Waste chicken eggshell as low-cost precursor for efficient synthesis of nitrogen-doped fluorescent carbon nanodots and their multi-functional applications

Abstract

A novel one-step top-down method is developed for the efficient synthesis of nitrogen-doped carbon nanodots (NCNDs) by using calcination treatment of waste chicken eggshell as resources. Based on the usage of urea as a dopant, the as-synthesized NCNDs are referred to as NCND 1 (no dopant) and NCND 2. The structural and composition analysis indicate that NCND 2 possesses a mean particle diameter of 2.6 nm and an amorphous carbon structure with a lattice spacing of 0.38 nm. Despite having a low N-content (∼4%), the as-prepared NCND 1 was also found to exhibit excellent photoluminescent properties, low cytotoxicity and could be effectively used for bioimaging, in conjunction with NCND 2. The multi-functional capability of NCND 2 is further demonstrated for stamping, printing, and forensic applications. This work may pave the way for employing carbonaceous waste materials as potential precursors in the synthesis of carbon nanomaterials for wide technological applications.

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Introduction

In recent years, fluorescent carbon-based nanomaterials such as carbon nanotubes,¹ fullerenes,² nanodiamond,³ graphene,⁴ and others⁵ have aroused tremendous research interest owing to their wide technological applications.⁶ Aside from the as-mentioned carbon nanomaterials, carbon nanodots (CNDs) have drawn the most extensive attention in the fields of optical imaging,⁷ optoelectronic devices,⁸ sensing,^9,10 and biotechnology.¹¹ This growing interest in CNDs, over traditional quantum dots, is due to their exceptional properties including high stability, low cytotoxicity as well as biocompatibility. To date, considerable approaches such as arc-discharge,¹² laser ablation,¹³ microemulsion,¹⁴ electrochemical synthesis,¹⁵ combustion,¹⁶ ultrasonic,¹⁷ microwave,¹⁸ organic pyrolysis,¹⁹ and hydrothermal^9a methods have been developed to prepare these versatile nanomaterials. On another note, nitrogen-doped CNDs (NCNDs) have emerged to complement CNDs in oxygen reduction reaction, (photo)catalysis, and CO₂ capture applications.²⁰ To incorporate nitrogen groups into the carbon framework, strategies such as ultrasonic treatment of glucose with aqueous ammonia,²¹ annealing of graphene oxide (GO) under ammonia atmosphere,²² and hydrothermal treatment of grass,²³ a mixture CCl₄ + NaNH₂,²⁴ and CCl₄ + diamine,²⁵ have been reported, recently. However, most of these methods rely heavily on complex and time-consuming processes, severe synthesis conditions, toxic chemicals, and high cost. In this context, it has become critical to search for alternative precursor materials avoiding the use of harmful solvents for rapid fabrication of NCNDs.

Indubitably, chicken eggs, the major source of proteins, are essentially consumed all across the world resulting in the production of significant solid waste, eggshell. Eggshell weighs approximately 10% of the total mass of hen egg and is commonly disposed in landfills without any pretreatment.²⁶ In china, for instance, it has been estimated that about 4 000 000 tones eggshell is generated annually and that will continue to grow in near future. After the pilot study on eggshell as a possible bone substitute,²⁷ in recent years, a great deal of attention has been paid for the application of eggshell as value-added products. The major applications included a starting material for calcium phosphate bioceramics,²⁸ an inexpensive adsorbent for removal of ionic pollutant from the aqueous solution,²⁶ a coating pigment for inkjet printing paper,²⁹ and a starting material to prepare active heterogeneous catalyst.³⁰ However, to the best of our knowledge, eggshell-derived fluorescent NCNDs are not reported to date.

In continuation of our high interest in carbon-based nanomaterials³¹ as well as in practicing green chemistry as paradigm for diverse applications,³² herein, for the first time, we present an easy, inexpensive, high-output calcination treatment of waste chicken eggshell to prepare water soluble fluorescent NCNDs (NCND 1 and NCND 2) yet avoiding any additives such as salts, acids, or bases. The as-prepared, metal free NCNDs exhibit good photostability, low toxicity, and multicolour fluorescence property in the T-cell acute lymphoblastic leukemia (CCRF-CEM) and human cervical cancer (HeLa) cell lines as well as in Zebrafish. In addition, the NCND 2 could be utilized as a very effective biocompatible fluorescent ink for stamping, printing, and forensic applications (Scheme 1).

Scheme 1 A schematic displaying the preparation of eggshell-derived photoluminescent NCNDs and their different applications in stamping, printing, and bioimaging.

Results and discussion

Characterization of NCNDs

TEM, XRD and AFM analysis. A typical transmission electron microscopy (TEM) image (Fig. 1(a)) shows that the as-prepared NCND 2 have a uniform dispersion without apparent aggregation and a mean particle diameter (D_p) of 2.6 nm. Further, the high resolution TEM (HRTEM) image (Fig. 1(b)) demonstrates that most NCND 2 are nearly spherical with a low carbon-lattice-structure.

Fig. 1 (a) TEM image of as-prepared NCND 2; scale bar 20 nm (inset: the corresponding size distribution histograms. (b) HRTEM image of NCND 2; scale bar 5 nm. (c) AFM image of NCND 2 on a silicon substrate. (d) The corresponding height-profile along the line in (c).

The X-ray diffraction (XRD) pattern of NCND 2 (Fig. S1†) showed a broad diffraction peak at 2θ = 22.3° having an interlayer d spacing of ∼0.38 nm, which agrees well with the HRTEM analysis and further confirms the presence of an amorphous carbon phase. Almost similar morphology and size distribution was recognized in case of NCND 1. Specifically, the TEM image revealed that NCND 1 with a size distribution of 1–10 nm have uniform dispersion, no aggregation and a D_p of ca. 3.2 nm (Fig. S2(a) and (b)†). In order to get more insight about size of NCND 1 and NCND 2, atomic force microscopy (AFM) was carried out. As can be seen in Fig. 1(c) and (d), the average height of as-synthesized NCND 2 is ∼2.2 nm. On the other hand, the height profile of labelled line in NCND 1 indicated an average height of ∼1.7 nm (Fig. S2(c) and (d)†).

FT-IR and SS ¹³C NMR analysis. The chemical and structural information about NCND 2 were evaluated by Fourier transform infrared (FT-IR) and solid state carbon-13 nuclear magnetic resonance (¹³C NMR) spectrum as shown in Fig. S3 and S4,† respectively. In particular, NCND 2 exhibited characteristic absorption bands of O–H/N–H stretching vibrations of amine groups at 3351 cm⁻¹, C–H stretching vibrations at 2982 and 2883 cm⁻¹, C–N stretching vibrations at 1436 cm⁻¹, C–O stretching vibrations at 1246 cm⁻¹, and C–H bending vibrations at 1064–1115 cm⁻¹.³³ The presence of significant carbonyl units, which combined with the hydroxyl groups on the surface, endows the NCND 2 with high water solubility as illustrated by the characteristic absorption peak at 1662 cm⁻¹. The ¹³C NMR spectrum of NCND 2 displayed a predominant carboxyl/amide carbon peak (160–173 ppm), a weaker aliphatic (16–30 ppm) and methyne (CH/CH–NH; 54–63 ppm) carbon peaks, and a much weaker aromatic peak at 127 ppm.²³ The FT-IR and ¹³C NMR spectra further support the contention that the as-prepared NCND 2 have nothing in common with a graphite-like structure.

XPS analysis. The X-ray photoelectron spectroscopy (XPS) was performed to determine the composition of the eggshell, NCND 1 and NCND 2. The full scan XPS spectra (Fig. S5†) reveal that the original eggshell predominantly confined oxygen, calcium and carbon together with a limited amount of nitrogen element. On the contrary, after calcination, the resultant NCND 2 displayed mainly three typical peaks of C1s, N1s, and O1s in relative atomic ratios of 60.0, 15.4, and 19.4%, respectively (Fig. 2; see Fig. S6† for NCND 1). In detail, the C1s spectrum can be deconvoluted into four peaks corresponding to C–C (284.3 eV), C–N (285.1 eV), C–O (286.1 eV), and C=O/C=N (288.6 eV) functional groups.³⁴ The N1s spectrum reveals three relative nitrogen species of C–N–C (399.7 eV), N–(C)₃ (400.4 eV), and N–H (401.5 eV), that may be bonded to the surface.³⁵ The spectrum of O1s exhibits two characteristic peaks at 531.6 and 533.0 eV, which can be attributed to C=O and C–OH/C–O–C groups, respectively.³⁶ Moreover, the nitrogen content in as-prepared NCND 2 can reach up to 15.4%, which is higher than that of N-doped carbon-based nanomaterials as reported previously.^22,23

Fig. 2 The XPS spectra of the NCND 2. (a) Survey spectrum; high resolution XPS of (b) C1s, (c) N1s, and (d) O1s spectrum.

UV-visible and fluorescence spectroscopy. The UV-vis absorption spectrum of NCND 2 exhibited a clear absorption band at 242 nm assisted with a shoulder peak at ca. 287 nm. When being excited at 360 nm, the NCND 2 exhibited a strong fluorescence peak centred at 469 nm, and exhibited blue fluorescence under UV light (Fig. 3(a)). It is worth pointing out here that the original eggshell powder was non-emissive under UV light indicating that the fluorescence might arises from the nanoparticles through radioactive recombination of excitons or due to the surface functional groups. The as-prepared NCND 2 gave a fluorescence quantum yield of 7.8% (Fig. 3(b)). In order to further explore their optical properties, the fluorescence of NCND 2 was examined at different excitation wavelengths. In particular, the NCND 2 exhibited an excitation dependent fluorescence behaviour (Fig. 3(c) and (d)). When the excitation wavelength was changed from 280 to 460 nm, the fluorescence peak was correspondingly shifted from 451 (blue) to 520 nm (green) and experienced a significant decrease in peak intensity, which is consistent with the previous report.³⁶ The corresponding Commission International d'Eclairage (CIE) coordinates of NCNDs as shown in Fig. 3(e) clearly illustrates the trend of this wavelength shift. The time-resolved fluorescence-decay measurements (Fig. 3(f)) indicated that NCND 2 have an average fluorescence life time (τ) of 11.3 ns which is quite longer than the reported values.³⁷ Such a longer lifetime further indicates that the N-containing auxochromic groups on NCND 2 surface are most responsible for luminescence properties³⁸ rather than the radioactive recombination of excitons.³⁹ The as-prepared NCND 1 showed almost similar fluorescence properties with marginal variations (Fig. S7†). This variation is most likely caused by the more effective electronic transition in NCND 2. To understand the role of N-dopant, urea, in the calcination treatment of eggshell, we compared the XPS data as well as photophysical properties of NCND 1 and NCND 2 and the results are summarized in Table S1.† These results clearly reveal that usage of urea certainly enhances the luminescence of NCND 2 to a reasonable extent. In particular, a careful examination of quantum yield values and atomic ratios of N1s in Table S1† demonstrates that the PL quantum yield and lifetime increase with increasing N-content. Specifically, NCND 2 indicated ∼a 4-fold enhancement in N-content relative to that of NCND 1. Despite this, both types of NCNDs share an excitation dependent emission origins and can be understood within the framework of surface traps or the so called auxochromes as described below.

Fig. 3 The characteristic optical properties of NCND 2. (a) The UV-vis absorption and fluorescence spectra of NCND 2 in aqueous solution (λ_ex = 360 nm); inset: digital photographs of NCND 2 solutions under daylight (left) and UV light (right). (b) Determination of fluorescence quantum yield of NCND 2. (c) Fluorescence spectra of NCND 2 solution at various excitation wavelengths. (d) The corresponding normalized fluorescence spectra of NCND 2. (e) The corresponding colour coordinate of NCND 2 excited from 280 to 460 nm. (f) A typical time-resolved fluorescence-decay curve of NCND 2 (λ_ex = 350 nm) measured at 450 nm showed an average life time (τ) of 11.3 ns.

It has been postulated that amino groups play dual role in the nanodots formation; as the precursor for N-doping and the surface passivation agents, which both greatly contribute to the photoluminescence enhancement of nanodots.¹⁸ If all surface states are assumed to be completely passivated, the emission will occur only through the radiative transition of sp² carbon (π–π* transition), which in turn, will result in an excitation independence due to single transition mode having certain energy.⁴⁰ Accordingly, in view of the excitation dependent emission property of NCND 2, it can be speculated that N elements are primarily existing as doping N in the core of the dot. This assumption may be further connected by temperature processing conditions (300 °C) as amino groups are unstable and tend to leave the surface at such high temperature.⁴⁰

In a parallel experiment, a small drop of NCND 2 solution (1.0 mg mL⁻¹) was placed on a glass slide and examined by fluorescence microscopy. When being excited at UV (300–385 nm), blue (450–480 nm), and green (510–550 nm) light, the NCND 2 film showed a spectacular response manifested in blue, green and red fluorescence, respectively, making them especially useful for bioimaging (Fig. S8†). The bright and colourful fluorescence may be attributed to the synergistic effect of the carbogenic core and the surface/molecular state of the NCND 2.

Properties and applications of NCNDs

Effect of light, salt and pH on NCNDs. When exposed to a high-brighten cold light (350 W) for 3 h (Fig. S9(a)†), the NCNDs exhibited excellent photostability over DAPI, a commonly used fluorescent dye. Interestingly, on prolonging the time (24 h), no photo-bleaching was recognized under ambient conditions. On another note, when dispersed in NaCl solution (0–500 mM), the fluorescence intensity did not change significantly, indicating that NCNDs could be stable in a medium of comparatively high ionic strength and aggregation (Fig. S9(b)†). This phenomenon is quite significant for NCNDs to be used under saline solutions for practical applications.

Interestingly, over a wide pH range (1–14), NCNDs exhibited a pH-dependent fluorescence behaviour (Fig. S9(c)†). In particular, the fluorescence intensity of NCND 1 decreased linearly from pH 1–13. On the other hand, NCND 2 exhibited striking fluorescence intensity at pH 9, presumably, due to the hydrolysis of amine functional groups. Such an irregular changes in the fluorescence intensity of NCND 2 may formulate them to be used as possible pH sensor in near future.

Cytotoxicity of NCNDs. The inherent cytotoxicity of NCNDs toward HeLa and CCRF-CEM cells was evaluated using MTT assay. It is noteworthy that the NCNDs are scarcely toxic towards HeLa cells even at high doses (1000 μg mL⁻¹) and longer incubation time (24 h). In this regime, the cell survival rate was above 90% as shown in Fig. S10.† However, the viability of CCRF-CEM cell line surprisingly declined by ∼25% upon addition of NCND 2 (1200 μg mL⁻¹).

Additional toxicity studies of NCND 2 were carried out on Zebrafish. Several healthy eggs were given the micro injection of NCND 2 and left until complete development (6 days post fertilization; 6 dpf). Notably, the NCND 2 (1000 μg mL⁻¹) did not show any peculiar impact on the survival of Zebrafish eggs during 6 dpf (Fig. S11†). The above experiments clearly indicate that as-prepared NCNDs are less toxic, safe, and ideal for cell imaging. In order to validate this, NCNDs were further used as a probe for confocal fluorescence imaging.

Applications of NCNDs in bioimaging

Fig. 4 and S12† demonstrate the confocal differential interference fluorescence images of HeLa and CCRF-CEM cells before and after the incubation with NCNDs. Specifically, after 6 h of incubation with NCNDs, the cells became brightly illuminated when excited at 405, 488, and 543 nm laser pulses. On the contrary, the DIC images did not show any visible fluorescence under control experiment. Reasonably, the observed multicolour images of NCNDs in living cell systems have root in their excitation dependent fluorescence behaviour (though stronger in NCND 2). It was found that NCNDs were clearly concentrated in the cell membrane and cytoplasm; whereas the cell nuclei were not infiltrated significantly indicating that NCNDs have good cell-permeability. In view of the possible interferences from out-of-focus light, leading to misinterpretation of the obtained results, the 3D reconstruct fluorescence and bright-field images of CCRF-CEM cells, HeLa cells and Zebrafish were accumulated along the z-direction (Fig. S13†). The results demonstrated a relatively homogeneous distribution as well as strong fluorescence intensities, which further confirmed that NCND 2 have good cell viability and permeability. Furthermore, the CLSM analysis indicated that NCND 2 are highly stable with no blinking and negligible bleaching.

Fig. 4 Confocal differential interference contrast/fluorescence images (all scale bars 20 μm) of CCRF-CEM (a and b) and HeLa cells (c and d). The images correspond to the control experiments (a and c) where no fluorescence appeared. The cells were incubated with (b) 1200 μg mL⁻¹ and (d) 1000 μg mL⁻¹ of NCND 2 in PBS (pH 7.4) at 37 °C for 6 h and were imaged under DIC, 405, 488, and 543 nm excitations, respectively.

The fluorescent NCND 2 were further investigated for multicolour bioimaging of Zebrafish (Danio rerio) using fluorescence microscopy. As can be seen in Fig. 5, during various stages of embryonic development (0–4 days post injection), NCND 2 exhibited blue, green, and red emission fluorescence, when observed through different channels. These merits certainly dictate the effectiveness of NCND 2 for sensing applications.

Fig. 5 The fluorescence microscopy images (scale bars 200 μm) of Zebrafish at different stages of embryonic development under white, UV (300–385 nm), blue (450–480 nm), green (510–550 nm) lights. (a) 1st day post-injection. (b) 4th day post-injection. (c) High resolution image of (b); scale bar 100 μm. The fluorescence microscope was equipped with Hg lamp as light source and DAPI (blue), fluorescein isothiocyanate (green) and propidium iodide (red) filters.

Applications of NCND 2 in stamping, printing and finger printing

Finally, we applied NCND 2 as a new type of biocompatible fluorescent ink for stamping, printing, and forensic applications. A commercial available paper upon which the NCND 2 adhered well with no background UV fluorescence was chosen as stamping and printing paper. The NCND 2–glycerol mixture was utilized as inks under UV, blue, and green light excitations (Fig. S14†). On the other hand, colourless aqueous solution of NCND 2 (1.0 × 10⁻³ mg mL⁻¹) was placed into empty cartridge and printed using a commercially available Canon PIXMA iP 2770 printer. Visible words, images, and patterns having strong fluorescence could be observed under UV illuminations (Fig. S15†), implying that NCND 2 could also be used for forensic applications. In this context, a NCND 2-formed fluorescent fingerprint on commercially available filter paper is shown in Fig. S16.†

Experimental

Materials and chemicals

Fresh chicken eggs were purchased from supermarket in Taiwan. Ethanol (99.9%), quinine sulfate (98%, suitable for fluorescence), paraformaldehyde, 4′-6-diamidino-2-phenylindole (DAPI), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, 98%), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich, USA. Sodium chloride (NaCl), calcium chloride (CaCl₂), magnesium chloride (MgCl₂), glucose, sodium phosphate monobasic (NaH₂PO₄), anhydrous sodium phosphate dibasic (Na₂HPO₄), tri-sodium phosphate (Na₃PO₄), and tris(hydroxymethyl)aminomethane (NH₂C(CH₂OH)₃) were purchased from J.T. Baker, USA. Other chemicals were of analytical grade and were used without further purification. For the cell culture studies, Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS) were purchased from Gibco-BRL, USA. Penicillin–streptomycin solution was purchased from Invitrogen, Carlsbad, USA. Phosphate buffer solutions were prepared with various pH values (pH 3–10). All aqueous solutions were prepared from a Milli-Q (MQ) ultrapure system (18.2 MΩ cm) in this study.

Preparation of NCND 1 and NCND 2

Considering the presence of eggshell membrane as potential impurity or interference,⁴¹ the eggshell was thoroughly washed several times with warm water followed by deionized water to remove impurities and interference material. This process enabled the lighter components to float and that were removed with ease. The eggshell was dried at 100 °C prior to grinding into fine powder. The dried eggshell powder (3.0 g) was placed in a Teflon-lined pressure vessel, sealed in a stainless steel autoclave (80 mL) capacity and calcinated at constant temperature (300 °C) for 2 h. After completing the reaction, the autoclave was cooled to the room temperature. The as-obtained brown powder was dispersed in ethanol, sonicated for 20 min, and filtered to remove large or agglomerated particles. The aqueous solution was then centrifuged (10 000 rpm, 20 min) to separate out the less fluorescence deposit. Finally, the supernatant containing NCND 1 was filtered through a 0.22 μm membrane and the as-obtained NCND 1 in 85.23% yield was used for further studies. A similar procedure was followed to prepare NCND 2 except urea (1.0 g) was mixed with eggshell powder (3.0 g) prior to calcination. The obtained yield of NCND 2 was evaluated to be 80.65%.

Characterization

TEM and HRTEM images were recorded on a JEOL JEM-2100 microscope operated at 200 kV. UV-visible (UV-vis) absorption spectroscopy was carried out using a JASCO V-570 instrument and the absorption maxima are expressed in nanometers (nm). Fluorescence spectroscopy measurements were carried out on a HITACHI F-4500 fluorescence spectrometer. AFM measurements were performed on a dimension Icon, Bruker Nano AFM. About 0.5 μL NCNDs solution was spotted onto a cleaned silicon wafer and dried in vacuum oven (∼12 h, 50 °C). Samples were imaged in air by tapping-mode AFM on a Nanoscope V controller with HAR1-200-10 tips (silicon cantilever, Bruker AFM Probes). The tip surface interaction was minimized by optimizing the scan set point. The XRD patterns were recorded using a Shimadzu XRD-600 diffractometer with Cu Kα radiation. The XPS was performed using ULVAC-PHI Quantera SXM spectrometer. Data were recorded using a monochromatized Al anode as the excitation source. The FT-IR spectrum was recorded on a Perkin-Elmer system 2000 (Perkin-Elmer, Fremont, CA, USA) by spotting the NCNDs solution (in ethanol) on a salt plate. The solid state carbon-13 nuclear magnetic resonance (SS ¹³C NMR) spectra were recorded on a Bruker Avance III 400 spectrometer (Billerica, MA, USA). The imaging of fluorescence emission was performed using an Olympus IX 71 (Tokyo, Japan) fluorescence microscope equipped with an Olympus E-620 camera. The fluorescence emission lifetime of NCNDs were analyzed with a FL920 time-correlated single photon counting module (Edinburgh Instruments EPL375, UK). Lifetime fitting was performed using a complex tri-exponential fitting function (SymPho-Time, PicoQuant).

Cell cultures and MTT assay

CCRF-CEM and HeLa cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Cell lines were separately cultured in RPMI 1640 medium supplemented with 10 vol% FBS and 1 vol% penicillin–streptomycin solution (5000 units mL⁻¹ penicillin G, 50 μg mL⁻¹ streptomycin sulfate in 0.85% NaCl) in a humidified CO₂ (5%) environment at 37 °C. The cells (10⁴ to 10⁵) were washed with phosphate buffered saline (PBS) and countered by a hemocytometer. The solution was then centrifuged at 1000 rpm for 5 min to precipitate out the dispersed cells at the bottom of the tube. After removing upper media, the cells were rinsed and re-dispersed in the same buffer solution for incubation. During experiment, the cells were kept in an ice bath at 4 °C.

The proliferation of cells in the presence of NCNDs were evaluated using a MTT assay, respectively. Initially, CCRF-CEM cells (4 × 10⁵ cells per well) and HeLa cells (5 × 10³ cells per well) were seeded into 96-well plates and incubated at 37 °C. After 24 h, the HeLa cells (50 μL) were incubated with different concentrations of NCNDs (0, 50, 100, 200, 500, and 1000 μg mL⁻¹) for 24 h. Likewise, the CCRF-CEM cells (50 μL) were incubated with NCNDs (0, 200, 400, 600, 800, 1000 and 12 000 μg mL⁻¹) for the same time. After incubation, the aliquot (75 μL) was removed, fresh medium (10% FBS) was added, and allowed to stand for 2 days for further cell growth. The MTT solution (10 μL, 5 mg mL⁻¹ in PBS) was then added to each well and incubated for an additional 2 h under identical conditions. Finally, medium was completely removed and the formazan crystal formed by the cellular reduction of MTT were dissolved in DMSO (150 μL) and thoroughly shaked for 15 min. The absorption at 570 nm was measured with background (pure DMSO) absorbance at 600 nm being substracted. Both were measured using a plate reader (Tecan Safire, Switzerland). The cell viability was expressed as A₅₇₀ treated cell/A₅₇₀ untreated cell × 100%.

In vitro and vivo fluorescence imaging

For fluorescence imaging, the CCRF-CEM and HeLa cells (1 × 10⁴ cells per well) were first seeded onto glass coverslips with cultured medium (1 mL, RPMI 1640 and 10% FBS) in 6-well plate. The plates were incubated in a humidified CO₂ (5%) environment at 37 °C for 12 h. The cells were washed with PBS followed by a serum-free medium (1 mL). Meanwhile, an aqueous solution of NCNDs were passed through a 0.22 μm membrane filter and diluted with deionized (DI) water. The NCNDs solution (500 μg mL⁻¹) was added to each well of the cells and mixed gently with the culture medium. After incubation for 6 h, the cells were rinsed three times with PBS in order to remove any nonbonding NCNDs. Finally, the cells were fixed on coverslips by making the use of 4% paraformaldehyde in PBS and allowed to stand for 20 min at room temperature. The fluorescence/differential interference contrast (DIC) images were recorded on a confocal laser scanning microscope (CLSM, LSM 700, Carl Zeiss, Jena, Germany) equipped with 40× (HeLa cells and Zebrafish) and 63× (CCRF-CEM cells) oil immersion lens and excited at 405, 488, and 543 nm wavelengths, respectively.

Zebrafish were kindly provided by the National Health Research Institute (NHRI), Taiwan. The stock solutions of NCND 2 were diluted to appropriate concentrations in DI water and sonicated until micro injection. Several healthy eggs were given the micro injection of NCND 2 (1.0 mg mL⁻¹) and left until complete development (6 days post fertilization; 6 dpf). For in vivo imaging experiments, the Zebrafish embryos were hatched and fixed on the cover slips with 1% agarose gel (Sigma, USA) to monitor the fluorescence of NCND 2 solution. Confocal slices were taken at 0.5 μm intervals, and a series of 2D live CCRF-CEM cells (HeLa cells, Zebrafish) fluorescence images were acquired at 512 × 512 pixels resolution and a section thicknesses of 14.0 (7.5, 75) μm, respectively. The 3D volume reconstruction from the Z-stack 2D images was carried out using Zen 2009 Light Edition software (Carl Zeiss, Germany).

Fluorescence quantum yield measurements

We used quinine sulfate in 0.1 M H₂SO₄ (refractive index (η) of 1.33, QY 0.54 at 340 nm) as the standard to determine the fluorescence QY of the as-prepared NCNDs. NCNDs were dissolved in ethanol (η = 1.36) and fluorescence spectra were recorded at excitation of 340 nm. By comparing the integrated PL intensities (excited at 340 nm) and the absorbance values (at 340 nm) of NCNDs with quinine sulfate, the QY of NCNDs were determined. Absolute values were calculated using the standard reference sample according to the following equation:
where Φ is the fluorescence QY, I is the integrated emission intensity, A is the optical density and η is the refractive index of the solvent. The subscripts s and x denote standard and sample respectively.

Patterning from NCND 2 solutions

For stamping, glycerol (50 μL) was added to an aqueous solution of NCND 2 (20 μL) and sonicated for 1 h in order to get a homogeneous mixture. The resulting mixture was directly utilized as rubber stamping ink on commercially available paper. For inkjet printing, colorless aqueous solution of NCND 2 (1.0 × 10⁻³ mg mL⁻¹) was filled into an empty ink cartridge of Canon PIXMA ip 2770 inkjet printer at room temperature. The printing process was realized in 15 min.

Statistical analysis

The comparisons between two groups of data was analyzed by one-tailed Student's t-test using statistical software (SPSS, Chicago, Ill, USA). A difference with P < 0.05 was considered statistically significant.

Conclusions

In conclusion, an eco-friendly calcination treatment of eggshell has been proven to be an effective strategy for accessing amphiphilic and fluorescently stable NCNDs for the first time. The excellent multicolor fluorescence properties combined with low cytotoxicity of NCNDs have been successfully used in Zebrafish bioimaging. Furthermore, the NCND 2 could be applied as biocompatible fluorescent ink for multicolor patterns in stamping, printing and forensic applications. This feature can be extended to high-throughput design in information encryption, storage, anti-counterfeiting, and optoelectronic fields. The synthesis and characterization of carbon nanodots utilizing bones, shells, and oysters, are currently in progress in our lab.

Acknowledgements

This work was supported by National Tsing Hua University (102N1807E1) and the Ministry of Science and Technology (NSC101-2113-M-007-006-MY3 and NSC 102-2621-M-007-001) of Taiwan. We also thank to Professor Yu-Fen Huang at the Department of Biomedical Engineering and Environmental Science, National Tsing Hua University, Taiwan for kindly providing CCRF-CEM and HeLa cell lines.

Notes and references

1. (a) K. Welsher, Z. Liu, S. P. Sherlock, J. T. Robinson, Z. Chen, D. Daranciang and H. Dai, Nat. Nanotechnol., 2009, 4, 773–780; (b) H. Tao, K. Yang, Z. Ma, J. Wan, Y. Zhang, Z. Kang and Z. Liu, Small, 2012, 2, 281–290.
2. J. Jeong, M. Cho, Y. T. Lim, N. W. Song and B. H. Chung, Angew. Chem., 2009, 121, 5400–5403 (Angew. Chem., Int. Ed., 2009, 48, 5296–5299).
3. A. Krueger, Adv. Mater., 2008, 20, 2445–2449.
4. (a) L. Tang, R. Ji, X. Cao, J. Lin, H. Jiang, X. Li, K. S. Teng, C. M. Luk, S. Zeng, J. Hao and S. P. Lau, ACS Nano, 2012, 6, 5102–5110; (b) Y. Dong, G. Li, N. Zhou, R. Wang, Y. Chi and G. Chen, Anal. Chem., 2012, 84, 8378–8382.
5. R. Leary and A. Westwood, Carbon, 2011, 49, 741–772.
6. (a) O. A. Shenderova, V. V. Zhirnov and D. W. Brenner, Crit. Rev. Solid State Mater. Sci., 2002, 27, 227–356; (b) Y. H. Ng, S. Ikeda, M. Matsumura and R. Amal, Energy Environ. Sci., 2012, 5, 9307–9318; (c) B.-T. Zhang, X. Zheng, H.-F. Li and J.-M. Lin, Anal. Chim. Acta, 2013, 784, 1–17; (d) Q. Li, S. Zhang, L. Dai and L.-s. Li, J. Am. Chem. Soc., 2012, 134, 18932–18935.
7. (a) X. GaO, L. Yang, J. A. Petros, F. F. Marshal, J. W. Simons and S. Nie, Curr. Opin. Biotechnol., 2005, 16, 63–72; (b) R. Hardman, Environ. Health Perspect., 2006, 114, 165–172; (c) J. K. Jaiswal and S. M. Simon, Trends Cell Biol., 2004, 14, 497–504.
8. X. Feng, V. Marcon, W. Pisula, M. R. Hansen, J. Kirkpatrick, F. Grozema, D. Andrienko, K. Kremer and K. Müllen, Nat. Mater., 2009, 8, 421–426.
9. (a) S. Zhu, Q. Meng, L. Wang, J. Zhang, Y. Song, H. Jin, K. Zhang, H. Sun, H. Wang and B. Yang, Angew. Chem., 2013, 125, 4045–4049 (Angew. Chem., Int. Ed., 2013, 52, 3953–3957); (b) Y. Dong, R. Wang, G. Li, C. Chen, Y. Chi and G. Chen, Anal. Chem., 2012, 84, 6220–6224.
10. (a) Y. Zhou, G. Xing, H. Chen, N. Ogawa and J.-M. Lin, Talanta, 2012, 99, 471–477; (b) Z. Lin, W. Xue, H. Chen and J.-M. Lin, Anal. Chem., 2011, 83, 8245–8251; (c) Z. Lin, W. Xue, H. Chen and J.-M. Lin, Chem. Commun., 2012, 48, 1051–1053.
11. (a) S.-T. Yang, L. Cao, P. G. Luo, F. Lu, X. Wang, H. Wang, M. J. Meziani, Y. Liu, G. Qi and Y.-P. Sun, J. Am. Chem. Soc., 2009, 131, 11308–11309; (b) C. Liu, P. Zhang, X. Zhai, F. Tian, W. Li, J. Yang, Y. Liu, H. Wang, W. Wang and W. Liu, Biomaterials, 2012, 33, 3604–3613.
12. X. Xu, R. Ray, Y. Gu, H. J. Ploehn, L. Gearheart, K. Raker and W. A. Scrivens, J. Am. Chem. Soc., 2004, 126, 12736–12737.
13. (a) S.-L. Hu, K.-Y. Niu, J. Sun, J. Yang, N.-Q. Zhao and X.-W. Du, J. Mater. Chem., 2009, 19, 484–488; (b) S. K. Yang, H. B. Zeng, H. P. Zhao, H. W. Zhang and W. P. Cai, J. Mater. Chem., 2011, 21, 4432–4436.
14. W. Kwon and S.-W. Rhee, Chem. Commun., 2012, 48, 5256–5258.
15. H. Li, X. He, Z. Kang, H. Huang, Y. Liu, J. Liu, S. Lian, C. H. A. Tsang, X. Yang and S.-T. Lee, Angew. Chem., 2010, 122, 4532–4536 (Angew. Chem., Int. Ed., 2010, 49, 4430–4434).
16. H. Liu, Y. Tao and C. Mao, Angew. Chem., 2007, 119, 6593–6595 (Angew. Chem., Int. Ed., 2007, 46, 6473–6475).
17. H. Li, X. He, Y. Liu, H. Huang, S. Lian, S.-T. Lee and Z. Kang, Carbon, 2011, 49, 605–609.
18. X. Zhai, P. Zhang, C. Liu, T. Bai, W. li, L. Dai and W. Liu, Chem. Commun., 2012, 48, 7955–7957.
19. (a) F. Wang, Z. Xie, H. Zhang, C. Y. Liu and Y. G. Zhang, Adv. Funct. Mater., 2011, 21, 1027–1031; (b) Z. Xie, F. Wang and C. Y. Liu, Adv. Mater., 2012, 24, 1716–1721.
20. (a) X. Fang, W. T. Zheng and J.-L. Kuo, RSC Adv., 2013, 3, 5498–5505; (b) C. H. Choi, W. T. Zheng, J. L. Kuo, C. H. Choi, M. W. Chung, H. C. Kwon, S. H. Park and S. I. Woo, J. Mater. Chem. A, 2013, 1, 3694–3699; (c) V. Chandra, S. U. Yu, S. H. Kim, Y. S. Yoon, D. Y. Kim, A. H. Kwon, M. Meyyappan and K. S. Kim, Chem. Commun., 2012, 48, 735–737.
21. Z. Ma, H. Ming, H. Huang, Y. Liu and Z. Kang, New J. Chem., 2012, 36, 861–864.
22. L. Lai, J. R. Potts, D. Zhan, L. Wang, C. K. Poh, C. Tang, H. Gong, Z. Shen, J. Lin and R. S. Ruoff, Energy Environ. Sci., 2012, 5, 7936–7942.
23. S. Liu, J. Tian, L. Wang, Y. Zhang, X. Qin, Y. Luo, A. M. Asiri, A. O. Al-Youbi and X. Sun, Adv. Mater., 2012, 24, 2037–2041.
24. Y.-Q. Zhang, D.-K. Ma, Y. Zhuang, X. Zhang, W. Chen, L.-L. Hong, Q.-X. Yan, K. Yu and S.-M. Huang, J. Mater. Chem., 2012, 22, 16714–16718.
25. Z. S. Qian, J. J. Ma, X. Y. Shan, H. Feng, L. X. Shao and J. R. Chen, Chem.–Eur. J., 2014, 20, 1–11.
26. W. T. Tsai, K. J. Hsien, H. C. Hsu, C. M. Lin, K. Y. Lin and C. H. Chiu, Bioresour. Technol., 2008, 99, 1623–1629.
27. L. Dupoirieux, D. Pourquier and F. Souyris, J. Craniomaxillofac. Surg., 1995, 23, 187–194.
28. C. Balázsi, F. Wéber, Z. Kövér, E. Horváth and C. Németh, J. Eur. Ceram. Soc., 2007, 27, 1601–1606.
29. S. J. Yoo, J. S. Hsieh, P. Zou and J. Kokoszka, Bioresour. Technol., 2009, 100, 6416–6421.
30. Z. Wei, C. Xu and B. Li, Bioresour. Technol., 2009, 100, 2883–2885.
31. (a) G. Gollavelli and Y.-C. Ling, Biomaterials, 2012, 33, 2532–2545; (b) M.-C. Wu, A. R. Deokar, J.-H. Liao, P.-Y. Shih and Y.-C. Ling, ACS Nano, 2013, 7, 1281–1290; (c) P. L. Lee, Y. K. Chiu, Y. C. Sun and Y.-C. Ling, Carbon, 2010, 48, 1397–1404; (d) J.-Y. Liu, H.-Y. Chang, Q.-D. Truong and Y.-C. Ling, J. Mater. Chem. C, 2013, 1, 1713–1716; (e) A. R. Deokar, L.-Y. Lin, C.-C. Chang and Y.-C. Ling, J. Mater. Chem. B, 2013, 1, 2639–2646; (f) G. Gollavelli and Y.-C. Ling, Biomaterials, 2014, 35, 4499–4507; (g) B. Garg, T. Bisht and Y.-C. Ling, Molecules, 2014, 19, 14582–14614; (h) B. Garg, T. Bisht and Y.-C. Ling, RSC Adv., 2014 10.1039/c4ra11205a.
32. (a) J.-Y. Liu, B. Garg and Y.-C. Ling, Green Chem., 2011, 13, 2029–2031; (b) B. Garg, S.-L. Lei, S.-C. Liu, T. Bisht, J.-Y. Liu and Y.-C. Ling, Anal. Chim. Acta, 2012, 757, 48–55; (c) B. Garg and Y.-C. Ling, Tetrahedron Lett., 2012, 53, 5674–5677; (d) B. Garg and Y.-C. Ling, Green Mater., 2013, 1, 47–61; (e) B. Garg and Y.-C. Ling, J. Chin. Chem. Soc., 2014, 61, 737–742; (f) B. Garg, L. Yan, T. Bisht, C. Zhu and Y.-C. Ling, RSC Adv., 2014, 4, 36344–36349.
33. (a) Y. Fang, S. Guo, D. Li, C. Zhu, W. Ren, S. Dong and E. Wang, ACS Nano, 2012, 6, 400–409; (b) Y. Zhang, C. Xing, D. Jiang and M. Chen, CrystEngComm, 2013, 15, 6305–6310.
34. S. Liu, J. Tian, L. Wang, Y. Luo, W. Lu and X. Sun, Biosens. Bioelectron., 2011, 26, 4491–4496.
35. Y. Zheng, Y. Jiao, L. Ge, M. Jaroniec and S. Z. Qiao, Angew. Chem., 2013, 125, 3192–3198 (Angew. Chem., Int. Ed., 2013, 52, 3110–3116).
36. S. Fleutot, J. C. Dupin, G. Renaudin and H. Martinez, Phys. Chem. Chem. Phys., 2011, 13, 17564–17578.
37. Z. L. Wu, P. Zhang, M. X. Gao, C. F. Liu, W. Wang, F. Leng and C. Z. Huang, J. Mater. Chem. B, 2013, 1, 2868–2873.
38. (a) S. W. Jin, D. H. Kim, G. H. Jun, S. H. Hong and S. Jeon, ACS Nano, 2013, 7, 1239–1245; (b) W. Kwon, G. Lee, S. Do, T. Joo and S. W. Rhee, Small, 2014, 10, 506–513.
39. (a) Y. M. Guo, Z. Wang, H. W. Shao and X. Y. Jiang, Carbon, 2013, 52, 583–589; (b) A. Jaiswal, S. S. Ghosh and A. Chattopadhyay, Chem. Commun., 2012, 48, 407–409.
40. X. Li, S. Zhang, S. A. Kulinich, Y. Liu and H. Zeng, Sci. Rep., 2014 DOI:10.1038/srep04976.
41. Q. Wang, X. Liu, L. Zhang and Y. Lv, Analyst, 2012, 137, 5392–5397.

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Footnote

† Electronic supplementary information (ESI) available: Characterization, absorption and fluorescence spectroscopic data, cytotoxicity, applications, and other additional data of NCND 1 and NCND 2. See DOI: 10.1039/c4ra10178b

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