Facile synthesis of stable colloidal suspension of amorphous carbon nanoparticles in aqueous medium and their characterization

Accepted Manuscript Facile synthesis of stable colloidal suspension of amorphous carbon nanoparticles in aqueous medium and their characterization Tirusew Tegafaw, In Taek Oh, Hyunsil Cha, Huan Yue, Xu Miao, Son Long Ho, Mohammad Yaseen Ahmad, Shanti Marasini, Adibehalsadat Ghazanfari, Hee-Kyung Kim, Kwon Seok Chae, Yongmin Chang, Gang Ho Lee PII:

S0022-3697(17)32509-X

DOI:

10.1016/j.jpcs.2018.04.031

Reference:

PCS 8547

To appear in:

Journal of Physics and Chemistry of Solids

Received Date: 28 December 2017 Revised Date:

9 March 2018

Accepted Date: 24 April 2018

Please cite this article as: T. Tegafaw, I.T. Oh, H. Cha, H. Yue, X. Miao, S.L. Ho, M.Y. Ahmad, S. Marasini, A. Ghazanfari, H.-K. Kim, K.S. Chae, Y. Chang, G.H. Lee, Facile synthesis of stable colloidal suspension of amorphous carbon nanoparticles in aqueous medium and their characterization, Journal of Physics and Chemistry of Solids (2018), doi: 10.1016/j.jpcs.2018.04.031. 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.

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Graphical Abstract

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Facile synthesis of stable colloidal suspension of amorphous carbon

nanoparticles

in

aqueous

medium

and

their

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characterization

Tirusew Tegafawa, In Taek Ohb, Hyunsil Chac, Huan Yuea, Xu Miaoa, Son Long Hoa, Mohammad Yaseen Ahmada, Shanti Marasinia, Adibehalsadat Ghazanfaria, Hee-Kyung Kimc,

a

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Kwon Seok Chaeb*, Yongmin Changc*, and Gang Ho Lee1a*

Department of Chemistry and Department of Nanoscience and Nanotechnology (DNN),

College of Natural Sciences, Kyungpook National University (KNU), Taegu 41566, South Korea

Department of Biology Education and DNN, Teacher’s College, KNU, Taegu 41566, South

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b

Korea c

Department of Molecular Medicine and Medical & Biological Engineering and DNN,

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School of Medicine, KNU and Hospital, Taegu 41566, South Korea

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Corresponding authors:

Gang Ho Lee, Tel: -82-53-950-5340, Fax: -82-53-950-6330, Email: [email protected] Yongmin Chang, Email: [email protected] Kwon Seok Chae, Email: [email protected]

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ACCEPTED MANUSCRIPT Abstract

In this study, amorphous carbon nanoparticles (CNPs) with an average diameter of 2.2 nm

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were synthesized by reducing dextrose (C6H12O6) with sodium hydroxide in an aqueous medium. The amorphous CNPs formed stable colloidal suspensions in water owing to the presence of hydrophilic functional groups on the nanoparticle surfaces. The amorphous CNPs

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exhibited ultraviolet (UV)-visible absorption (λabs) at 267 nm and emission (λem) at 453 nm under UV irradiation; thus, the colloidal suspension appeared sky-blue in color under UV

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irradiation. The amorphous CNPs were paramagnetic with weak magnetization at room temperature and exhibited small longitudinal (r1) and transverse (r2) water proton relaxivities of 0.036 and 0.068 s-1mM-1, respectively. The amorphous CNPs exhibited no cellular toxicity up to the measured carbon concentration of 500 µM and presented fluorescence microscopy

applications.

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images on a micrometer scale, thus demonstrating their utility in fluorescence bio-imaging

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Keywords: Amorphous carbon nanoparticle; Magnetic property; Optical property; Cellular

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toxicity; Fluorescence microscopy image

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ACCEPTED MANUSCRIPT 1. Introduction Carbon, a non-toxic element, is one of the key elements in living organisms. Thus, realizing the biomedical applications of various chemical forms of carbon is a challenging

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task. Carbon has various allotropes such as diamond, graphite, C60, C70, graphene, nanotubes, nanoribbons, and amorphous carbon [1,2]. Among these, amorphous carbon nanoparticles (CNPs) are particularly attractive materials for biomedical applications because of their small

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size, facile large scale synthesis, low cost production, and easy surface modification [3-7]. In several studies, CNPs with defect structures exhibited ferromagnetism and nearly

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temperature-independent saturation magnetization and coercivity up to room temperature, while bulk carbon was non-magnetic [8-12]. CNPs also showed particle-size-dependent absorption and emission properties in the ultraviolet (UV)-visible region, similar to that of quantum dots (QDs) [13-17]. The magnetic properties of CNPs make them useful for

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magnetic resonance imaging (MRI), and their optical properties may be applied in fluorescence bio-imaging. Among application studies, fluorescence bio-imaging applications of CNPs have been intensively investigated [15-31]. The optical properties of CNPs have also

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been exploited for sensing metal ions [30,32-35] and bacterial pathogens [36]. Furthermore, CNPs have been applied in delivery systems for drugs, siRNA, DNA, and membrane-

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impermeable molecules [27-29]. They had also been applied for adsorption of organic dyes [37], and improving the photocatalytic activities of metal oxides [38-40] and electrochemical hydrogen storage of metal oxides [41]. However, limited research has been performed on the magnetic properties of CNPs; moreover, water proton relaxivities have also not been measured so far. In this study, we developed a simple one-step synthesis of amorphous CNPs in a basic aqueous medium that form stable colloidal suspensions without any additional surface 3

ACCEPTED MANUSCRIPT modification. Dextrose was used as the carbon source. We investigated the water proton relaxivities, magnetic properties, UV-visible absorption and emission properties, in vitro

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cellular toxicity, and micrometer-scale fluorescence microscopy images of the CNPs.

2. Experimental 2.1. Chemicals

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Dextrose (or D-glucose) (C6H12O6) (≥ 99.5%) and NaOH (99.99%) were purchased from Sigma-Aldrich, USA, and used as received. Triple-distilled water was used to synthesize the

2.2. Synthesis of amorphous CNPs

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amorphous CNPs and prepare the nanoparticle solutions.

Stable colloidal suspensions of amorphous CNPs were synthesized as shown in Fig. 1.

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Two separate solutions were prepared: (1) a dextrose solution made of 7 g of dextrose in 20 mL of triple-distilled water in a three-necked round bottom flask and (2) a NaOH solution made of 2 g of NaOH in 10 mL of triple-distilled water in a beaker. The dextrose solution was

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magnetically stirred at room temperature under atmospheric conditions until the dextrose completely dissolved in the triple-distilled water. Then, the NaOH solution was added to the

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dextrose solution via a syringe while maintaining a solution pH of 9. The mixture solution was heated to 90 oC and magnetically stirred at this temperature for 2 h. The product solution was cooled to room temperature. To remove unreacted dextrose, Na+, and OH-, the product solution was dialyzed against triple-distilled water using a dialysis tube (Cellu·Sep® H1, MWCO = ~ 2,000 Da) for 24 h. The triple-distilled water was replaced with fresh water, and the dialysis process was repeated three times. One half of the sample was dispersed in tripledistilled water to prepare a suspension, and the other half was dried in air to obtain a powder 4

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2.3. General characterization

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A high-resolution transmission electron microscope (HRTEM) (FEI, Titan G2 ChemiSTEM CS Probe) operating at 200 kV was used to measure the particle diameter (d) and morphology of the CNPs. A drop of the sample solution dispersed in triple-distilled water

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was placed on a carbon film supported by a 200-mesh copper grid using a micropipette (Eppendorf, 2-20 µL), and allowed to dry in air at room temperature. The copper grid with

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the CNPs was then mounted inside a vacuum chamber for measurement.

A multi-purpose powder X-ray diffraction (XRD) machine (Philips, X’PERT PRO MRD) with unfiltered CuKα radiation (λ = 0.154184 nm) was used to determine the crystal structure of the CNPs. The samples were scanned in the 2θ range of 15 - 100˚ in 0.033˚ steps.

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A Fourier transform-infrared (FT-IR) absorption spectrometer (Mattson Instruments, Inc., Galaxy 7020A) was used to record the FT-IR absorption spectra of the CNPs between 400 and 4000 cm-1. A pellet of the powder sample in KBr was prepared for measurements.

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An elemental analysis instrument (ThermoFisher, Flash 2000) was used to analyze both the powder and solution samples. From these analyses, the chemical composition (C, H, and

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O) of the CNPs and the carbon concentration in the solution sample were estimated. The magnetic properties of the powder sample were measured using a vibrating sample magnetometer (Lake Shore, 7407-S). Approximately 20 mg of the powder sample was used, and the magnetization (M) versus applied field (H) curve was recorded between -2.0 and 2.0 T at 297 K. From the M – H curve, the magnetism and M values of the sample were determined. The optical properties of the colloidal suspension of the CNPs in water were measured by 5

ACCEPTED MANUSCRIPT recording the UV-visible absorption spectra using a UV-visible absorption spectrophotometer (Agilent Technologies, Cary-Series) and photoluminescence (PL) spectra using a PL spectrometer (Agilent Technologies, Cary Eclipse Fluorescence Spectrophotometer). A quartz

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cuvette with two optically clear sides (Sigma-Aldrich, 3 mL) was filled with the sample solution to record the UV-visible absorption spectra, and a quartz cuvette with four optically clear sides was filled with the sample solution to record the PL spectra. 13

C nuclear magnetic resonance (NMR) spectra were recorded using a 500

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The 1H and

MHz NMR spectrometer (Bruker, Avance III 500 MHz) to obtain more information on the

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CNPs. For measurements, ~60 mg of CNPs was dissolved in 2 mL of D2O.

2.4. Measurement of water proton relaxivities

A 1.5 T MRI instrument (GE Medical System, Signa Advantage 1.5 T) equipped with a

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knee coil (EXTREM) was used to measure the longitudinal (T1) and transverse (T2) water proton relaxation times. The original concentrated sample solution was diluted with tripledistilled water to prepare a series of samples with different carbon concentrations (1.0, 0.5,

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0.25, 0.125, and 0.0625mM). The relaxation times were then determined using these diluted solutions. The longitudinal (r1) and transverse (r2) water proton relaxivities of the sample

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solution were estimated from the slopes of 1/T1 and 1/T2, respectively, plotted versus the carbon concentration. The T1 relaxation time measurements were conducted using an inversion recovery method. In this method, the inversion time (TI) was varied at 1.5 T, and the MR images were acquired for 35 different TI values in the range from 50 to 1,750 ms. The T1 relaxation times were then obtained from the nonlinear least-squares fit to the measured signal intensities at various TI values. For the measurement of T2 relaxation times, the Carr-Purcell-Meiboom-Gill pulse sequence was used in multiple spin-echo measurements. 6

ACCEPTED MANUSCRIPT Then, 34 images were acquired at 34 different echo time (TE) values in the range from 10 to 1,900 ms. The T2 relaxation times were obtained from the nonlinear least-squares fit to the mean pixel values for the multiple spin-echo measurements at various TE values. The

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parameters used for the measurements were as follows: the external MR field (H) = 1.5 tesla, the temperature (T) = 22 °C, the number of acquisitions (NEX) = 1, the field of view (FOV) = 16 cm, the phase FOV = 0.5, the matrix size = 256 × 128, the slice thickness = 5 mm, the

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pixel spacing = 0.625 mm, the pixel band width = 122.10 Hz, and the repetition time (TR) =

2.5. Measurement of cell viability

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2,000 ms.

A CellTiter-Glo luminescent cell viability assay (Promega, USA) was used to measure the cellular toxicity of the CNPs. In this assay, the intracellular adenosine triphosphate was

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quantified using a luminometer (Perkin-Elmer, Victor 3). Human prostate cancer (DU145) and normal mouse hepatocyte (NCTC1469) cell lines were used. Cells were seeded on a 24well cell culture plate and incubated for 24 h (5 × 104 cell density, 500 µL cells/well, 5% CO2,

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and 37 °C). Dulbecco modified Eagle medium and Roswell Park Memorial Institute 1640 were used as the culture media for the NCTC1469 and DU145 cells, respectively. Five test

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solutions (10, 50, 100, 200, and 500 µM carbon) were prepared by diluting the original sample solution with a sterile phosphate-buffered saline solution. Approximately 2 µL of each test solution was added to the cells, and the treated cells were then incubated for 48 h. The cell viabilities were determined twice to obtain the average value and normalized with respect to that of the carbon-free control cells.

2.6. Fluorescence microscopy image measurements 7

ACCEPTED MANUSCRIPT A solution of amorphous CNPs was dropped onto a glass slide, and fluorescence microscopy images were captured on a micrometer scale using a fluorescence microscope

3. Results and discussion 3.1. Product yield, particle diameter (d), and crystal structure

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(Olympus, IX 51) at an excitation wavelength (λex) of 370 nm with a mercury lamp.

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The estimated production yield was ~22%. HRTEM images show that the amorphous CNPs were nearly monodisperse in particle diameter (Fig. 2a). The estimated average particle

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diameter (davg) was 2.2 nm from a log-normal function fit to the observed particle size distribution (Fig. 2b). A photograph of the original concentrated aqueous sample solution (2.65 M carbon) is shown in Fig. 2c, revealing the excellent colloidal dispersion of the CNPs in water. A photograph of the powder sample is shown in Fig. 2d. To confirm the colloidal

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dispersion, laser light was passed through both the diluted sample solution and the tripledistilled water as a control. Light scattering was observed only in the sample solution, confirming the colloidal nature of the dispersion, i.e., the Tyndall effect (Fig. 2e).

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The XRD pattern of the as-synthesized powder sample is shown in Fig. 3. The peaks were broad, indicating the amorphous nature of the sample. Two very broad peaks were observed

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at 2θ = 19o and ~38o, which were assigned to the C (002) and C (004), respectively, consistent with the reported data [42,43]. The results are summarized in Table 1.

3.2. FT-IR absorption spectral results As shown in Fig. 4a, the C=C stretching at 1,574 and 1,390 cm-1, corresponding to the Gand D-bands [44,45], respectively, appeared in the FT-IR absorption spectrum of the sample (bottom spectrum in Fig. 4a), as previously observed in other CNPs [15,16,44,46]. These 8

ACCEPTED MANUSCRIPT bands did not appear in the spectrum of free dextrose (top spectrum in Fig. 4a) [47] because of no C=C bond as shown in Fig. 4b, supporting the formation of CNPs. The G-band in the FT-IR absorption spectrum arose from the symmetry broken sp2-carbons on the CNP surfaces

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[44] owing to their conjugation with hydrophilic functional groups such as –OH and –COOH, whereas the D-band resulted from the defect sp2-carbons on the CNP surfaces, making CNPs paramagnetic as discussed later. The high intensity peaks for O-H stretching at 3,320 cm-1 and C-O stretching at 1,067 cm-1 indicate the existence of large amounts of OH groups on the

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CNP surfaces. In addition, the very small COOH stretching peak at 1,759 cm-1 indicates the

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presence of a small number of COOH groups on the CNP surfaces. These OH and COOH groups engender the CNPs with excellent colloidal stability in water. The C-H stretching band at 2922 cm-1 indicated the presence of aliphatic carbons in the CNPs.

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3.3. CNP composition

By elemental analysis, the estimated composition of the powder sample was 45.67% C, 5.38% H, and 48.95% O by weight, or 31.10% C, 43.92% H, and 24.98% O in atomic percent

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(Table 1). This confirmed the presence of a large number of OH groups on the CNP surfaces. The estimated composition of the solution sample was 3.10% C, 11.90% H, and 84.90% O by

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weight, or 1.48% C, 68.21% H, and 30.31% O in atomic percent. Based on these data, the estimated carbon concentration of the sample solution was determined to be 2.65 M.

3.4. 1H and 13C NMR spectra 1

H and 13C NMR spectra were recorded to obtain more information on the composition of

CNPs. As shown in Fig. 5a, the 1H peaks appeared between 0.5 and 4.5 ppm, indicating that sp3-carbons formed the majority in CNPs. As shown in the expanded scale in Fig. 5a, the 9

ACCEPTED MANUSCRIPT sharp peaks are attributed to H atoms bonded to sp3-carbons, whereas the broad peaks can be assigned to H atoms in –OH groups. As shown in Fig. 5b, the 13C peaks between 10 and 80 ppm resulted from sp3-carbons, whereas the peak at 180.158 ppm was the result of –COOH,

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which was weakly observed in the FT-IR absorption spectrum (Fig. 4a). The sp2-carbons that used to appear between 100 and 150 ppm were not detected at the present signal-to-noise

below.

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3.5. UV-visible absorption and emission properties

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ratio, indicating a low amount. This is consistent with a low CNP quantum yield as discussed

The UV-visible absorption and PL spectra of the amorphous CNPs in water are shown in Figs. 6a and 5b, respectively. The UV-visible absorption spectrum of water was also recorded for reference (bottom spectrum in Fig. 6a). The amorphous CNPs showed an absorption

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maximum (λabs) at 267 nm and an emission maximum (λem) at 453 nm (λex = 370 nm) (Table 1). PL spectra were measured at several excitation wavelengths (Fig. 6b), but excitation wavelength-dependent emission behavior was not observed except for emission intensity

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because the particle diameter was nearly monodispersed, as can be seen in the HRTEM image (Fig. 2a). This result is consistent with the observations reported elsewhere [17].

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Photographs of a diluted sample solution before and during UV irradiation (λex = 365 nm) are shown in Fig. 6c, revealing fluorescence in the sky-blue region under UV irradiation. This result would be extremely useful for fluorescence imaging. Therefore, the CNPs were applied to fluorescence microscopy as discussed in Section 3.8. The estimated quantum yield (QY) of the CNPs at λex = 365 nm was ~9% and is consistent with the results of previous studies [17,30]. For measurements, fluorescein was used as a reference with a QY of 95% at λex = 465 nm in a 0.1 M NaOH aqueous solution. 10

ACCEPTED MANUSCRIPT For calculations, the following formula was used: QYCNP = QYfluorescein ⅹ (PLI/AI)CNP ⅹ (AI/PLI)fluorescein ⅹ (ηCNP/ηfluorescein)2 in which PL (labeled as PLI) and UV-visible absorption (labeled as AI) intensities were estimated from the corresponding peak areas in the PL and

aqueous CNP and fluorescein solutions.

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3.6. Magnetic properties and water proton relaxivities

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UV-visible absorption spectra, respectively, and the refractive index η was 1.33 for both the

The r1 and r2 values of the amorphous CNPs estimated from the slopes of 1/T1 and 1/T2

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plotted against carbon concentration in Fig. 7 were 0.036 and 0.068 s-1mM-1, respectively (Table 1). These small r1 and r2 values are not suitable for use as MRI contrast agents. The small r1 value results from the orbital contribution in the magnetic moment of carbon, and the small r2 value occurs owing to the weak magnetization of the amorphous CNPs at room

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temperature [48] as shown in the M-H curve (Fig. 8), which indicates that the amorphous CNPs were paramagnetic. For example, the M value of the amorphous CNPs was only 0.37 emu/g at 2.0 T (Table 1). Pure carbon is non-magnetic. Therefore, the small M value results

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from defect structures, as has been previously observed in graphite and CNPs in which even higher magnetizations and ferromagnetism were observed owing to their larger amount of

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defect structures [8-12]. Because the r2 value increases with an increase in the M value of the chemicals [49] (here, the r1 value cannot be improved because of the orbital component in the magnetic moment of carbon as mentioned above [48]), more defect structures should be incorporated into the CNPs to obtain a higher r2 value so that they can be used as a T2 MRI contrast agent.

3.7. In vitro cytotoxicity results 11

ACCEPTED MANUSCRIPT Biocompatibility of the amorphous CNPs was verified by assessing their in vitro cytotoxicity using DU145 and NCTC1469 cell lines. The amorphous CNPs were non-toxic up to the measured carbon concentration of 500 µM in both cell lines (Fig. 9), which

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demonstrated their biocompatibility.

3.8. Fluorescence microscopy imaging

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Fluorescence microscopy images were obtained using a dispersed sample solution on a glass slide before (Fig. 10a) and under (Fig. 10b) UV irradiation (λex = 370 nm) with a

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mercury lamp. As shown in Fig. 10b, the fluorescence image of the amorphous CNPs was observed on a micrometer scale. Considering that the amorphous CNPs form stable colloidal suspensions in water (Fig. 2c), show non-toxicity (Fig. 9), and present fluorescence microscopy images on a micrometer scale (Fig. 10b), the amorphous CNPs are useful for

4. Conclusion

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fluorescence bio-imaging, similar to QDs and organic dyes.

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In summary, we prepared amorphous CNPs by reducing dextrose with NaOH in an aqueous medium. The estimated production yield was ~22%. We characterized their water

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proton relaxivities, magnetic properties, optical properties, and in vitro cellular cytotoxicity, and obtained micrometer-scale fluorescence microscopy images. The results revealed the following:

(1) The CNPs were amorphous and nearly monodisperse with an average diameter of 2.2 nm. The CNPs also formed stable colloidal suspensions in water. (2) The CNPs were non-toxic at the measured carbon concentrations of up to 500 µM. (3) The CNPs were paramagnetic and exhibited weak magnetization at room temperature. 12

ACCEPTED MANUSCRIPT Consequently, their r2 value was small, i.e., 0.068 s-1mM-1. Their r1 value was small as well, i.e., 0.036 s-1mM-1, owing to the orbital contribution in the magnetic moment of carbon.

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(4) The CNPs appeared sky-blue in color under UV irradiation and exhibited absorption at λabs = 267 nm and emission at λem = 453 nm. The estimated quantum yield at λex = 365 nm was ~9%.

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be used as fluorescence bio-imaging agents.

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(5) Fluorescence microscopy images on a micrometer scale demonstrated that the CNPs can

Acknowledgments

This study was supported by the Basic Science Research Program (Grant No. 2017R1A2B3003214 to YC and 2016R1D1A3B01007622 to GHL) and the Basic Research

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Laboratory (BRL) Program (Grant No. 2013R1A4A1069507) of the National Research Foundation funded by the Ministry of Education, Science, and Technology. The authors wish

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to thank the Korea Basic Science Institute for providing their XRD diffractometer.

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ACCEPTED MANUSCRIPT living cells, Int. J. Nanomed. 12 (2017) 7375-7391. [31] F. Wu, H. Su, X. Zhu, K. Wang, Z. Zhang, W.-K. Wong, Near-infrared emissive lanthanide hybridized carbon quantum dots for bioimaging applications, J. Mater. Chem.

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for sensitive and selective detection of mercury(II) ions, Anal. Chem. 84 (2012) 5351– 5357.

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[33] Y. Dong, R. Wang, G. Li, C. Chen, Y. Chi, G. Chen, Polyamine-functionalized carbon quantum dots as fluorescent probes for selective and sensitive detection of copper ions, Anal. Chem. 84 (2012) 6220–6224.

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functionalized carbon nanodots: a novel chemiluminescence probe for selective detection of iron(III) ions, RSC Adv. 4 (2014) 45768–45771. [35] V. Roshni, D. Ottoor, Synthesis of carbon nanoparticles using one step green approach

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and their application as mercuric ion sensor, J. Lumin. 161 (2015) 117-122. [36] H. Safardoust-Hojaghan, M. Salavati-Niasari, O. Amiri, M. Hassanpour, Preparation of

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highly luminescent nitrogen doped graphene quantum dots and their application as a probe for detection of Staphylococcus aureus and E. coli, J. Mol. Liq. 241 (2017) 11141119.

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carbon material as a strong protonic acid, Angew. Chem. Int. Ed. 43 (2004) 2955-2958. [43] N. Tsubouchi, C. Xu, Y. Ohtsuka, Carbon crystallization during high-temperature pyrolysis of coals and the enhancement by calcium, Energy Fuels 17 (2003) 1119-1125.

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[44] J. H. Kaufman, S. Metin, Symmetry breaking in nitrogen-doped amorphous carbon: infrared observation of the Raman-active G and D bands, Phys. Rev. B 39 (1989)

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ACCEPTED MANUSCRIPT [48] R. B. Lauffer, Paramagnetic metal complexes as water proton relaxation agents for NMR imaging: theory and design, Chem. Rev. 87 (1987) 901-927. [49] A. Roch, R. N. Muller, P. Gillis, Theory of proton relaxation induced by

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superparamagnetic particles, J. Chem. Phys. 110 (1999) 5403-5411.

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Table 1

(Atomic %) C

2.2

amorphous

~22

31.10

H

43.92

O

24.98

λabs

λem

(nm)

(nm)

267

453

Color

Water proton relaxivity

QY at 365 nm (%)

Sky-blue

~9

20

r1

r2

(s-1mM-1)

(s-1mM-1)

0.036

0.068

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(%)

Optical property

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Composition

TE D

(nm)

Yield

EP

Structure

AC C

davg

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Summary of the observed properties of synthesized amorphous CNPs. Magnetic property

Magnetism

M (emu/g) at 2.0 T

paramagnetic

0.37

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Figures and captions

Triple-distilled water

Cooling to room temp.

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+

Dissolve

Add NaOH solution

Room temp., pH = ~ 9 magnetic stirring, in air

90 oC, 2 h

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Dextrose

AC C

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TE D

Fig. 1. One-step synthesis of amorphous CNPs that form stable colloidal suspensions in water.

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Dialysis Water-soluble amorphous CNP

8

(b)

7

davg = 2.2 nm

6 5 4 3 2.0

2.4 2.8 d (nm)

3.2

3.6

4.0

TE D

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SC

1.6

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Number of nanoparticles

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Fig. 2. (a) HRTEM image (arrows indicate the amorphous CNPs), (b) log-normal function fit

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to the observed particle diameter distribution, (c) photograph of aqueous sample suspension, (d) photograph of a powder sample, and (e) laser light scattering of a CNP suspension,

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confirming its colloidal nature, i.e., the Tyndall effect (left: triple-distilled water; right: diluted sample solution).

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Intensity (Arb. Unit)

C (002)

60 2θ (degree)

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40

80

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20

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C (004)

100

Fig. 3. Powder XRD pattern of CNPs revealing their amorphous structure. The assignments

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are Miller indices (hkl).

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(a)

Transmittance (%)

Dextrose

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Sample 1759 COOH

1067 C-O 1390 1574 C=C (D) C=C (G)

2922 C-H

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3320 O-H 4000 3000 2000 1000 -1 Wavenumber (cm )

HO

(b)

O

OH

TE D

OH

OH OH

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Fig. 4. (a) FT-IR absorption spectra of amorphous CNPs (bottom spectrum: G and D indicate G- and D-bands, respectively) and free dextrose (top spectrum) and (b) chemical structure of

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dextrose.

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TE D

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in D2O.

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Fig. 5. (a) 1H NMR spectrum (inset is expanded scale) and (b) 13C NMR spectrum of CNPs

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Sample Water

200

(b)

300 400 500 600 Wavelength (nm)

700

λem = 453 nm

λex =

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250 nm

300

EP

300 nm 350 nm 370 nm

400 500 Wavelength (nm)

600

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Emission Intensity (Arb. Units)

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λabs = 267 nm

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(a)

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Absorbance (Arb. Units)

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Fig. 6. (a) UV-visible absorption spectra, (b) PL spectra of colloidal suspension of amorphous CNPs in water (λex = 250, 300, 350, and 370 nm) and (c) photographs of corresponding diluted sample solution before (left) and under (right) 365 nm UV irradiation by mercury lamp.

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-1

-1

r2 = 0.068 s mM

1.8

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1/T1

-1

1/T (s )

1.6 1.4

1/T2

1.2

-1

-1

1.0

0.0

0.2

0.4

0.6

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r1 = 0.036 s mM

0.8

1.0

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Carbon concentration (mM)

Fig. 7. Plots of 1/T1 and 1/T2 versus carbon concentration (slopes correspond to r1 and r2

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values, respectively).

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0.4

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0.0 -0.2

T = 298 K

-0.4 -10k

0 H (Oe)

10k

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-20k

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M (emu/g)

0.2

20k

Fig. 8. Curve of magnetization (M) versus applied field (H) for amorphous CNP powder

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sample at T = 298 K.

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DU145 NCTC1469

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100 75 50 25 0

10 50 100 200 500 Carbon concentration (µM)

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0

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Cell viability (%)

125

AC C

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Fig. 9. In vitro cell viability of aqueous sample solution in DU145 and NCTC1469 cell lines.

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Fig. 10. Fluorescence microscopy images of sample dispersed on a glass slide: (a) before and (b) under UV irradiation by mercury lamp at λex = 370 nm. The sky-blue fluorescence image

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shown in (b) is a result of the amorphous CNPs.

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Highlights Title: Facile synthesis and characterization of stable colloidal suspension of amorphous carbon nanoparticles in aqueous medium

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Authors: Tirusew Tegafaw, In Taek Oh, Hyunsil Cha, Huan Yue, Xu Miao, Son Long Ho, Ahmad Mohammad Yaseen, Shanti Marasini, Adibehalsadat Ghazanfari, Hee-Kyung Kim,

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Kwon Seok Chae, Yongmin Chang, and Gang Ho Lee

• The amorphous CNPs with an average diameter of 2.2 nm were synthesized in an aqueous

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medium.

• The CNPs were non-toxic and formed stable colloidal suspensions in water. • The CNPs were paramagnetic and exhibited weak magnetization at room temperature. • The CNPs exhibited small water proton relaxivities.

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applications.

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• Fluorescence microscopy demonstrated the utility of the CNPs in fluorescence bio-imaging