Accepted Manuscript Title: Facile synthesis of nitrogen-doped carbon dots for Fe3+ sensing and cellular imaging Author: Xiaojuan Gong Wenjing Lu Man Chin Paau Qin Hu Xin Wu Shaomin Shuang Chuan Dong Martin M.F. Choi PII: DOI: Reference:

S0003-2670(14)01483-4 http://dx.doi.org/doi:10.1016/j.aca.2014.12.045 ACA 233651

To appear in:

Analytica Chimica Acta

Received date: Revised date: Accepted date:

13-10-2014 19-12-2014 25-12-2014

Please cite this article as: Xiaojuan Gong, Wenjing Lu, Man Chin Paau, Qin Hu, Xin Wu, Shaomin Shuang, Chuan Dong, Martin M.F.Choi, Facile synthesis of nitrogendoped carbon dots for Fe3+ sensing and cellular imaging, Analytica Chimica Acta http://dx.doi.org/10.1016/j.aca.2014.12.045 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.

Facile synthesis of nitrogen-doped carbon dots for Fe3+ sensing and cellular imaging Xiaojuan Gonga,1, Wenjing Lua, Man Chin Paaub, Qin Hub, Xin Wua, Shaomin Shuanga, Chuan Donga,*, Martin M. F. Choib,2,** a

Institute of Environmental Science, and School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006, China b Partner State Key Laboratory of Environmental and Biological Analysis, and Department of Chemistry, Hong Kong Baptist University, 224 Waterloo Road, Kowloon Tong, Hong Kong SAR, China

Highlights ·Fast synthesis of nitrogen-doped carbon dots (N-CDs) by microwave method.

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·Optimization of synthesis of N-CDs. ·Fluorescence sensing of Fe3+ by N-CDs.

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Graphical abstractGraphic abstract

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·Cell imaging and detecting Fe3+ in biosystem by N-CDs.

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ABSTRACT A fast and facile approach to synthesize highly nitrogen (N)-doped carbon dots (N-CDs) by

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microwave-assisted pyrolysis of chitosan, acetic acid and 1,2-ethylenediamine as the carbon source,

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condensation agent and N-dopant, respectively is reported. The obtained N-CDs are fully characterized by elemental analysis, transmission electron microscopy, high-resolution transmission

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electron microscopy, Fourier transform infrared spectroscopy, Raman spectroscopy, X-ray diffraction pattern, X-ray photoelectron spectroscopy, UV-vis absorption, and photoluminescence

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spectroscopy. Doping N heteroatoms benefits the generation of N-CDs with stronger fluorescence

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emission. As the emission of N-CDs is efficiently quenched by Fe3+, the as-prepared N-CDs is employed as a highly sensitive and selective probe for Fe3+ detection. The detection limit can reach

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as low as 10 ppb and the linear range is 0.010–1.8 ppm Fe3+. The as-synthesized N-CDs have been successfully applied for cell imaging and detecting Fe3+ in biosystem.

Keywords: Nitrogen-doped carbon dots, Chitosan, EDA, Fe3+ detection, Cell image ∗ ∗∗

1

Corresponding author. Tel: +86-351-7018613; fax: +86-351-7018613. Corresponding author. Fax: +852-34117348. E-mail addresses: [email protected] (C. Dong); [email protected] (M.M.F. Choi). Exchange student on visit to Hong Kong Baptist University.

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Present address: Acadia Divinity College, Acadia University, 15 University Avenue, Wolfville, Nova Scotia, B4P 2R6, Canada

1. Introduction Emerging from carbon nanomaterials, fluorescent carbon dots (CDs) have been recently attracted considerable attention owing to their superior physical and chemical properties [1,2]. Unlike the conventional semiconductor quantum dots (QDs) containing toxic heavy metal elements and chalcogens, CDs are mainly composed of non-toxic C, O, and N elements, and are thus superior in the aspects of good water solubility, outstanding photoluminescence (PL) properties, high quantum yield (Φs), large stokes shifts, robust chemical inertness, ease of functionalization, low cytotoxicity, and excellent biocompatibility [3-6]. Most CDs reported so far are hydrophilic in nature and appear to be a promising alternative to semiconductor QDs in the fields of photocatalysis [7,8], biological labeling [9,10], drug and gene delivery [11,12], chemosensor and biosensor [13-15], fluorescent ink

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[16], and biomedicine [9]. Therefore, it is worthwhile to continue to search for superior synthesis methods of CDs.

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Up to now, extensive efforts have been devoted to synthesize CDs and the synthesis methods can

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be generally classified into top-down and bottom-up approaches [1]. “Top-down” methods involve breaking down larger mass carbon materials into individual nanoparticles (NPs) through arc-

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discharge single-walled carbon nanotubes [2], laser ablation of graphite [6], electrochemical

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oxidation of graphite and multiwalled carbon nanotubes [17,18], carbonizing polymerized resols on

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silica spheres [19], commercially activated carbon and lampblack [20-22], and chemical oxidation of oxide graphene [23]. Complicated synthesis conditions, time and energy consumption, expensive

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starting materials and/or instruments, and difficulty in preparation of large quantities and high

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quality of CDs are often problematic for the “top-down” approaches [24]. By contrast, “bottom-up” approaches allow the preparation of nanostructures with less defects and more homogeneous

polymerization,

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chemical compositions, which involve preparation of CDs from precursors through dehydration, carbonization,

and

passivation

[19,25-27].

Many

precursors

including

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carbohydrates and organic matters such as tea, coffee, pomelo peel, rice, and orange juice have been

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used for preparation of CDs through the hydrothermal routes [28-32]. These methods avoid tedious post-treatment processes and use of a large amount of strong acid for passivation. Although many CDs have been prepared, their broad photoluminescence (PL) emission (λem) profiles, low Φs, and

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difficulties in large-scale preparation remain to be overcome [33,34]. As such, the development of an economical, facile, effective, scalable and green synthetic route to produce strong fluorescent CDs on a large scale for practical application is still needed. In the present work, we report a facile, economical and effective approach to synthesize strong fluorescent CDs by microwave-assisted pyrolysis of chitosan, acetic acid and 1,2-ethylenediamine (EDA) as the precursors. Chitosan rich in carbon can serve as an excellent carbon source [35-37]. Acetic acid promotes the dissolution of chitosan and provides more carboxyl groups for

condensation with EDA. EDA containing nitrogen (N) atoms can function as N-dopants for CDs [38-40]. The resulting N-doped CDs (N-CDs) possess excellent water-solubility and are non-toxic. The synthesis method is cost-effective and fast and can be completed within 15 min using a domestic microwave oven [40,41]. Herein, the effects of the precursors chitosan, acetic acid and EDA on the Φs of N-CDs have been carefully investigated and optimized. It is found that doping more N atoms into CDs can greatly increase the Φs of CDs. The as-synthesized N-CDs can significantly improve Φs and is higher than that of some other N-doped CDs [31,35,36,42-45]. The obtained N-CDs solution exhibits homogeneous phase without any noticeable precipitation at ambient conditions for six months, indicating their long-term colloidal stability. The as-prepared NCDs display excellent stability under various external conditions including pH, ionic strength and

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cation. These N-CDs have been demonstrated as an effective fluorescent probe for sensitive and selective detection of Fe3+ with a detection limit as low as 10 ppb, which is much lower than other

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previous reported values [39,46-49] and is lower than the maximum allowable level (0.3 ppm) for Fe3+ in drinking water permitted by the United States Environmental Protection Agency. Its

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potential applications in bioimaging and intracellular Fe3+ monitoring have been explored. The effects of various metal ions on the PL of N-CDs in cellular environment were for the first time

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investigated. The results demonstrate that N-CDs can be applied for detecting Fe3+ in biosystem and

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shows almost no interference from other metal ions. Our as-synthesized N-CDs could open up more

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analytical applications in bioimaging, biosensing, and biomedicine.

2. Experimental

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2.1. Materials

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Chitosan, potassium bromide, and EDA were obtained from Aldrich (Milwaukee, WI, USA). Glacial acetic acid was from Fisher Chemicals (Fair Lawn, NJ, USA). Dimethyl sulfoxide (DMSO), modified

Eagle's

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Dulbecco's

medium

(DMEM),

fetal

bovine

serum

(FBS),

trypsin,

ethylenediaminetetraacetic acid (EDTA), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

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bromide (MTT) were purchased from Solarbio (Beijing, China). All reagents of analytical reagent grade or above were used as received without further purification. Aqueous solutions were prepared

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with distilled deionized (DDI) water.

2.2. Apparatus The elemental analysis was carried out on an Elementar Analysensysteme vario EL cube elemental analyzer (Hanau, Germany). Analyses were performed in triplicate and the average values were obtained. The transmission electron microscopic (TEM) and high-resolution transmission electron microscopic (HRTEM) images were acquired on a JEOL JEM-2100

transmission electron microscopy (Tokyo, Japan) with an accelerating voltage of 300 kV. The FTIR spectra were performed on a Perkin-Elmer Paragon 1000 FTIR spectrometer (Waltham, MA, USA). The Raman spectra were acquired on a Horiba JY HR800 spectrometer (Palaiseau, France) at excitation (λex) 633 nm. The X-ray diffraction (XRD) patterns were recorded on a Bruker D8 Advance powder X-ray diffractometer (Bremen, Germany) with CuKα radiation operating at 40 kV and 40 mA. The data were collected from 2θ = 10‒70o at a scan rate of 0.03o per step and 2 s per point. The X-ray photoelectron spectra (XPS) were acquired on an AXIS ULTRA DLD X-ray photoelectron spectrometer (Kratos, Tokyo, Japan) with AlKα radiation operating at 1486.6 eV. Spectra were processed by the Case XPS v.2.3.12 software using a peak-fitting routine with symmetrical Gaussian-Lorentzian functions. The UV-vis spectra were performed on a Varian Cary

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300 Scan UV-vis absorption spectrophotometer at 200–650 nm. The PL spectra were recorded on a Varian Cary Eclipse spectrofluorometer (Palo Alto, CA, USA).

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Nanosecond fluorescence lifetime experiments were performed using a S920 time-correlated single-photon counting (TCSPC) system under right-angle sample geometry. An Edinburgh EPL

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405 nm picosecond diode laser with a repetition rate of 2 MHz (Livingston, UK) was used to excite the samples. The fluorescence was collected by a photomultiplier tube (Hamamatsu H5783p)

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connected to a Becker & Hickl SPC-130TCSPC board (Berlin, Germany). The time constant of the

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N

instrument response function was 100 ns.

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2.3. Procedures

1.00 g chitosan was thoroughly mixed with 10.0 mL 8.0% glacial acetic acid, and 5.0 mL EDA

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in a 100-mL beaker. The mixture was heated in a domestic microwave (700 W) for 15 min during

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which the solution changed from opaque to dark brown, indicating the carbonization of the reactants. The reaction mixture was cooled down to room temperature and followed with addition

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of 30 mL ultrapure water to dissolve the N-CDs product. The obtained dark brown solution was centrifuged at 13000 rpm for 15 min. The clear supernatant brown solution was removed and

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dialyzed through a dialysis membrane with MWCO of 500–1000 Da (Spectrum Laboratories, Rancho Dominguez, CA, USA) in a 2 L DDI water with stirring and recharging with fresh DDI

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water every 24 h over a course of 3 days. Finally, a clear and reddish brown aqueous solution containing N-CDs was lyophilized to obtain the dry N-CDs product. Similarly, CDs without EDA (N-doping) were prepared as described above.

2.4. MTT assay For the cell cytotoxicity test, human renal carcinoma786-0 cells were first plated on a Costar® 96-well cell culture cluster and cultured at 37oC with 5.0% CO2 in air for 3 h to adhere cells onto

the surface. The well without cells and treatment with N-CDs, N-CDs/Fe2+ and N-CDs/Fe3+ was taken as the zero sets. The medium was then changed with 100 µL of fresh DMEM supplemented with 10% FBS containing N-CDs, N-CDs/Fe2+ and N-CDs/Fe3+, and the cells were allowed to grow for another 48 h. At least five parallel samples were performed in each group. Cells not treated with N-CDs, N-CDs/Fe2+ and N-CDs/Fe3+were taken as the controls. After adding 20 µL of 5.0 mg/mL MTT reagent into every well, the cells were further incubated for 4 h. The culture medium with MTT was removed and 150 µL of DMSO was added. The resulting mixture was shaken for ca. 10 min at room temperature. The optical density (OD) of the mixture was measured at 490 nm with a SunRise microplate reader (Tecan Austria GmbH, Grödig, Austria). The cell viability was estimated as: Cell viability (%) = (ODTreated/ODControl) × 100%, where ODControl and ODTreated were obtained in

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the absence and presence of N-CDs, N-CDs/Fe2+and N-CDs/Fe3+, respectively. The concentrations

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of Fe2+ and Fe3+ used were 0.10 mM.

2.5. Cellular imaging

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The human renal carcinoma 786-0 cells were cultured in DMEM supplemented with 10% FBS and the cells were seeded in the culture dish and cultured with 0.30 mg/mL N-CDs/10mMFe2+ and

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0.30 mg/mL N-CDs/10mMFe3+, respectively at 37oC. After incubation for 20 h, the 786-0 cells

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were harvest using 0.25% trypsin/0.020% EDTA, washed three times (1.0 mL each) with pH 7.4

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phosphate buffered saline (PBS comprising 137 mM NaCl, 2.7 mM KCl, 8.0 mM Na2HPO4, and

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2.0 mM KH2PO4) and kept in PBS for optical imaging by a Olympus FV1000 confocal microscope (Tokyo, Japan) with 20× and 40× objective. In addition, Ca2+, Mg2+, Hg2+, Al3+, Fe2+, and Fe3+ (0.10

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mM) were added respectively to the culture dish with 786-0 cells which had been previously

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incubated with 0.30 mg/mL N-CDs for 20 h. Then the dynamic 786-0 cell images and fluorescence

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intensity were monitored at various reaction times (0.0, 5.0 and 40 min).

3. Results and discussion

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3.1. Optimization of synthesis of N-CDs The synthetic method can be used to prepare different types of N-CDs by tuning the precursors

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conditions as depicted in Fig. S1 and Table S1. In our preliminary experiments, the OH (from chitosan), COOH (from acetic acid), and NH2 (from EDA) moieties were found crucial for the formation of N-CDs. First, if the precursor contains only OH and COOH groups, the Φs of the asprepared N-CDs is only 6.80%. However, if NH2 groups are added, the Φs of the N-CDs are above 7.00%. The highest Φs is 20.10% for N-CDs prepared with 1.00 g chitosan, 10.0 mL of 8.0% acetic acid and 5.0 mL of EDA. In essence, chitosan, acetic acid and EDA are essential for condensation, polymerization and further carbonization. Reaction time is another important parameter for N-CDs

synthesis. Fig. S2 and Table S1 depict the Φs of N-CDs synthesized at various reaction conditions. As the reaction time proceeds from short to long, the polymer-like N-CDs are converted into carbogenic N-CDs. In a modest reaction time, polymer-like CDs are formed and the PL arises from the surface/molecule state (perhaps owing to amide-containing fluorophores). In a short or long reaction time, owing to further carbonization, partial carbogenic CDs are formed and the PL is derived from the synergistic effect of the carbogenic core and the surface/molecule state. The carbogenic core plays a greater role in CDs no matter the synthesis time decreases or increases. The optimal reaction time is found to be 15 min as it produces the highest Φs. 3.2. Characterization of N-CDs

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In order to further study the effect of EDA, N-CDs and CDs were synthesized (vide supra). Table S2A summarizes the elemental analysis of CDs and N-CDs. The elemental content of CDs

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differs significantly from N-CDs. Higher N and C and less O content are found for N-CDs. For ease of comparison, the elemental contents are expressed in terms of relative number of atom as depicted

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in Table S2B. The empirical formula for CDs and N-CDs are approximately C22H47N6O20 and C25H47N8O8, respectively. CDs and N-CDs contain the same number of H atom. However, the

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numbers of C, N and O atoms in N-CDs differ significantly from that of CDs. More O atoms are

N

found in CDs whereas N-CDs contain more C and N atoms. In other words, the O atoms were

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replaced by the C and N atoms in N-CDs after addition with EDA, indicating that EDA can promote

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carbonization of chitosan and the N originated from EDA is doped into N-CDs. TEM has been used extensively as a powerful tool in the study of NPs from which the

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morphology and size can be identified. Fig. 1A and C show the representative TEM images of CDs

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and N-CDs, respectively. Both CDs and N-CDs are mostly of spherical morphology and disperse rather evenly on the TEM grid surface. The corresponding histograms obtained by statistical

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analysis of approximately 100 particles using the ImageJ software are displayed in Fig. 1B and D. The particle size distributions of CDs and N-CDs are 1.20–4.30 nm and 2.75–6.75 nm with average

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diameters of 2.48 ± 0.40 nm and 4.27 ± 0.50nm, respectively. In brief, doping more N into CDs could facilitate the growth of carbon core, allowing the formation of larger of N-CDs (≥ 4 nm). In

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addition, a magnified TEM image of N-CDs (Inset of Fig. 1C) and the representative HRTEM image of N-CDs (Fig. S3) show that the lattice spacing is ca. 0.21 nm which is consistent with the (100) facet of graphitic structure. Figure 1 FTIR spectra, Raman spectra and XRD patterns were recorded to confirm the higher content of N in N-CDs than that of CDs. Fig. S4A depicts the IR spectra of CDs (spectrum a) and N-CDs (spectrum b). A broad absorption peak attributing to the O-H stretching (～3347–3581 cm-1) and

sharp absorption peaks corresponding to C-H (2937 cm-1), C-O (1150 cm-1), and C-O-C stretching (1025 cm-1) are found for both CDs and N-CDs. These functional groups are believed to be derived from chitosan as their carbon sources. The IR spectrum of chitosan possessing these functionalities is displayed in Fig. S4B. In addition, the IR spectra of CDs and N-CDs possessing distinctive absorption peaks at 1650 cm-1 are attributed to the N-H vibrations in chitosan, which are present in abundance and common in other CDs synthesized by chitosan [35-37]. The IR spectrum of N-CDs possesses an absorption peak at 1565 cm-1, indicating the formation of C=C unsaturated bonds in the carbon cores which are consistent with other fluorescent CDs [50,51]. More importantly, the characteristic absorption peaks amido CON-H bending (1560 and 3290 cm-1) and amido CO-N (1369 cm-1) stretching are identified in N-CDs, suggesting the presence of amido functionalities on

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N-CDs. Fig. S5 depicts the Raman spectra of (a) CDs and (b) N-CDs. Two predominant peaks are observed at 1330 and 1557 cm-1 corresponding to the D and G bands of CDs, respectively. The

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magnitude of D band is slightly larger than that of the G band, indicating that CDs have some defects. The intensity ratio of the D and G band (ID/IG) is a measurement of the extent of disorder as

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well as the ratio of sp3/sp2 carbons [52]. The ID and IG of CDs and N-CDs were calculated with a baseline correction at the Raman bands of 1363 and 1630, and 1386 and 1548 cm-1, respectively. In

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our case, the relative intensity of the “disorder” D band to the crystalline G-band (ID/IG) for CDs

N

and N-CDs are 1.06 and 1.18, respectively. The large ratio indicates the defect of N-CDs with a

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partially disordered crystal structure arising from the sp2 carbon core. The D and G bands of N-CDs

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are not very obvious attributing to the fact that high fluorescence of N-CDs may disturb the Raman characterization. Moreover, N-CDs have high degree of graphitization and more layers of graphite

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which are consistent with the literature [53-56]. This finding agrees well with the TEM analysis

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(vide supra) that N-CDs are larger. The XRD analysis was conducted to compare the crystallinity of chitosan, CDs and N-CDs. Chitosan has a main crystallinity peak at 2θ = 20o and two amorphous

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humps at around 11o and 40o (Fig. S6A). After microwave-assisted carbonization, the crystallinity of chitosan was diminished. Fig. S6B displays the XRD patterns of (a) CDs and (b) N-CDs. CDs

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possess a sharp peak at 22o and a broad peak at 42o, revealing an amorphous carbon phase and partial graphitization of CDs. However, N-CDs display a broad peak centered at 23o (0.34 nm),

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attributing to the highly disordered carbon atoms and high degree of graphitization. This result is in good agreement with the Raman spectrum (vide supra). In summary, the pyrolysis of chitosan and doping more N heteroatoms into CDs should have taken place at high temperature under microwave irradiation, resulting in the formation of high degree graphitization of N-CDs with surface-attached amine/amido moieties. These functional groups are potential linkers for attachment of therapeutic moieties for targeted drug delivery. Figure 2

XPS were acquired to gain further insight into the surface functional groups and elemental states of CDs and N-CDs. Fig. 2A and C depict the survey scan of CDs and N-CDs, respectively. Both CDs show three apparent peaks centered at 285.0, 400.5, and 532.0 eV corresponding to C1s, N1s, and O1s, respectively. The peaks associated with C1s and N1s of N-CDs are much more intense than that of CDs, indicating that higher contents of C and N in CDs. These results further confirm more carbonization and the incorporation of heteroatoms N from EDA into N-CDs, concurring with the elemental analysis (vide supra). Fig. 2B and D illustrate the C1s XPS spectra of CDs and NCDs, respectively. For CDs, the C1s spectrum is deconvoluted into four peaks at 284.6, 286.2, 287.9, and 289.0 eV corresponding to C=C, C-O, C=O, and O-C=O, respectively [57-59]. For NCDs, additional peaks at 283.4, 283.9, and 285.2 eV associated with C-H, C-N-C, and C-N are

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observed [57,60,61]. Both CDs and N-CDs show slightly different O1s spectra (Fig. S7A and B). The O1s of CDs spectrum is deconvoluted into two peaks at 532.7 and 531.7 eV corresponding to

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C=O and O=C-O groups, respectively whereas the O1s spectrum of N-CDs exhibits two peaks at 531.5 and 530.3 eV corresponding to the C-N-O and C-OH groups, respectively. The N1s spectrum

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(Fig. S7C) of N-CDs shows three peaks at 399.5, 400.5, and 401.5 eV attributing to the C-N-C, N(C)3, and N-H functionalities, respectively. Again, these confirm the doping of N onto the surface of

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the as-synthesized N-CDs. In summary, the XPS data show the presence of C=C, C-O, C=O, and O-

N

C=O surface-functionalities on CDs whereas the C=C, C-O, C=O, O-C=O, C-H, C-N-C, and C-

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NH2 surface-functionalities are on N-CDs. Both XPS and IR confirm the surface of N-CDs is co-

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doped with more N atoms.

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3.3. Photoluminescence properties of CDs

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In quest of exploring the spectral properties of the as-prepared CDs, the UV-vis absorption and PL spectra were acquired and depicted in Fig. 3. For the CDs, an absorption peak at ca. 279 nm

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corresponding to the n → π* transition of C=O bond (spectrum a in Fig. 3A) is observed [62,63]. For N-CDs (spectrum b in Fig. 3A), in addition to the n → π* transition at 270 nm, a prominent

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broad absorption peak at 330–340 nm is found, probably attributing to the formation of excited defect surface states induced by the N heteroatoms [64-66]. Fig. 3B and C display the PL spectra of

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CDs and N-CDs under various λex, respectively. The λem peak of CDs remains constant at ca. 422– 428 nm when λex is 280–340 nm; however, it is red-shifted from 429 to 540 nm when λex moves from 360 to 480 nm. Similarly, the λem peak of N-CDs is kept constant at ca. 417–421 nm when λex is 280–340 nm and the λem peak is red-shifted from 423 to 540 nm when λex moves from 360 to 480 nm. The λex-independent PL spectra are probably attributed to the π* → π transitions of the graphitic structure of the carbon cores whereas the λex-dependent PL spectra are derived from the π* → n transitions (surface states) of the surface-attached functionalities (C=O/C-NH2). The λex-dependent

PL spectra are bathochromically shifted with the increase in λex, indicating that the PL band can be tuned by adjusting λex. The λex-dependent PL behavior is common with most CDs. This means that λem can be tuned by just controlling λex without changing CDs. The emission intensities of the NCDs are much stronger than that of the CDs. Obviously, doping more N into the CDs surface could introduce surface states with a concomitant effect on enhancing the PL of CDs. Figure 3 The Φs of CDs and N-CDs were determined at λex/λem of 340/370–650 nm. The Φs are 7.32% and 20.1% for CDs (Fig. S8A) and N-CDs (Fig. S8B), respectively using quinine sulfate (Fig. S8C) as the reference. The Φs of N-CDs is about 3 times of CDs, indicating that doping more N into CDs could greatly improve its Φs. These results are consistent with other reports that CDs contain

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heteroatoms such as N could improve Φs [31]. It is obvious that N-CDs emit stronger than CDs as seen by the photographic images in the insets of Fig. 3B and C. Since N-CDs possess strong and

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stable PL, it is further explored its applications in sensing and bioimaging (vide infra).

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3.4. Stability of N-CDs

The effects of ionic strength (in terms of the concentration of KCl) and pH on the PL stability of

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N-CDs were investigated. The PL intensity and spectral feature of N-CDs do not change much

N

under different concentrations of KCl (Fig. S9A) which is beneficial since it is necessary for N-CDs

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to be used in the presence of various salt concentrations in practical applications. Fig. S9B displays

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the effect of pH on the PL intensity of N-CDs. The PL intensity increases with the increase in pH and reaches the highest at pH 5.0–7.0; however, the PL intensity drops significantly when the pH is

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higher than 11.0. Since the PL intensity is highest and maintains fairly constant at the physiological

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pH 7.0–7.5, it has potential for application in cellular imaging (vide infra). The dry N-CDs powder sample could be repeatedly re-dispersed in water without any aggregation, which is advantageous

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for preservation and transportation. The obtained N-CDs solution exhibits homogeneous phase without any noticeable precipitation at ambient conditions for six months, indicating their long-term

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colloidal stability.

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3.5. Sensing of Fe3+ The PL quenching effect of various cations on N-CDs was investigated. Different metal ions

including K+, Ni2+, Ca2+, Fe2+, Al3+, Zn2+, Cu2+, Mg2+, Co2+, Pb2+, Hg2+, Ba2+, Na+, Ag+, Cd2+, Cr3+, and Fe3+ of each at a concentration of 10 mM were reacted with a N-CDs solution (0.10 mg/mL). Fig. 4A displays the relative change in PL intensity of N-CDs in the presence of various metal ions. Fe2+, Cu2+, Pb2+, Hg2+, Ag+, Cd2+, and Cr3+ ions cause the slight PL changes (defined as the relative change of PL intensity in 60–80% as compared to blank) which can be attributed to the nonspecific

interactions between the carboxylic and/or amine groups and the metal ions [67]. Among these metal ions, Fe3+ displays the strongest PL quenching effect on N-CDs, attributing to the special coordination between the Fe3+ ions and the phenolic hydroxyl and/or amine groups of N-CDs which has been widely used for the detection of Fe3+ ions or colored reactions in traditional organic chemistry [67,68]. Such a specific fluorescence quenching effect may originate from the strong interactions between Fe3+ ions and the surface groups of N-CDs which transfer the photoelectrons from N-CDs to Fe3+ ions [39,69]. These results clearly demonstrate that the N-CDs-based Fe3+ sensor is highly selective to Fe3+ over the other metal ions. Figure 4 This PL quenching may contribute to the nonradiative electron-transfer that involves partial

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transfer of an electron in the excited state to the d orbital of Fe3+ [67]. To obtain further insight into the PL quenching mechanism, TCSPC was used to study the exciton behavior of N-CDs in the

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absence and presence of Fe3+ (Fig. S10A). The average lifetime of N-CDs is 7.28 ns and then decreases to 4.83 ns after reacting with Fe3+. The reduced lifetime indicates an ultrafast N-CDs/Fe3+

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electron-transfer process and leads to quenching. Fig. S10B shows the fluorescence quenching of N-CDs at various concentrations of Fe3+ ions. Fig. S10C displays the Stern-Volmer plot of N-CDs

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with increasing concentration of Fe3+ (F0/F against concentration of Fe3+), where Fo and F are the

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PL intensities of N-CDs at λex/λem of 340/421 nm in the absence and presence of Fe3+, respectively.

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The quenching efficiency is fitted by the Stern-Volmer equation, F0/F = 1 + Ksv[Q], where Ksv is the

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Stern-Volmer quenching constant and [Q] is the Fe3+ concentration. The Ksv is calculated to be 2.8 × 103 L/mol with a correlation coefficient r2 of 0.993. The Fo/F curve is linearly related to the

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concentration of Fe3+ in the range 0.010–1.80 ppm, indicating their excellent sensing properties in

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the detection of trace Fe3+. The limit of detection (LOD) and limit of quantification (LOQ) were calculated by taking the PL intensity equal to 3 times the standard deviation of the intensity at the

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blank (n = 10) divided by the slope of the calibration graph, and three times the LOD, respectively. The LOD and LOQ of the proposed sensor were determined as 10 and 30 ppb, respectively. The

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LOD is much lower than some other reported values [39,46-49] and lower than the maximum allowable level (0.3 ppm) for Fe3+ in drinking water permitted by the United States Environmental

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Protection Agency. Fig. 4B displays the visual fluorescence change of an N-CDs solution (0.10 mg/mL) by Fe3+ (280 ppm) under UV irradiation. It is very obvious that the blue emission disappears after adding Fe3+ to the N-CD solution. Fe3+ ions are indispensable for a large number of living systems and play an important role in many biochemical processes. The detection of Fe3+ ions through a visible fluorescent method would be of considerable benefit.

3.6. Biocompatibility and application of N-CDs in cell imaging

The toxicity of CDs is a natural concern because of their potential for bioimaging and nanoscale dimensions. Toxicity studies have been conducted by various research groups. The reports are many at the moment and CDs appear to have low toxicity [32,34,39,50,70]. Herein, it is crucial to assess the toxicity of N-CDs, N-CDs/0.10 mMFe2+ and N-CDs/0.10 mMFe3+ to the human renal cell carcinoma 786-0 cells by the MTT assay. Fig. 5A depicts the cell viability studies under various concentrations of N-CDs, N-CDs/Fe2+, and N-CDs/Fe3+. The cell viability gradually decreases with the increase in the concentration of N-CDs, N-CDs/Fe2+, and N-CDs/Fe3+; fortunately, the decreases are very small. The survival rates are higher than 82% even under a high concentration (100 µg/mL) of N-CDs, N-CDs/Fe2+, and N-CDs/Fe3+. In addition, under the same concentration, the cell viability of N-CDs is higher than that of N-CDs/Fe3+, and N-CDs/Fe2+. These results demonstrate

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that N-CDs display low toxicity to the 786-0 cells, inferring its potential use in bioimaging of live cells or other biomedical applications.

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Figure 5

It has been reported that water-soluble fluorescent CDs are ideal cell imaging probes with

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minimal cytotoxicity [4,10,23,27,39,70]. Herein, 786-0 cells were used to explore the potential of our N-CDs as a bioimaging agent. These cells were initially incubated with N-CDs/Fe2+ and N-

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CDs/Fe3+ for 20 h, respectively. The cellular uptake of N-CDs was then observed by laser scanning

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confocal microscopic (LSCM) as depicted in Fig. 5B. The bright-field images of the 786-0 cells

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incubated with (i) N-CDs/Fe2+ and (ii) N-CDs/Fe3+ (first panels in Fig. 5B) indicate clearly the

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normal morphology of the cells, verifying that N-CDs/Fe2+ and N-CDs/Fe3+ are biocompatible and possess minimum toxicity to the cells. The cells display blue (second panels), green (fourth panels),

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and red (fifth panels in Fig. 5B) emissions when they are excited with 405, 488, and 543 nm lasers,

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respectively. The merged images (the third panels in Fig. 5B) of the first and second panels and Fig. S11 demonstrate the ability of N-CDs to penetrate into the cell membrane without any further

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surface passivation. This observation demonstrates N-CDs potential as a bioimaging agent for living cells. The cells incubated with N-CDs/Fe2+ emit stronger than that of N-CDs/Fe3+, attributing

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to the fact that Fe3+ could quench N-CDs in aqueous solution (vide supra). To assess the dynamic effect of N-CDs fluorescence by different metal ions including (i) Ca2+,

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(ii) Mg2+, (iii) Hg2+, (iv) Al3+, (v) Fe2+, and (vi) Fe3+ on 786-0 cells, the time-dependent fluorescence images and signals at λex/λem of 543/650±25 nm were acquired and displayed in Fig. 6. Fig. 6A shows the LSCM images of the 786-0 cells initially incubated with N-CDs (0.30 mg/mL) at 37oC for 20 h and then exposed to metal ions (0.10 mM in the culture solution) at time = 0.0 (left panels), 5.0 min (middle panels) and 40 min (right panels), respectively. All the cell images produce very nice red emissions at all the time intervals. It seems that these metal ions (except Fe3+) do not affect the red emission of N-CDs in the cells. By contrast, the red emissions from the cells

incubated with Fe3+ diminish as the time passes by (Fig. 6A(vi)). Fig. S12 depicts the LSCM images of 786-0 cells incubated with N-CDs and exposed to Fe3+ at various times. The cell images were taken at λex/λem of 405/422 ± 25, 488/500 ± 25 and 543/650 ± 25 nm, respectively. All the blue, green and red cell emissions diminish with the increase in incubation times with Fe3+, which are consistent with the results in Fig. 6A(vi). Fig. 6B displays the PL intensity of the cell red emissions after administered with various metal ions (Ca2+, Mg2+, Hg2+, Al3+, Fe2+, and Fe3+). The stars in the figure indicate the moment for injection of metal ions (the dips). All these metal ions do not induce any PL change of the cells after 1 h. However, the PL intensity drops quickly after adding Fe3+ to the 786-0 cells. Again, this demonstrates that Fe3+ can quench N-CDs effectively under the cell environment. As such, N-CDs can be an effective probe for monitoring Fe3+ in

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biosystem. Figure 6

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4. Conclusion

In summary, a low-cost, facile, and high efficient method for fabrication of N-CDs with a Φs as

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high as ca. 20.1% has been developed. The effect of EDA on the synthesis of N-CDs was studied by (HR)TEM, FTIR, Raman, XRD, UV absorption, PL spectroscopy, and XPS. It is found that adding

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EDA favors the formation of N-CDs with stronger emissions. Moreover, N-CDs show good

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sensitivity and selectivity to Fe3+ and can serve as an effective probe for PL detection of Fe3+ with a

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detection limit as low as 10 ppb. Furthermore, N-CDs could be utilized as a reagent capable of

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detecting Fe3+ in biosystem. Combining its simple and fast synthetic method, favorable optical properties, low cytotoxicity and ease of labelling, it is anticipated that N-CDs could have potential

Acknowledgements

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applications in biological labelling, disease diagnosis and biosensors.

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Financial supports from the Hundred Talent Programme of Shanxi Province, HKBU Faculty Research Grant (FRG1/13-14/039) and National Science Foundation of China (21175086) are

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gratefully acknowledged. We would express our sincere thanks to Ms Winnie Y.K. Wu of the Institute of Advanced Materials for taking the TEM images and XPS. The TEM was supported by

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the Special Equipment Grant from the University Grants Committee of the Hong Kong Special Administrative Region, China (Grant SEG_HKBU06).

Appendix A. Supplemental material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.aca. References

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Figures And Tables:

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Fig. 1. (A) TEM image and (B) particle size distribution histogram of CDs. (C) TEM image and (D) particle size distribution histogram of N-CDs. The insets depict the magnified TEM images of CDs and N-CDs. Fig. 2. (A) XPS survey scan and (B) C1s XPS of CDs. (C) XPS survey scan and (D) C1s XPS of NCDs. Fig. 3. (A) UV-vis absorption spectra of (a) CDs and (b) N-CDs. Spectra are offset for ease of comparison. (B) and (C) are PL spectra of CDs and N-CDs at λex 280–480 nm, respectively. The insets display the images of CDs (0.15 mg/mL) and N-CDs (0.15 mg/mL) under daylight and UV irradiation.

Fig. 4. (A) The relative change in PL intensity of N-CDs (0.10 mg/mL) after reacting with different metal ions (10 mM). (B) Photographic images of N-CDs (0.10 mg/mL) before and after adding Fe3+ (280 ppm) under daylight (left panel) and UV irradiation (right panel). Fig. 5. (A) Cytotoxicity test of 786-0 cells treated with various concentrations of N-CDs, NCDs/0.10 mM Fe2+, and N-CDs/0.10 mM Fe3+. The cell viabilities are normalized to the control (water). The error bars represent the standard deviation of three independent measurements. (B) LSCM images of 786-0 cells incubated with (i) 0.30 mg/mL N-CDs/10 mMFe2+ and (ii) 0.30 mg/mL N-CDs/10 mMFe3+ at 37oC for 20 h. The first left panels show the bright-field images of 786-0 cells. The second, fourth, and fifth panels are cell images taken at λex/λem of 405/422 ± 25, 488/500 ± 25. and 543/650 ± 25 nm, respectively. The third panels are the merged images of the first and second panels.

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Fig. 6. (A) LSCM images of 786-0 cells cultured in 0.30 mg/mL N-CDs at 37oC for 20 h and then subjected to various metal ions: (i) Ca2+, (ii) Mg2+, (iii) Hg2+, (iv) Al3+, (v) Fe2+, and (vi) Fe3+ in the culture solutions at time = 0.0 (left panels) and 5.0 min (middle panels) and 40 min (right panels). The images are taken at λex/λem: 543/650 ± 25 nm. (B) The PL fluctuation curves of 786-0 cells from 0.0 to 60 min. The labelled with asterisks represent the metal ions injection into the culture solution.