-CDs confirms from FTIR analysis, suggests that both the elements (C and N)

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originate from M. liliiflora flower. The plausible formation mechanism of N-CDs from the M. liliiflora flower as described as follows: The predominant phytoconstituents of M. liliiflora flower are essential oils (trans-α-Farnesene, -Cadinene and etc) [24, 25] and volatile components (1,8-cineole, Farnesol, Sabinene, -pinene, α-pinene, Camphor and etc) [26] which

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are taken in to reason for the formation of N-CDs [57, 58]. Fig. S3 shows the plausible formation

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mechanism of N-CDs by a simple hydrothermal method. During the hydrothermal process, the

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phytoconstituents of M. liliiflora flower involves several steps including dehydration, polymerization, and carbonization. Initially, the hydroxyl group present in phytoconstituents will

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undergo dehydration at 100 C which leads to furfural derivatives. The nitrogen groups in

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phytoconstituents might react with carbonyl groups of phytoconstituents that will form stable

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complex. Further, the polymerization and condensation of furfural derivatives will produce water-soluble polymers. The resulted polymers of furfural derivatives and nitrogen with carbonyl

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groups stable complex will undergo the carbonization that end up in N-CDs with good optical

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properties [45, 57, 58].

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3.2. Optical properties of the N-CDs Optical properties of the synthesized N-CDs were determined by UV-Vis and fluorescence

spectroscopy analysis. The UV-Vis spectrum (Fig. S4) showed two eminent broad peaks around 275 and 315 nm attributed to the –* transition of the C=C bond and n–* transition of the 13

C=O/C=N bond, respectively [59–62]. Fluorescence excitation spectrum of N-CDs (Fig. S5a) displays a maximum fluorescence at an excitation wavelength of about 340 nm. The temperature-dependent fluorescence emission spectra are shown in Fig. S5b. The fluorescence

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intensity increases with increasing the reaction temperature from 160 to 240 C. The excitation wavelength was varied to find out the maximum fluorescence for the synthesized N-CDs at 240 C. Fig. S5c shows the fluorescence spectra of the N-CDs excited at wavelengths ranging from 280 to 400 nm. The fluorescence intensity increases with increasing the excitation wavelength

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and then decreased. Upon excitation at 340 nm, N-CDs emitted strong fluorescence centered at

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405 nm with full width at half maximum as narrow as 65 nm. Further increasing the excitation

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wavelength from 340 to 400 nm, the fluorescence emission intensity gradually decreased.

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Furthermore, the fluorescence emission of red-shifts (407–465 nm) is observed when the excitation wavelength is varied from 380 to 400 nm. Such excitation-dependent fluorescence

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behavior is related to the different surface defects and different surface states of the N-CDs [63,

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64]. Fig. S5d illustrates the chart diagram of excitation-dependent emission wavelength, in which the fluorescence emission wavelength slightly decreases when the excitation wavelength is

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changed from 280 to 310 and then gradually increased. This fluorescence behaviour is perhaps

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attributed to the small variation of particle sizes and the distribution of surface emissive trap sites [65]. QY is one of the most important features to determine suitable applications. The QY of the

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synthesized N-CDs is measured to be 11% using quinine sulphate as a reference. The photographs of the aqueous solution of N-CDs under daylight as well as UV light as

shown in Fig. S6a. The aqueous solution of N-CDs is colourless under daylight but show bright blue fluorescence under UV light with an excitation wavelength of 365 nm. The bright blue 14

colour can be observed in the naked eye under UV light irradiation even after 6-months of storage owing to the prolonged fluorescence intensity of N-CDs. Furthermore, the fluorescence of the N-CDs was examined by fluorescence spectrometer which is shown in Fig. S6b. As shown

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in the spectra, there is no significant difference in the fluorescence intensities of both before and after 6-months storage of N-CDs. These results suggested that the synthesized N-CDs possess excellent fluorescence durability (stability). 3.3. Applications of the N-CDs

M. liliiflora derived N-CDs were applied to diverse applications including selective and

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sensitive detection of Fe3+ ions, live cell imaging and fluorescent ink without any chemical

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modifications because of their excellent water solubility/dispersibility, high cytocompatibility,

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easily washable and durable fluorescence with high QY. To identify selective fluorescence

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sensing of the Fe3+ ion by the fluorescence N-CDs probe, the quenching performance was carried

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out with 1 mM concentration of different metal ions which is presented in Fig. 4a. The N-CDs

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exhibit a strong characteristic fluorescence emission peak at 405 nm under an excitation of 340 nm (blank, absence of metal ions). The difference in fluorescence intensities of the N-CDs by the

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addition of metal ions was observed in the spectrum. Among them, the fluorescence of the NCDs is nearly zero in the presence of a Fe3+ ion, revealing that the N-CDs is sensitive towards

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Fe3+ ions. The fast and excellent quenching of the N-CDs was observed immediately after the

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addition of Fe3+ ion and is due to the strong interaction between the functional groups of N-CDs and Fe3+ ions. Fe3+-N-CDs complex will be formed during the addition of Fe3+ ion into the NCDs solution which leads to fluorescence quenching by an efficient energy transfer between Fe3+ and N-CDs. Adsorption affinities are clear from the (F0-F)/F0 plot in the presence of 1 mM concentration of different metal ions as shown in Fig. 4b. Where, F indicates the fluorescence 15

intensity in presence of metal ions and F0 indicates the fluorescence intensity in absence of metal ions (blank). Among the studied metal ions, Fe3+ ion in the presence of N-CDs shows high (F0F)/F0 value which is due to strong adsorption affinity between the Fe3+ and N-CDs [66]. The

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fluorescence of N-CDs was quenched by the addition of Fe3+ ion under UV light with an excitation wavelength of about 365 nm and is clear from an inset of Fig. 4b. The fluorescence intensities of N-CDs in the presence of different concentrations of Fe3+ ions are examined to evaluate the limit of detection (LOD). The fluorescence intensity was continuously decreased

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upon increasing the concentration of Fe3+ ion from 0 to 250 M which is shown in Fig. 4c. The synthesized N-CDs having many hydroxyl and carboxylic functional groups on the surface

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which increase towards the complete complexation as the concentration of Fe3+ ion increases and charge or electron transfer occur within the complex [67, 68]. Thus, the fluorescence keeps on

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quenching by the incessant addition of Fe3+ ion. Fig. 5d shows the relationship between the (F0-

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F)/F0 and concentration of the Fe3+ ion. The kinetic plot shows that the fluorescence quenching of

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Fe3+ ions is relatively fast as well as a good linear relationship and diverted curve observed in the low and high concentrations, respectively. The prediction of LOD was complicated, therefore the

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fluorescence quenching experiment was conducted with a low concentration of Fe3+ ion from 0

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to 25 M (inset Fig. 4c). As shown in an inset Fig. 4d, the plot of (F0-F)/F0 against 0 to 25 M concentration of Fe3+ ion exhibits a good linear relationship with the R2 value of 0.999. The LOD

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value for the detection of Fe3+ was calculated as 1.2 M using the equation (2). The auspicious quenching ability of Fe3+ ions may arise from the strong adsorption affinity and highly efficient energy transfer between the N-CDs and Fe3+ ions [69–71]. The results suggest that the fluorescence N-CDs probe might be useful for the selective and sensitive detection of the Fe3+ 16

ion from the industrial wastewater due to its wide range of detection ability.

3σ S

(2)

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LOD =

where, σ is the standard deviation (SD) of the signals (n = 4) and S is the slope of the linear calibration plot.

In addition, the zeta potential measurements were carried out for both the synthesized N-

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CDs and Fe3+-N-CDs complex to estimate the electronic charge on their surface which is shown

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in Fig. S7. The obtained zeta potentials are around −36 (Fig. S6a) and 18 mV (Fig. S6b) for N-

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CDs and Fe3+-N-CDs complex, respectively. The oxygen-containing functional groups such as

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carbonyl and hydroxyls on the surface of the synthesized N-CDs which is reacted with Fe3+ ions

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and made a Fe3+-N-CDs complex. Hence, the negative value of zeta potential shifted to a

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positive value, and the resulted in stable complex. The results suggest that the synthesized NCDs exhibits the vast number of oxygen-containing functional on the surface which easily react

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with Fe3+ ions and resulting fluorescence turn-off (quenching). The oxygen-containing functional groups on the surface react with Fe3+ ions forms the Fe3+-N-CDs complex which was determined

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by the FTIR spectroscopy. Fig. S8 shows the ATR-FTIR spectrum of the Fe3+-N-CDs complex.

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In this spectrum, the adsorption band intensity of carboxyl/hydroxyl and nitrogen-containing functional groups considerably decreased compared to the synthesized N-CDs. This might be due to complexation of Fe3+ ions with surface carboxyl/hydroxyl and nitrogen-containing functional groups of N-CDs. Especially, absence of the adsorption band of oxygen-containing functional groups such as carboxyl and hydroxyl in the spectrum strongly suggest that the stable 17

complexed with Fe3+ ions. Thus, non-radiative electron transfer is possible from the N-CDs surface during the complex formation between Fe3+ ions and N-CDs, resulting in attendant

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complete fluorescence quenching (turn-off) of N-CDs [72].

Considering their durable fluorescence and excellent water dispersibility, the synthesized N-CDs were also utilized as a fluorescent probe for live cell imaging. Prior to considering the utility of the N-CDs as fluorescent probes for real biological system, the biocompatibility of N-CDs is very important. Hence, the in vitro cytotoxicity of synthesized N-CDs was evaluated by MTT

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assay against Clone 9 hepatocytes. The Clone 9 hepatocytes were incubated for 24 h with

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different concentrations of N-CDs (0−100 μg mL−1), the N-CDs loaded Clone 9 hepatocytes

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show the absence of significant difference in the cell viability (%) compared to the control

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sample clear from Fig. S9. The cytotoxicity was negligibly increased by increasing the

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concentration of N-CDs and the viability of the cells is greater than 97% even at high

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concentration of N-CDs (100 μg mL−1) which demonstrate that N-CDs did not induce the cell death. The results suggest that the N-CDs possess good biocompatibility against Clone 9

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hepatocytes [73]. Thus, the synthesized N-CDs could be used as a fluorescent probe in the real

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biological system and clinical research. The synthesized N-CDs with biocompatibility was applied for Clone 9 hepatocytes live cell

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imaging. Fig. 5 show the fluorescence confocal microscopy images of Clone 9 hepatocytes in the presence and absence of N-CDs/Fe3+-N-CDs complex. The N-CDs display bright multi-colour fluorescence such as blue, green, and red in Clone 9 hepatocytes in different excitation wavelengths of 405, 488 and 555 nm laser pulses, respectively, owing to their high QY. As a result of multi-colour emission, a wide range of excitation wavelength can be applied to cellular 18

imaging agent in the biological system [74, 75]. The appropriate amount of ferric (Fe3+) ion solution was mixed with the multi-fluorescent N-CDs. Afterward, turn-off the fluorescence of NCDs due to the Fe3+ ions more actively react with surface functional groups of N-CDs which

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make complex between them. The made metal complexes would comfort charge-transfer and restrict the recombination of excitons, resulting in the fluorescence quenching behavior. The turn-off fluorescence Fe3+-N-CDs complex used as a probe for live cell imaging. A similar procedure was adopted for the cell culture and imaging as the plain N-CDs. The Fe3+-N-CDs complex treated Clone 9 hepatocytes display a nearly zero fluorescence which might be used to

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detect the cell in the biological systems. The untreated Clone 9 hepatocytes (absence of N-CDs)

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as a control did not show any fluorescence at any excitations. As a conclusion from this result,

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the synthesized N-CDs could be internalized successfully in the Clone 9 hepatocytes and it can

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be employed for multi-colour fluorescence imaging with good biocompatibility.

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On the other hand, the aqueous solution of synthesized N-CDs was used as a fluorescent ink. The aqueous solution of N-CDs was injected into a vacant pen and directly used as ink without

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the need of any pretreatment to write fluorescent words. The handwritten filter paper under UV

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light without any coagulation is shown in Fig. 6. The photograph displays the text and pattern which are highly distinct from the background. Hence, the result suggests that the green

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synthesized N-CDs ink could be an alternative for traditional fluorescent pens due to their excellent properties such as durable and permanent fluorescence, pollution free, biocompatible and easy washable [76–78].

19

Conclusion Durable fluorescence N-CDs have been successfully synthesized from a green source of M. liliiflora flower by an economical hydrothermal method. M. liliiflora flower serving as both

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carbon and nitrogen source, which is different from the traditional synthesis method using two reactants. The synthesized N-CDs potentially utilized as a fluorescent probe for various applications including selective and sensitive detection of Fe3+ ion and live cell imaging without any chemical modifications, owing to their excellent monodispersibility, good biocompatibility and bright fluorescence as well as excellent fluorescence durability with high QY of 11%. On the

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other hand, the N-CDs has been directly used as a fluorescent ink without any coagulation within

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the pen based on their stable fluorescence, eco-friendly and easily washable. Thus, the

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synthesized N-CDs could be applied in three different field: as a fluorescence sensor for the

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detection of Fe3+ in the industrial wastewater; as a fluorescence probe for cellular imaging in real

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biological system and clinical application; and as a fluorescent ink without any pretreatment of

Acknowledgments

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the sample which could be an alternative for traditional fluorescent pens.

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This research was supported by the Nano Material Technology Development Program of the

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Korean National Research Foundation (NRF) funded by the Korean Ministry of Education, Science, and Technology (grant number 2012M3A7B4049675). This work was also supported

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by the National Research Foundation of Korea (NRF) grant funded by the Priority Research Centers Program (grant number 2014R1A6A1031189).

20

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Fig. 1 Schematic illustration for the synthesis and applications of fluorescent N-CDs.

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Fig. 2 (a and b) HRTEM images with a different magnification and (c) AFM topography image

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of fluorescent N-CDs.

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(b)

C1s

Intensity (Counts)

400000 300000

100000 0

O1s N1s

OKL

200000

80000

Raw C=C C-C C-O-C/O-C=O C-N/C-OH Base line Fitted

60000 40000 20000 0

1200

1000

800

600

400

200

0

290

Intensity (Counts)

27000

15000

284

282

24000 21000

O1s

Raw H-O-H C=O C-OH/C-O-C Base line Fitted

18000 15000

A

13000

(d)

N

Raw C-N-C (C)3-N Base line Fitted

14000

286

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N1s

12000

406

404

402

400

M

Intensity (Counts)

(c)

288

Binding Energy (eV)

Binding Energy (eV) 16000

C1s

100000

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(a)

500000

CKL

Intensity (Counts)

600000

398

396

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Binding Energy (eV)

12000 538

536

534

532

530

528

Binding Energy (eV)

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Fig. 3 XPS (a) survey scan and the corresponding deconvolution spectra of (b) C 1s peak, (c) N

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1s peak and (d) O 1s peak of fluorescent N-CDs.

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200

100

0.8 0.6 0.4 0.2

500

550

300

200

200

100

350

400

450

500

550

Wavelength (nm)

(d)

0.4 0.3 0.2

400

050 M 150 M 250 M

450

500

550

600

Cu

3+

Fe

2+

2+

Hg

2+

Ni

Pb

0.15

0.05

2

R = 0.999 0.00 0

0.0

0

0.10

5

10

15

20

25

3+

Concentration of Fe (M)

50

100

150

200 3+

Concentration of Fe

250

(M)

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Wavelength (nm)

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0.1

Blank 100 M 200 M

2+

Zn

2+

Co

A

100

0 350

0.5

Blank 10 M 20 M 25 M

300

3+

2+

Metal Ions (1 mM)

-0.2

(F0-F)/F0

Intensity (Counts)

Intensity (Counts)

(c)

Ca

600

Wavelength (nm) 400

2+

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450

Cr

Cd

Al

N

400

2+

3+

0.0

0 350

(b)

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300

1.0

(F0-F)/F 0

Blank 3+ Al 2+ Ca 2+ Cd 2+ Co 3+ Cr 2+ Cu 3+ Fe 2+ Hg 2+ Ni 2+ Pb 2+ Zn

(a)

(F0-F)/F0

Intensity (Counts)

400

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Fig. 4 (a) Fluorescence emission spectra of N-CDs in presence of various metal ions (1000 μM). (b) The fluorescence intensity ratio (F0-F)/F0 of the N-CDs in the presence of various individual

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metal ions. (c) The fluorescence emission spectra of N-CDs in the presence of increasing Fe3+ concentrations (0–250 μM). (d) The relationship between fluorescence intensity and

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concentrations of Fe3+ from 0 to 250 μM. Inset (c) The fluorescence emission spectra of N-CDs

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in the presence of increasing Fe3+ concentrations (0–25 μM) and inset (d) is Stern-Volmer plot of the quenching of the fluorescence of N-CDs by addition of Fe3+ ions from 0 to 25 μM (SD: 0.00273; n=4).

29

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Fig. 5 Confocal fluorescent microscopy image of Clone 9 hepatocytes in presence and absence

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of N-CDs and Clone 9 hepatocytes in presence N-CDs-Fe3+ complex for 24 h incubation time at

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three different excitation wavelength such as 405 (Blue), 488 (Green) and 555 nm (Red).

30

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Fig. 6 The photographic image of the fluorescent pattern and text is written on a filter paper

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under a UV light with a wavelength of 365 nm using the aqueous solution of N-CDs.

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A

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79.

31