Preparation of nitrogen-doped carbon dots with a high fluorescence quantum yield for the highly sensitive detection of Cu2+ ions, drawing anti-counterfeit patterns and imaging live cells

NEW CARBON MATERIALS Volume 34, Issue 4, Aug 2019 Online English edition of the Chinese language journal Cite this article as: New Carbon Materials, 2019, 34(4): 390-402

RESEARCH PAPER

Preparation of nitrogen-doped carbon dots with a high fluorescence quantum yield for the highly sensitive detection of Cu2+ ions, drawing anti-counterfeit patterns and imaging live cells Wen Liu1,*, Rong Zhang1, Yu Kang1, Xiao-ying Zhang2, Hao-jiang Wang1, Li-hong Li1, Hai-peng Diao1, Wen-long Wei3,* 1

School of Basic Medical Sciences, Shanxi Medical University, Taiyuan 030001, China;

2

School of Shanxi provincial Traditional Chinese medicine, Taiyuan 030012, China;

3

Department of Chemistry and Chemical Engineering, Taiyuan University of Technology, Taiyuan 030024, China

Abstract:

Water-soluble luminescent nitrogen-doped CDs (N-CDs) having a high QY of 29.83% were prepared by a one-step

microwave-assisted pyrolysis method using ammonium citrate and triethylenetetramine as the precursors. The microstructure, optical properties and biocompatibility of the N-CDs were investigated. Results indicate that they exhibit a strong fluorescence, favorable biocompatibility and excellent optical stability. Their photoluminescence is quenched by Cu2+ ions through the formation of a complex between the N-CDs and Cu2+, which can be used for Cu2+ detection. Compared with other CD-based nanomaterials, the N-CDs show outstanding optical properties, and excellent selectivity and sensitivity for Cu2+ detection with a linear range of 0.01-11 μM and a detection limit of 4.5 nM. The selectivity for Cu2+ detection is so good that the N-CDs have been successfully used in Cu2+ detection for real water samples. They have also been used to draw fluorescent patterns and image living cells. Key Words: Carbon dots; Highly fluorescence quantum yield; Cu2+ sensor; Fluorescent patterns; Cells imaging

1 Introduction With the progress of science and technology, fluorescent carbon

nitrogen-doped CDs (N-CDs) were fabricated to improve the

materials have attracted much attention. Fluorescent carbon materials

fluorescence quantum yields of CDs since nitrogen atoms have five

are widely used in life marker diagnosis, drug delivery and

valence electrons and a comparable atomic size for bonding with

fluorescence detection, and biomimetic and fluorescence material

carbon atoms

carbon leads to energy level transition due to different light excitated

QYs of the CDs

by photon

[1, 2]

. Because of the traditional quantum dots are the

semiconductors mainly consisting of the group IIB-VIA, IIIA-VA or IVA-VIA elements, which have biosecurity and environmental pollution risks

[3]

[12, 13]

. As well known, covalent bond can increase the

[14]

. Furthermore, nitrogen doping for CDs can

effectively control its intrinsic properties, such as the surface and local chemical reactivity and electronic properties [15-17]. The carbon dots have rich surface groups by the chemical

. Carbon dots (CDs) have attracted increasing

interaction, including coordination, π-π interaction and covalent bond.

attention as they were serendipitously discovered in 2004, which is

The surface modification of CDs can not only control and regulate the

fascinating fluorescent nanomaterials [4]. The CDs are a new type of

surface properties of the CDs for increasing its diversified

fluorescent materials, which have the advantages such as good

applications, but also increase the QYs of the CDs. The

biocompatibility, small toxicity, easy availability of raw materials,

photoluminescence of the CDs could be quenched by the electron

low cost, high degree of fluorescence adjustable, easy to be

acceptor or the electron donor molecule, indicating that the CDs are

functionalized, excellent light stability and high anti-bleaching

good electron acceptors or electron donors, the CDs can be used to

ability[5-8].

measure certain specific ions by using this property

[18-20]

. As well

2+

At present, the syntheses of CDs have two types of methods, the

known, Cu is essential for all living systems, which plays a critical

top-down and bottom-up methods. However, the methods usually

role in some pathological and physiological processes. But, it is

involve complex processes, harsh synthesis conditions and low

harmful with exposure with body for long-term at high concentrations.

fluorescence quantum yields (QYs) of <10%

[9-11]

. Currently,

Copper have a significant impact on the function and development of

Received date: 01 Jun 2019; Revised date: 01 Aug 2019 *Corresponding author. E-mail: [email protected]；[email protected] Copyright©2019, Institute of Coal Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-5805(19)60021-1

Wen Liu et al. / New Carbon Materials, 2019, 34(4): 390-402

the immune system and the central nervous system, which is the main element as the composition of hemocyanin

[21, 22]

. And Cu2+ can cause

many diseases including Alzheimer and Wilson’s diseases

[23, 24]

size distribution of as-prepared N-CDs was obtained by counting about 100 particles from the TEM image. Functional groups and

. On

elemental characterization of as-prepared CDs were performed by a

the basis of the U.S. environmental protection policy, the Cu2+ upper

Thermo EscaLab-250xi X-ray photoelectron spectrometer using 300

limit is 20 μM in drinking water [25]. So, it is an extremely urgent need

W Al-Kα radiations. Fourier Transform Infrared Spectroscopy spectra

to detect Cu2+ from the environment with a high selectivity and

were collected by a Varian FTIR-640 spectrometer, the samples were

2+

sensitivity. The excess Cu can inhibit the activity of protein, leading

diluted with KBr and pressed into the disk. Fluorescence photographs

to tissue necrosis. The extensive use of Cu2+ and its derivatives can

of the N-CDs under 365 nm UV light were obtained with a Shanghai

also cause environmental pollution

[26]

, so quantitative determination

Jia-Peng ZF-6 Ultraviolet Analyzer. In addition, UV-Visible

of Cu2+ is very important. For the past few years, nanomaterials have

absorption spectroscopy was performed by a Shanghai Mapada

2+

been employed for sensing Cu . Huang et al.

[27]

reported the g-C3N4

UV-Vis

6100

spectrophotometer.

All

fluorescence

spectral

nanofilms as a fluorescence sensor for rapid, selective and sensitive

measurements for the N-CDs were carried out using a Varian

sensing Cu2+. Liu et al.

Cary-Eclipse fluorescence spectrophotometer in a 1cm×1cm quartz

[9]

applied polymer nanomaterials for

label-free selective and sensitive detection of Cu2+. As novel

cell.

2+

nanomaterials, CDs have also been used for constructing Cu sensors. Ganiga et al.

[28]

reported that the N-CDs were used for Cu2+ sensors

and its detection limit is 10 μM. But, as a new type of nanomaterial probe, the CDs did not show obvious advantages over other nanomaterial-based

fluorescence

probes

and

other

organic

fluorescence probes in terms of selectivity and sensitivity, mainly due to the fact that the obtained CDs did not show satisfied fluorescence activities, leading to low detection sensitivities. Apparently, the development of specially CD fluorescence probes with high QYs is important for the applications of the fluorescent CDs. In this work, high QY N-CDs are synthesized by a one-step microwave assisted pyrolysis method, which is a facile, fast and low cost

preparation

route,

using

ammonium

citrate

and

triethylenetetramine as reaction reagents. The as-prepared N-CDs have highly selective and ultrasensitive Cu2+ detection. Moreover, the synthesized N-CDs can be applied for patterning and detection of Cu2+ ions in living cells.

2 Experimental 2.1. Materials AgNO3, KCl, NaCl, CaCl2, CoCl2, CdCl2, FeCl2, CuCl2, NiCl2, Hg(NO3)2, MnCl2, MgCl2, ZnCl2, PbCl2, AlCl3, CrCl3, FeCl3, NaH2PO4 and Na2HPO4 were purchased from Shanghai Aladdin Reagent Corporation. Ammonium citrate and triethylenetetramine were obtained from Sigma-Aldrich Reagent Corporation. 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was purchased from Beijing Solarbio Science and Technology Corporation. All chemicals were analytical grade. Ultrapure water (≥ 19 MΩ·cm) were used throughout the experiment and obtained from a Smart-N system (Heal Force, China).

2.2. Instruments The X-ray diffraction (XRD) patterns were obtained using a D8 X-ray powder diffractometer (Bruker, Germany) with Cu Kα radiation operating at 40 kV. The data were collected from 2θ=10-70o by a scan rate of 0.03o per step. The transmission electron microscopic (TEM) images were obtained by a TECNAI G2 F20 electron microscope (FEI, America) at 200 kV. The samples for TEM image were prepared on a super-thin carbon-coated copper grid. The

2.3. Synthesis of the N-CDs In

this

work,

the

N-CDs

were

obtained

by

the

microwave-assisted pyrolysis method. Firstly, ammonium citrate (2.4322 g) was dissolved in 10 mL ultrapure water, and then 1.46 mL triethylenetetramine was added to the solution in the beaker. The mixture was stirred to form a homogeneous solution. Secondly, the beaker was placed in the 800 W microwave oven (Galanz, China) and heated for 2 min using high power. After most of water was evaporated to form a uniform yellow gel, 5 mL of ultrapure water was added into the beaker, and then continuously heated for 1 min in the microwave oven. This process was repeated about 3 times until the color of the gel changed into orange. Lastly, the gel was dispersed in distilled water (5 mL). The suspension of the N-CDs was centrifuged at 15 000 rpm for 10 min, and filtered by a 0.22 μm millipore filter and subjected to dialysis (MWCO=500-1000 D) against distilled water for 48 h.

2.4. QY measurement The QY of as-synthesized N-CDs was determined by the established procedure. Quinine sulfate was used as a standard (QY=0.54) in the measurement. In order to minimize the re-absorption effects, absorbencies were kept under 0.1 in the 1 cm cuvette at the 360 nm wavelength

[29-31]

. The QY of the as-prepared

N-CDs was determined using the following equation:

Q = QR 

I AR n2   I R A nR2

Where Q is the QY, I is the measured integrated fluorescent emission intensity, A and n are the absorbance and the solvent refractive index, respectively.

2.5. Detection of Cu2+ ion The detection of Cu2+ ion was carried out at room temperature in a 0.01 M pH=7.4 PBS buffered saline. In a typical assay, 20 μL of the N-CDs (0.2 mg/mL) was added into 2 mL PBS buffer, followed by the addition the different concentrations of Cu2+. The selectivity of Cu2+ sensing was determined by adding other metal cation ions (including other 16 metal ions) instead of Cu2+ and performed with

Wen Liu et al. / New Carbon Materials, 2019, 34(4): 390-402

the same method as the detection of Cu2+ ion. The concentrations of

2.8. Cell culture and imaging

metal ions were 100 μM. The fluorescence spectra were collected from fluorescence emissions at 450 nm, which were performed at an excitation wavelength of 360 nm. The present method was examined by the tap water samples for real water sample analysis to evaluate the N-CDs-based sensor for 2+

Cu

Human hepatoma cells (SMMC-7721) were routinely cultured containing 5% CO2 at 37 °C under a humidified atmosphere in the

ion detection in an artificial system. The real samples from tap

water were centrifuged at 13000 rpm for 10 min, and filtered through a 0.22 μm micron filter. Aliquots (1000 μL) of this water sample were spiked with the Cu2+ standard solutions. And then the spiked water samples were diluted by PBS to 2 mL, which contained the as-prepared N-CDs and analyzed using the above mentioned method.

DMEM medium. SMMC-7721 cells were subcultured onto the glass bottom Petri dish, and allowed to grow for attachment within 24 h. The DMEM mediums containing 1.0 mg/mL N-CDs were used for culturing the SMMC-7721 cells. The DMEM mediums were removed after incubation for 3 h. The SMMC-7721 cells were washed using the PBS buffer three times, and then kept in the PBS for fluorescence imaging using a confocal laser scanning microscope (CLSM) (Olympus, Japan). Additionally, N-CDs-stained SMMC-7721 cells were treated with different concentrations of Cu2+ ion (50 μM and 100 μM) in 2 mL pH=7.4 PBS for additional 1 h.

2.6. MTT assay The human hepatoma cells (SMMC-7721) were purchased from the cell bank of the Chinese Academy of Sciences. The biocompatibility and cytotoxicity of the N-CDs were tested using MTT assay. Firstly, human hepatoma cells (SMMC-7721) were cultured in a 96-well microtiter plate with 5% CO2 atmosphere at 37 °C for 12 h. The wells of cells were taken as a blank group without treatment with the N-CDs. Secondly, various concentrations of the N-CDs (0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8 mg/mL in DMEM medium, at pH=7.4) were added into the wells and cultured for 24 h and 48 h. Thirdly, the cells, adding 20 μL 5.0 mg/mL MTT into every well, were cultured for 4 h. The MTT reagent was removed, and then the cells were washed with the PBS buffer (0.1M pH=7.4) for three times and added 150 μL of DMSO into every wells. The absorbance was obtained at 490 nm with a micro-plate reader (ELx808, Biotek).

2.7. Patterning

3 Results and discussion 3.1. The synthesis and characterization of the N-CDs As illustrated in Fig. 1, the luminescent N-CDs with a high fluorescence QY were synthesized from ammonium citrate and triethylenetetrmaine by a simple, convenient and rapid green route of microwave-assisted pyrolysis method. The N-CDs can be easily dispersed with a transparent appearance in an aqueous solution. As depicted in Fig. 2, various optical determinations are employed to characterize the as-synthesized CDs and the reaction conditions (reaction time, temperature and ratios) are optimized to get good PL properties. As shown in the Fig. 3a, there is a broad diffraction peak centered at 2θ=25°, which suggest the amorphous nature of the N-CDs. The transmission electron microscopy (TEM) image of the N-CDs is shown in Fig. 3b. It can be seen that the as-synthesized

The N-CDs were applied for drawing fluorescent patterns as the

N-CDs are monodispersed and with a spherical morphology. The

fluorescent carbon ink. Commercially available papers were chosen

corresponding particle size distribution of the N-CDs is shown in Fig.

as the patterning materials which showed no UV fluorescence

3c. The particle size distribution shows that diameters of

background. The N-CDs solution (0.5 mg/mL) was added into the pen

as-synthesized N-CDs are mainly distributed in the range of 6 to 8 nm

and coated with papers for drawing fluorescence patterns, and then

through counting about one hundred particles of the N-CDs.

dried at room temperature. Blue fluorescence patterns were obtained under 365 nm UV light.

Fig. 1 Schematic of the fabrication and sensing process of Cu2+ along with the photograph of the corresponding sample under 365nm UV lamp excitation.

Wen Liu et al. / New Carbon Materials, 2019, 34(4): 390-402

Fig. 2 PL spectra of (a) the N-CDs synthesized at different reaction times, (b) the N-CDs synthesized at different powers, (c) the different molar ratios of ammonium citrate to triethylenetetraine and (d) the N-CDs synthesized with different reaction methods (λex=360 nm). The concentration of the N-CDs is 0.2 mg/mL.

Fig. 3 (a) XRD pattern of the N-CDs, (b) TEM image of the N-CDs, scale bar is 10 nm, (c) the diameter distribution of the N-CDs and (d) FTIR spectrum of the N-CDs.

Wen Liu et al. / New Carbon Materials, 2019, 34(4): 390-402

Fig. 4 (a) XPS spectrum of the N-CDs, (b-d): High-resolution C1s, O1s and N1s peaks of the N-CDs, respectively. The functional groups of the as-prepared of N-CDs are identified

associated with C-OH/C-O-C. The results of functional groups

by the FTIR spectrum in Fig. 3d. The broad absorption bands at 3420

identified and characterized by the XPS are in good consistent with

and 3274 cm-1 indicate the stretching vibration peaks of O-H and N-H,

the results of FTIR. The results indicate that the N-CDs contained

respectively. The significant absorption peak at 1648 cm-1 represents

oxygen and nitrogen functional groups, which imply that the

the C=O stretching vibration. The absorption peak at 1108 cm-1

as-prepared carbon nanomaterial has good water solubility favorable

corresponds to the C-O group stretching vibration. The characteristic

for its further applications and modifications.

-1

-1

absorption bands at 2856-3091 cm and 1551 cm exhibit the C-H stretching vibration and the C=C stretching vibrations of the obtained

3.2. The optical properties of the N-CDs

-1

the N-CDs, respectively. The absorption peaks at 1265 and 1438 cm

are assigned to stretching vibrations of C-N-C and C-N, respectively. The absorption peak at 1369 cm-1 is attributed to the stretching modes of C-O-C. Moreover, the surface elemental analysis of the N-CDs is performed by the XPS. In Fig.4a, three typical strong peaks of XPS spectrum (287.7, 399.6 and 533.5 eV) are ascribed C1s, N1s and O1s, respectively. The elemental composition of the N-CDs is analyzed by XPS, the results demonstrate that the as-prepared N-CDs are mainly composed of C (70.05 %), N (9.74 %) and O (20.20 %). The content of N (9.74 %) is higher than that of the N-CDs reported in the references (4.23 %, 6.88 %)

[9, 32]

, which is ascribed to surface

passivation of carbon nanoparticles. The high resolution XPS spectrum of C1s is shown in Fig. 4b, which exhibits four main peaks. The binding energy peaks at 284.6 and 285.4 eV are assigned to C=C bond and C-N, while the peaks around 286.5 and 288.1 eV to C-OH and C=O, respectively. As shown in Fig. 4c, the two peaks at 399.2 and 400.5 eV from the N1s spectrum are assigned to C-N-C and N-H, respectively. The O1s XPS spectrum is shown in Fig. 4d, the binding energy peak at 531.3 eV is attributed to C=O, the peak at 532.4 eV is

In Fig. 5, the optical properties of as-synthetized N-CDs are studied. As shown in Fig. 5a, the UV-Visible absorption spectrum of the N-CDs exhibits strong absorption peaks at 240 nm and 360 nm, which are attributed to the π-π* transitions of the aromatic C=C sp2 and the trapping of excited energy of the surface states, respectively [33-35]

. From the inset in Fig. 5a, under the radiation of 365 nm UV

light, very bright blue fluorescence of the N-CDs is observed. In Fig. 5b, the PL spectra of N-CDs show that the optimum excitation peak of the N-CDs at 360 nm is in accordance with the UV-Visible absorption spectrum (Fig. 5a). In Fig.5b, the emission spectra show that the maximum emission wavelength is shown at 450 nm under the 360 nm excitation wavelength. Fig. 5c shows a detailed investigation for PL of the N-CDs under different excitation wavelengths. With the increased excitation wavelength from 300 to 410 nm, the fluorescence emission peak remains unchanged at 450 nm. The excitation-independent PL behavior of as-prepared N-CDs is considered to be originated from less surface defects and size effect, which is reported in the related literatures

[36-38]

. The wavelength

independent characteristic of the N-doped CDs is valuable for up and

Wen Liu et al. / New Carbon Materials, 2019, 34(4): 390-402

down conversion cell imaging where the undesired auto fluorescence (such as tissue auto fluorescence) can be avoided [39]. As shown in Fig. 5d, quinine sulfate is used as a standard (QY=54%), the QY of the N-CDs is calculated to be about 29.83%, demonstrating the good fluorescence characteristics. The long-time photochemical stability is dominant critical for the N-CDs in various environments. So the PL stability of the as-synthesized fluorescent N-CDs is studied. In Fig. 6a, the response of as-synthesized N-CDs to a wide range pH values (from 2.0 to 12.0) is studied. The PL intensity of as-synthesized N-CDs enhances in an acidic solution, and the PL intensity decreases in an alkaline solution, which is possibly ascribed to lots of the hydrophilic carboxyl on the surface of the N-CDs. As depicted in Fig. 6b, the effect of ionic strength on the PL stability of the N-CDs is also studied. However, the PL intensity of the N-CDs remains almost unchanged with the concentration of NaCl from 0 to 1.0 M, which is valuable since it is important in the practical applications in the presence of the salt in different concentrations. The result indicates that the as-synthetized N-CDs can be applied at high salt concentrations since they can keep stable and strong PL under extreme environmental conditions. Additionally, the PL intensity does not appear significantly changed under UV irradiation for several hours (in Fig. 6c), manifesting the good photo stability of the as-prepared luminescent nanomaterial. Similar result is obtained when the as-prepared N-CDs is stored for 2 months (Fig. 6d), indicating the favorable photo stability of the as-prepared N-CDs. The above results manifest that the as-prepared

commercial purpose and meaningful applications.

3.3. The response of the N-CDs to Cu2+ In this experiment, to evaluate the sensitivity and selectivity of the N-CDs in the sensing system, the PL intensity of as-prepared N-CDs with different metal cations are studied under 360 nm excitation at a pH value of 7.40. As shown in Fig. 7a, a much lowered PL intensity is seen for the N-CDs with an addition of Cu2+ ions (100 μM), while either a slight change or no change in the PL intensity are displayed for other metal ions (100 μM). It’s not hard to see that Cu2+ ion shows the strongest fluorescence quenching effect on the N-CDs among these metal ions, which indicates that the N-CDs can be developed as an excellent fluorescence Cu2+ sensor. The response of the N-CDs to Cu2+ ions is ascribed to the cupric amine complex formation between Cu2+ ions and amine groups on the N-CDs surface, which leads to the fluorescence quenching through an inner filter effect

[14, 40]

. To further research the fluorescence quenching

mechanism, as shown in Fig. 7b, the UV-Visible spectrum of the N-CD solution was analyzed in the absence and presence of Cu2+. The UV-Visible absorption results indicate that the N-CDs have two absorption bands centered at 240 and 360 nm. However, the addition of Cu2+ (100 μM) into the N-CD solution results in some changes, namely the absorption band centered at 360 nm occurs a blue shift and the absorption band centered at 240 nm disappears, which illustrate that the new substance is produced.

N-CDs have excellent photo stability and are a good candidate for the

Fig. 5 (a) Absorbance spectra of the N-CDs (0.2 mg/mL). Inset: photographs of the corresponding samples under sunlight and UV irradiation, (b) Excitation and emission spectra of the N-CDs (0.2 mg/mL), (c) PL spectra recorded for progressively excitation wavelengths from 300 to 410 nm in a 10 nm increment, the concentration of the N-CDs is 0.2 mg/mL and (d) Plots of integrated PL intensity against absorbance of quinine sulfate and N-CDs at a λex value of 360 nm.

Wen Liu et al. / New Carbon Materials, 2019, 34(4): 390-402

Fig. 6 (a) Effect of pH value on the PL intensity of the N-CDs, (b) Effect of ionic strength on the PL intensity of the N-CDs (The ionic strength is controlled by NaCl), (c, d) Effect of time on the PL intensity at 360 nm of the N-CDs. The concentration of the N-CDs is 0.2 mg/mL. Table 1 A comparison of different CD-based fluorescent probes for Cu2+ detection. Methods

Starting materials

LOD (nM)

Liner ranges

Refs.

Pyrolysis

Citric acid and BPEI

6

0.01-1.1 μM

[14]

Pyrolysis and microwave-assisted

Leeks

50

0.01-10 μM

[41]

Hydrothermal

Bamboo leaves

115

0.333-6606 μM

[43]

Pyrolysis and hydrothermal

Prawn shells

5

0.01-1.1 μM

[44]

Exothermic reaction

Ethylene diamine

1.0×104

10-400 μM

[28]

Microwave-assisted method

Thiourea

50

0.2-25 μM

[45]

Hydrothermal

Carbon nano-onions

20

0.01-75 μM

[46]

4.5

0.01-11 μM

This work

Ammonium citrate and Microwave-assisted method triethylenetetramine

N-CDs at λex/λem of 350 and 450 nm in the absence and presence of Fig. 7c shows the PL quenching of the N-CDs at different 2+

Cu2+, respectively. And it can be fitted to the linear S-V equation over

concentrations of Cu ions from 0 to 100 μM. The PL intensity of the

a concentration range of 0.01-11.00 μM with a correlation coefficient

N-CDs decreased progressively with increasing the concentration of

(R2) of 0.998 (the inset of Fig. 7d), while the plot cannot be fitted to a

Cu2+ ions, indicating that the sensing system of the N-CDs is sensitive

linear equation over the whole range of the concentration of Cu2+,

2+

to the Cu

ions. As depicted in Fig. 7d, the quenching efficiency of

the N-CDs is fitted in low concentrations by the Stern-Volmer equation: F0/F=1+Ksv[Q], where F0 and F are the PL intensities of the

manifesting that both the dynamic and static quenching processes exist in this N-CD sensor system [41, 42]. By taking the fluorescence intensity of the N-CDs equal to three

Wen Liu et al. / New Carbon Materials, 2019, 34(4): 390-402

times the standard deviation of the PL intensity at the blank (n = 11),

was collected and analyzed with the N-CD-based Cu2+ sensing

the detection limit (LOD) of the N-CD sensor is calculated to be 4.5

system. A standard addition method is used for this detection. In

nM (S/N = 3), which is lower than that of other reported CDs-based

detail,

Cu2+ sensors (as shown in Table 1).

auto-fluorescence, the tap water sample was filtered through the 0.22

to

remove

particulate

materials

that

may

have

μm millipore filtration.

3.4. Real sample analysis To further evaluate the applicability, the real sample tap water

Fig. 7 (a) Effect of different metal ions (100 μM) on F/F0 of the N-CD solution at 450 nm, (b) UV-Vis absorption spectra of the N-CDs and N-CDs+Cu2+ mixture, Inset: photographs of the corresponding samples under UV irradiation, (c) The PL spectra of the N-CD solution in the presence of different concentrations of Cu2+, (d) Plot of F0/F versus different concentrations of Cu2+ ions where F0/F is the PL intensities of the N-CDs in the absence and presence of Cu2+. Inset: The Stern-Volmer plot of the N-CDs at various concentrations of Cu2+ ions (0.01-11 μM). The concentration of the N-CDs is 0.2 mg/mL.

Fig. 8 The N-CD-based Cu2+ sensor in the tap water samples. (a) The PL spectra of the N-CD solution in the presence of different concentrations of Cu2+ and (b) The good linearity in the range of 0.01-10 μM.

Wen Liu et al. / New Carbon Materials, 2019, 34(4): 390-402

Fig. 9 Different photographs of the patterns using as-prepared N-CDs under (a-c) UV light excitation and (d-f) sun light.

3.5. patterning Up to now, fluorescein, carbon nanotubes, polymer composites and

luminescent

semiconductor

quantum

dots

have

been

patterned[47,48]. Whereas, luminescent patterning using CDs are still relatively rare. Here, as-prepared N-CDs are applied for hand-drawn patterns as fluorescent inks. The aqueous solution of as-synthesized N-CDs is used for coating the commercially available papers (no background fluorescence), which is dried under room temperature. As shown in Fig. 9, the blue fluorescent patterns are seen under 365 nm excitation for the patterns coated with the N-CDs under the sun light. After three months for drawing fluorescent patterns, bright blue Fig. 10 Viability (%) of SMMC-7721 cells after 24 and 48 h treatments with the N-CDs calculated from MTT assay.

fluorescent patterns coated with the N-CDs are almost invariable. So, the “vis-invisible” fluorescent properties of as-prepared N-CDs may provide the application in anti-counterfeit fields.

Tap water samples were mixed with the N-CD solutions and spiked with Cu2+ standard solutions from 0 to 10 μM. It is shown that the PL intensity of the N-CDs decreases with increasing the Cu2+ concentration in the standard solutions (Fig. 8a). The calibration curve of real water samples for determining Cu2+ is obtained in Fig.8b. The N-CD-based sensor provides a good linear response (R2=0.9928) to Cu2+ in spiked samples, which demonstrating its excellent selectivity for real sample detection. The result validates that our N-CD sensor is capable of detecting Cu2+ in real environmental water samples. Thus, the N-CD-based Cu2+ sensor provides a simple, green, reliable and sensitive strategy for Cu2+ detection in real samples.

3.6. Cell cytotoxicity assay Because of the stable optical properties and nanoscale dimensions of the N-CDs, it has the potential application for biologic imaging. For effective and obvious biological imaging, the need for selective fluorescent labeling not only needs to has optical advantageous properties, but also has a low cytotoxicity

[49-52]

. The

cell cytotoxicity of as-prepared N-CDs is tested by the MTT assay in SMMC-7721 cells. As shown in Fig. 10, SMMC-7721 cells were cultured with as-synthesized N-CDs (concentration ranging from 0.2 mg/mL to 1.8 mg/mL) for 24 and 48 h. Even to cultured cells at a high concentration of 1.8 mg/mL for 48 h under the above

Wen Liu et al. / New Carbon Materials, 2019, 34(4): 390-402

experimental conditions, more than 85% of cells are viable, the

fluorescence emissions. In addition, using SMMC-7721 cells as a

results demonstrate that as-prepared N-CDs have a low toxicity.

model, we also study the Cu2+-dependent fluorescence imaging of the

3.7. Cu2+-dependent intracellular fluorescence imaging As depicted in Fig. 11, in order to explore the Cu2+-dependent intracellular

fluorescence

imaging

of

as-prepared

N-CDs,

SMMC-7721 cells were used as a model by a laser scanning confocal microscope (LSCM). In Fig. 11a, the SMMC-7721 cells incubated with the N-CDs and N-CDs/Cu2+ for the bright-field images (the first left column in Fig. 11a) display the normal cell morphology, indicating that as-prepared N-CDs and N-CDs/Cu2+ have a negligible toxicity to and a good biocompatible with the SMMC-7721 cells. As depicted in Fig. 11b, it is apparent that the transfected SMMC-7721 cells show quite bright fluorescence due to the excellent fluorescence property of the N-CDs, verifying that as-prepared N-CDs have been successful internalized into the SMMC-7721 cells. In other words, the

N-CDs. SMMC-7721 cells are cultured for 3 h in a DMEM medium (containing 0.5 mg/mL N-CDs ), and then cultured with Cu2+ (50 μM and 100 μM) for 10 min. In Fig. 11, the stained brightness is gradually weakened with increasing Cu2+ (the brightness of the SMMC-7721 cells incubated with the N-CDs/Cu2+ at the third line is weaker than that of the N-CDs/Cu2+ at the second line in Fig. 11). Moreover, the every two-channel fluorescence images are overlapped well for the same area (As shown in Fig. 11c). The intracellular fluorescence of the N-CDs/Cu2+ is weaker than that of the N-CDs, indicating that Cu2+ could quench the fluorescence of the N-CDs in aqueous solutions. As is well known, the Cu2+ is very important in metabolic processes and in the living systems. As such, the above results display that as-prepared N-CDs can be applied in imaging living cells and can be a useful probe for sensing Cu2+ in biosystems.

cells are excited with 405 nm laser when they display quite blue

Fig. 11 Laser scanning confocal microscopy images of SMMC-7721 cells incubated with 0.50 mg/mL N-CDs in the absence and presence of different concentrations of Cu2+ ions. (a) The first left column shows the bright-field images of SMMC-7721 cells, (b) The second column is cell images taken at λex/λem of 405/450 ±25 nm and (c) The third column is the merged images of the first and second columns.

4 Conclusions We have reported a convenient, simple and rapid green method to prepare highly fluorescent N-CDs using ammonium citrate and triethylenetetramine as the precursors and demonstrated their applications in sensing, patterning and cell imaging. The as-prepared N-CDs have an outstanding optical stability, and the fabricated

N-CDs as a novel probe, have been applied to sensitive detection of Cu2+ ions with a detection limit (LOD) as low as 4.5 nM. Owing to their low cytotoxicity and strong fluorescence, the obtained N-CDs can be effectively utilized as cell imaging reagents. Above all, easy cellular uptake behaviors verify the fabricated N-CDs are promising for the sensing of Cu2+ in living cells. Herein, an extreme simple, rapid and convenient strategy is demonstrated to produce the N-CDs a high QY for versatile applications.

Wen Liu et al. / New Carbon Materials, 2019, 34(4): 390-402

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