Green and economical synthesis of nitrogen-doped carbon dots from vegetables for sensing and imaging applications

Abstract

Fluorescent carbon-based nanomaterials have attracted tremendous concern owing to their unique properties of chemical stability, excellent biocompatibility, tunable excitation and emission spectra, low toxicity and photostability. Herein, a green, simple and low-cost approach was present to obtain nitrogen-doped carbon dots (N-doped C-dots) with the quantum yield of 37.5% using vegetables as the sole carbon source through facile one-pot hydrothermal treatment without additional solvents. The as-prepared N-doped C-dots are fully characterized by high-resolution transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, UV-vis absorption, and fluorescence spectroscopy. The synthesized N-doped C-dots displayed excellent water solubility and stability in a wide range of pH and ionic strength. As the emission of N-doped C-dots is efficiently quenched by Cu²⁺, the C-dots can serve as a suitable sensing platform for label-free sensitive and selective detection of Cu(II) ions with a detection limit of 9.98 nM. The cell viability results of HeLa cells proved the low toxicity of C-dots. Further, the imaging of Escherichia coli (E. coli) cells and HeLa cells demonstrated the biolabeling potential in vivo of the synthesized C-dots with low toxicity and good biocompatibility.

---

1. Introduction

Growing applications of nanomaterials have drawn increasing attention due to their size-dependent tunable electronic, optical, magnetic and mechanical properties.¹ Among the attractive nanomaterials, fluorescent carbon-based materials have aroused enormous excitement because of their superiority in water solubility, chemical inertness,² biocompatibility,³ low toxicity,⁴ ease of functionalization and resistance to photobleaching.^5,6 The novel properties make them very attractive in diverse applications, ranging from energy conversion and storage to biomedical imaging.⁷ Zhu et al.⁸ proposed a new method for the synthesis of graphene quantum dots (GQDs) in large scale and GQDs were demonstrated to be an eco-friendly material as well as excellent biolabeling agents. Liu et al.⁹ reported the biocompatible N-doped GQDs as efficient two-photon fluorescent probes for cellular and deep-tissue imaging. Wang et al.¹⁰ used C-dots to label U87 cells and discovered the U87 cells became brightly illuminated under the microscope. The study of Yang group¹¹ showed that C-dots exhibited excellent biocompatibility and the capacity to specifically target the cells overexpressing the folate receptor. Given that, fluorescent C-dots have exceptional advantages over traditional organic dyes and semiconductor quantum dots in applications within biological systems.¹² However, many of the conventional synthesis methods for these materials are expensive, sophisticated, and tedious.¹³ Besides, the low quantum yield (QY) of as-prepared C-dots limits the range of their applications in most cases. Meanwhile, heteroatom doping carbon nanomaterials are inspiring intensive research interests, which can effectively tune their intrinsic properties, including optical characteristics, surface and local chemical features.^14–16 Thus, it is highly desired to develop simple and green approaches to obtain high-fluorescent doping C-dots.

Copper ion, the most abundant essential transition metal ion in human body except zinc and iron ions, plays a critical role in a variety of fundamental biological processes in living organism.¹⁷ Many physiological functions could be affected by the concentration of Cu²⁺ in the body, such as enzyme activity and cell metabolism, while excess Cu²⁺ in the body is related to gastrointestinal disturbance and damage to liver and kidneys.^18–20 Imbalance of copper homeostasis leads to many pathological sequences, such as Alzheimer's, Menkes, and Wilson's diseases.^21,22 Beyond that, copper ion is also commonly found in waste streams of many industrial processes and has been known to cause severe health problems.^23,24 Currently available methods, such as atomic absorption spectroscopy and inductively coupled plasma mass spectroscopy,²⁵ are complicated and sample-destructive for determination of copper levels in both biological and environmental samples. Therefore, fluorescent nanomaterial-based sensors with their superiority have been proposed for measuring the level of copper.

Recently, much effort has been put into developing green methodologies for preparing nanomaterials from natural precursors which are widely available and harmless to the environment. In this work, vegetables are selected as raw materials which are inexpensive and easily available. We report a facile, low-cost, and environmental-friendly method to obtain high-fluorescent N-doped C-dots without requirement of severe synthetic conditions. Their emission properties, quantum yield, stability, solubility and toxicity were investigated in detail. In addition, the selective and sensitive determination of Cu²⁺ was performed by using unmodified N-doped C-dots as fluorescent probe. More importantly, we further demonstrate that these N-doped C-dots can serve as effective fluorescent probes for E. coli cell and HeLa cell imaging.

2. Experimental

2.1 Materials

Natural green pakchoi was picked from the home garden and washed several times by distilled and doubly distilled water before juicing. Tris(hydroxymethyl)methyl amino methane (Aladdin, Shanghai, China) and acetic acid (Sinopharm Chemical Reagent Co., Ltd.) were prepared for buffer solutions of different pH values with the concentration of 10 mM. Quinine sulfate (98%, suitable for fluorescence) was supplied by Aladdin for quantum yield evaluation. CuCl₂, NaCl, KCl, CaCl₂, CoCl₂, BaCl₂, FeCl₃, Hg(NO₃)₂, AgNO₃, Bi(NO₃)₃, Pb(NO₃)₂, Al(NO₃)₃, MgSO₄, FeSO₄, MnSO₄, ZnSO₄ were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All the reagents were of analytical grade and used without further purification. Doubly distilled water was used throughout.

2.2 Apparatus and characterization

High-resolution TEM (HRTEM) images were taken on a JEOL JEM 2100 (Japan) microscope at an acceleration voltage of 200 kV. Fourier transform infrared (FT-IR) spectra were performed on Spectrum 400F spectrometer (PerkinElmer, USA). X-ray photoelectron spectroscopy (XPS) was conducted on PHI Quantera spectrometer equipped with Al Kα excitation. Binding energy was calibrated with C 1s of 284.8 eV. The UV absorption spectra were obtained by a TU-1810 spectrophotometer (Puxi Analytic Instrument Ltd., Beijing, China). Fluorescence measurements were recorded on a Cary Eclipse fluorescence spectrophotometer (VARIAN, USA) using a quartz cuvette of 2 mm path length. A PHS-2C(A) pF-meter (Shanghai Dapu Instruments Co., Ltd. Shanghai, China) was used for measuring pH in the experiment.

2.3 Synthesis of C-dots

The N-doped C-dots was typically fabricated by one-pot hydrothermal process (Fig. 1) as follows. Specifically, 60 mL pakchoi juice was transferred into a 100 mL Teflon-lined autoclave and heated at 150 °C for 12 h and then cooled to room temperature naturally. The product was collected by removing the large particles through filtration with a 0.22 μm filter membrane and then centrifugation at 12 000 rpm for 20 min. The as-prepared N-doped C-dots were stored at 4 °C for further characterization and use.

Fig. 1 Schematic diagram of the formation of C-dots.

2.4 Quantum yield measurements

Fluorescence quantum yield (QY) of the resultant N-doped C-dots was determined using quinine sulfate (0.1 M H₂SO₄ as solvent; QY = 0.54) as standard according to the following equation:⁶

Q_x = Q_std(I_x/I_std)(A_std/A_x)(η_x/η_std)² (1)

where the subscripts std and x denote standard and test samples respectively, Q refers to the quantum yield, I is the integrated emission intensity, A stands for the absorbance, which should be less than 0.1 at and above the excitation wavelength in the 10 mm cuvette in order to minimise re-absorption effects, and η is the refractive index of the solvent.

2.5 Metal ion sensing capability

Various metal ion sources have been used, such as CuCl₂, NaCl, KCl, CaCl₂, CoCl₂, BaCl₂, FeCl₃, Hg(NO₃)₂, AgNO₃, Bi(NO₃)₃, Pb(NO₃)₂, Al(NO₃)₃, MgSO₄, FeSO₄, MnSO₄, and ZnSO₄. N-doped C-dots solution was used to detect the metal ion (a calculated amount of ions of 50 μM). The spectra were recorded with excited wavelength at 380 nm for all of the fluorescence spectra.

2.6 N-doped C-dots-based Cu²⁺ detection

The dose-dependent response of N-doped C-dots to Cu²⁺ was investigated. Variable amount of Cu²⁺, certain concentration of N-doped C-dots, and Tris-HAc buffer (10 mM, pH 7.4) were added to a 1.5 mL volumetric flask, and mixed thoroughly. The corresponding fluorescence spectra were recorded from 390 to 600 nm upon 380 nm excitation. Both excitation and emission slit widths were set as 5 nm.

2.7 Cell culture

HeLa cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U mL⁻¹ penicillin, and 100 U mL⁻¹ streptomycin. Cell culture was maintained at 37 °C in a humidified condition of 95% air and 5% CO₂ in culture medium.

2.8 Cell viability assay

The cytotoxicity of N-doped C-dots was assessed through MTT assay. HeLa cells were seeded in 96-well plates at a density of 5 × 10⁴ cells mL⁻¹. After 24 h attachment, the cells were incubated with 0, 50, 100, 200, 400, 800, 1200, and 2000 μg mL⁻¹ N-doped C-dots for another 24 h. Afterward, 20 μL of 5 mg mL⁻¹ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution was added to every cell well, and the cells were further incubated for 24 h. Then, the culture medium was removed by MTT, followed by the addition of 150 μL of dimethyl sulfoxide (DMSO). The mixture was shaken for 5 min at room temperature. The optical density (OD) of the mixture was measured at 570 nm. The cell viability was estimated using the following equation:

Cell viability (%) = (OD_treatment/OD_control) × 100% (2)

where OD_control was obtained in the absence of N-doped C-dots, and OD_treatment was obtained in the presence of N-doped C-dots.

2.9 In vitro cellular uptake

E. coli cells were cultured at 37 °C overnight, and washed by PBS buffer (10 mM, pH = 7.4) with centrifugation at 8000 rpm for 10 min. The cells were diluted to a suitable concentration. Then cell suspension was treated with carbon dots for 18 h. After the cell suspension was washed in PBS three times, cell images were taken with a Laser Scanning Confocal Microscope Leica TCS SP8 (Germany) with the excitation wavelength of 488 nm and 405 nm, respectively.

An aliquot (0.1 mL) of the C-dots suspension was added to the HeLa cell culture and then the cells were incubated at 37 °C for 8 h. The excess C-dots were removed by washing with PBS buffer solution (10 mM, pH = 7.4) for 3 times, and then, the cells were fixed on the slide for analysis by a laser confocal fluorescence microscope.

3. Results and discussion

3.1 Synthesis and characterization of N-doped C-dots

Pakchoi juice was used as the sole carbon source without addition of other solvent through one-pot hydrothermal carbonization to obtain the luminescent N-doped C-dots. This approach was environmentally green and the raw material was cheap and easily available. After hydrothermal treatment, the non-fluorescence solution turned from green to clear yellow, which symbolized the formation of C-dots. The particle size and morphology of N-doped C-dots were observed by the high-resolution transmission electron microscopy (HRTEM). The TEM images (Fig. 2) indicated that the as-prepared N-doped C-dots were well dispersed and showed a narrow size distribution between 1.0 and 3.0 nm with the average size of 1.8 nm.

Fig. 2 (A) TEM images of N-doped C-dots (inset: corresponding HRTEM image). (B) Particle size distribution histogram of N-doped C-dots.

The surface functional groups of resultant N-doped C-dots were identified by FT-IR spectrum. Fig. 3 showed characteristic absorption bands of O–H and N–H stretching vibrations at 3377 cm⁻¹.²⁶ Further, the band of the ester group at 1730 cm⁻¹, and the peak of the C=O group at 1667 cm⁻¹ appeared.²⁷ The peak at 1385 cm⁻¹ was assigned to C–H and N–H bending vibrations,²⁸ and the characteristic absorption bands of C–OH stretching at 1154 cm⁻¹ and 1046 cm⁻¹ were also observed.

Fig. 3 FT-IR spectrum of N-doped C-dots.

To elucidate the components of the current C-dots, XPS survey spectra were performed. As illustrated in Fig. 4A, the XPS spectrum displayed three peaks at 284.8, 399.9, and 532.0 eV, revealing the presence of C 1s, N 1s, and O 1s, which indicated the formation of N-doped C-dots. The four peaks at 284.6, 285.6, 287.7 and 288.6 eV of Fig. 4B were assigned to C–C, C–N, C–O, and C=O, respectively.²⁹ The O 1s spectrum (Fig. 4C) exhibited that two fitted peaks at 531.7 and 533.1 eV were ascribed to C=O and C–OH/C–O–C groups,³⁰ respectively. In addition, the N 1s spectrum was shown in the inset of Fig. 4D. N1 at 399.8 eV could be assigned to C–N–C bonds, and N2 at 401.0 eV mainly corresponded to N–H chemical bonds, implying that N atoms had been partly doped into the C-dots.³¹ In general, the FT-IR data showed satisfactory agreement with XPS results, intensively suggesting that the C-dots in this work were equipped with characteristic functional groups including –COOH, –OH and –NH₂, thereby facilitating their excellent water solubility without any further modification. These functional groups improved the hydrophilicity and stability of the N-doped C-dots in the aqueous system, which accounted for the great potential of the material for sensing in aqueous samples.

Fig. 4 (A) Survey XPS spectra of C-dots. High-resolution (B) C 1s, (C) O 1s and (D) N 1s region of C-dots.

Multi-spectral methods were employed to detect the remarkable optical properties of the aqueous dispersion of the as prepared N-doped C-dots. The UV-vis absorption spectrum (Fig. 5) showed that the synthesized N-doped carbon dots had a strong absorption band centered at 281 nm, assigning to the n–π* transition of C=O bond.^32–34 Even without any surface passivation, the as-prepared N-doped C-dots exhibited excellent water solubility. The fluorescence property of N-doped C-dots was monitored by a wide range of wavelengths. It was observed that the emission wavelength of N-doped C-dots was obviously red-shifted from 463 nm to 534 nm in the excitation range of 300–480 nm (Fig. 6), and the fluorescence spectra of N-doped C-dots revealed a strong emission peak centered on 473 nm with the excitation of 380 nm. The size dependent absorption is associated with a red shift in the emission spectrum. The quantum yield of N-doped C-dots was calculated to be 37.5% (quinine sulfate as the reference), significantly higher than some of the biomass-based C-dots previously reported,^35–38 which was competent for acting as fluorescence probe. The high quantum yield is possibly due to the existence of nitrogen-containing functional groups, which are generally excellent auxochromes.³⁵ In addition, the effects of ionic strength (in terms of the concentration of NaCl) and pH on the fluorescence stability of N-doped C-dots were investigated. The stability results towards extreme pH and ionic strengths of the present N-doped C-dots showed that the fluorescence of synthesized N-doped C-dots possessed fantastic stability in the wide range of different pH conditions (Fig. 7), and the fluorescence intensities remained constant with the increasing NaCl concentration (Fig. 8), which would be beneficial for use in the presence of various salt concentrations in practical applications. Due to the excellent stability as shown above, the as-prepared N-doped C-dots were further explored its applications in sensing and bioimaging.

Fig. 5 UV absorption spectrum of N-doped C-dots. The inset shows N-doped C-dots in day light and UV light, respectively.

Fig. 6 Fluorescence emission spectra of N-doped C-dots.

Fig. 7 Fluorescence intensity change of N-doped C-dots at different pH values.

Fig. 8 Fluorescence response of N-doped C-dots in 10 mM pH 7.4 Tris-HAc buffer solution of various ionic strengths.

3.2 Metal ion sensing capability of N-doped C-dots

C-dots can serve as a sensing candidate because the oxygen-containing groups on the surface of C-dots can coordinate with transition metal ions.³⁹ We explored the feasibility of using N-doped C-dots as fluorescent probe for quantitative determination of metal ion. The effect of metal ions on the luminescence of C-dots was inspected by measuring the fluorescence intensities in the presence and absence of representative metal ions (Cu²⁺, Co²⁺, Ba²⁺, Fe²⁺, Fe³⁺, Hg²⁺, Ag⁺, Bi³⁺, Pb²⁺, Al³⁺, Mg²⁺, Mn²⁺, Na⁺, K⁺, Ca²⁺, and Zn²⁺). Upon the addition of 50 μM of each metal ion, only Cu²⁺ induced instantaneous and noticeable intensity decrement while other metal ions showed either no or slight change in the fluorescence intensity relative to the free C-dots (Fig. 9). The functional groups on the surface of the C-dots are responsible for the affinity towards Cu²⁺ ions and this leads to the fluorescence quenching of the C-dots through electron or energy transfer.⁴⁰ These results suggested that C-dots could be developed as an efficient fluorescence sensor for Cu²⁺. Initially, the fluorescence intensity response for C-dots–Cu²⁺ system towards different pH values was carried out from 2 to 10. As shown in Fig. 10, the fluorescence intensity decrease more obviously in the buffer solution of pH 7 than other pH conditions. Accordingly, physiological pH 7.4 approximately to 7 was chosen as the optimal pH value. Then, fluorescence titration experiments were performed to investigate the sensitivity of the C-dots toward Cu²⁺. It was clear that the fluorescence intensity of C-dots decreased progressively with increasing concentration of Cu²⁺, revealing that this system was sensitive to Cu²⁺. Upon the addition of various concentrations of Cu²⁺ to a fixed concentration of C-dots solution, the fluorescence intensity changes were investigated upon the excitation at 380 nm. Fig. 11 exhibited the relationship between the fluorescence intensity of C-dots and the concentration of Cu²⁺, where the fluorescence intensity can be seen to decrease regularly with increasing concentration of Cu²⁺. The relationship is described by the Stern–Volmer equation:

F₀/F = 1 + K_SV[Q] (3)

where F₀ and F are the fluorescence intensities of C-dots in the absence and presence of Cu²⁺, respectively, [Q] is the concentration of Cu²⁺, and K_SV is the Stern–Volmer constant. The calibration curve in Fig. 12 showed a linear relationship (R² = 0.991) of (F₀/F) versus the concentration of Cu²⁺ over the range from 0 to 100 nM. The detection limit for Cu²⁺ was calculated to be 9.98 nM (S/N = 3), which was comparable to other reported values using alternative methods.^27,41 The results demonstrated that C-dots could serve as sensitive and selective probe for the determination of Cu²⁺.

Fig. 9 Selectivity of synthesized N-doped C-dots-based sensor for Cu²⁺ over other metal ions. The concentration of metal ions was 50 μM.

Fig. 10 Copper ion response towards N-doped C-dots in the pH range from 2 to 10.

Fig. 11 Fluorescence response of N-doped C-dots on various concentrations of Cu²⁺ (from top to bottom, 0, 0.005, 0.010, 0.025, 0.050, 0.075, 0.10, 0.50, 0.75, 5.0, 10, 25, 50, 75, 250, 500, 750 μM). Inset: FL emission spectra of N-doped C-dots in the presence of Cu²⁺ with concentration ranging from 0 to 750 μM.

Fig. 12 The dependence of F₀/F on different concentrations of Cu²⁺ within the range of 0–100 nM.

3.3 Toxicity of the N-doped C-dots and its bioimaging application

For effective bioimaging, it is required that the selected fluorescent marker has optical merits and low cytotoxicity at the same time. To evaluate the cytotoxicity of C-dots, the viability of HeLa cells treated with C-dots was measured by the methyl thiazolyl tetrazolium (MTT) method. As shown in Fig. 13, HeLa cells were incubated with different doses of C-dots for 24 h, respectively. Consequently, the viability remained greater than 95% even when the cells were incubated with an ultrahigh concentration (2000 μg mL⁻¹) of C-dots for 24 h, demonstrating that the obtained C-dots were scarcely toxic toward the cells (without any further functionalization).

Fig. 13 Cell viability assays of the cells treated with different concentrations of the N-doped C-dots.

E. coli cells and HeLa cells were treated with carbon dots respectively and then observed under a fluorescence microscope to prove the viability of C-dots as an ideal bioimaging agent. Fig. 14 showed photographs of the E. coli cells and HeLa cells captured by a laser scanning confocal microscope. It was obvious that the transfected E. coli cells and HeLa cells became quite bright owing to the strong fluorescence from the N-doped C-dots, indicating a large amount of N-doped C-dots had been internalized into the cells. Bright blue (Fig. 14A and C) and green (Fig. 14B and D) fluorescence could be observed under 405 nm and 488 nm excitation, respectively. The experiment demonstrated that the as-prepared C-dots could serve as a promising candidate for in vivo imaging due to their low toxicity and good biocompatibility. This multicolor emission shows a great advantage of the C-dots over other labeling agents, because it gives us much space to choose the wavelength for observation in vitro.

Fig. 14 Images of cells labeled with N-doped C-dots. E. coli cells: fluorescence with 405 nm (A) and 488 nm (B) excitation. Scale bar: 25 μm. HeLa cells: fluorescence with 405 nm (C) and 488 nm (D) excitation. Scale bar: 30 μm.

4. Conclusions

In summary, N-doped C-dots were prepared through a one-pot hydrothermal treatment of green and inexpensive vegetables as the sole carbon source without additives. The synthesized N-doped C-dots had a narrow size distribution of 1–3 nm. They had high quantum yield, good aqueous monodispersity, high photostability, and were suitable for use in solutions with wide range of pH value and ionic strength. Also, they were used as fluorescent probes to detect Cu²⁺ with a detection limit of 9.98 nM. In addition, they possessed low toxicity and perfect biocompatibility and were used as optical imaging agents to E. coli cells and HeLa cells. The imaging results may promote the clinical application of the C-dots. They have high potential for bioimaging both in vitro and in vivo and applications in biomedical research such as intracellular detection, drug delivery, etc. The low cost, biocompatibility, and low-toxicity of the N-doped C-dots and their distinct optical properties indicate that numerous possible applications of C-dots will be explored with further research.

Acknowledgements

We gratefully acknowledge the financial support of National Natural Science Foundation of China (30970696, 21172056), Key Project of Henan Ministry of Education (14A150018), Key Programs of Henan for Science and Technology Development (142102310273), PCSIRT (IRT1061), the Program for Innovative Research Team in University of Henan Province (2012IRTSTHN006), and the analysis support from the Instrumental Analysis Center of Tsinghua University.

Notes and references

1. V. Biju, Chem. Soc. Rev., 2014, 43, 744.
2. S. Ghosh, A. M. Chizhik, N. Karedla, M. O. Dekaliuk, I. Gregor, H. Schuhmann, M. Seibt, K. Bodensiek, I. A. T. Schaap, O. Schulz, A. P. Demchenko, J. Enderlein and A. I. Chizhik, Nano Lett., 2014, 14, 5656.
3. W. Li, Z. Zhang, B. Kong, S. Feng, J. Wang, L. Wang, J. Yang, F. Zhang, P. Wu and D. Zhao, Angew. Chem., Int. Ed., 2013, 52, 8151.
4. L. Wang, S. J. Zhu, H. Y. Wang, S. N. Qu, Y. L. Zhang, J. H. Zhang, Q. D. Chen, H. L. Xu, W. Han, B. Yang and H. B. Sun, ACS Nano, 2014, 8, 2541.
5. H. T. Li, Z. H. Kang, Y. Liu and S. T. Lee, J. Mater. Chem., 2012, 22, 24230.
6. S. J. Zhu, Q. N. Meng, L. Wang, J. H. Zhang, Y. B. Song, H. Jin, K. Zhang, H. C. Sun, H. Y. Wang and B. Yang, Angew. Chem., Int. Ed., 2013, 52, 3953.
7. Z. Li, Q. Sun, Y. Zhu, B. E. Tan, Z. P. Xu and S. X. Dou, J. Mater. Chem. B, 2014, 2, 2793.
8. S. J. Zhu, J. H. Zhang, C. Y. Qiao, S. J. Tang, Y. F. Li, W. J. Yuan, B. Li, L. Tian, F. Liu, R. Hu, H. N. Gao, H. T. Wei, H. Zhang, H. C. Sun and B. Yang, Chem. Commun., 2011, 47, 6858.
9. Q. Liu, B. D. Guo, Z. Y. Rao, B. H. Zhang and J. R. Gong, Nano Lett., 2013, 13, 2436.
10. L. Wang, Y. Yin, A. Jain and H. S. Zhou, Langmuir, 2014, 30, 14270.
11. X. D. Yang, X. Yang, Z. Y. Li, S. Y. Li, Y. X. Han, Y. Chen, X. Y. Bu, C. Y. Su, H. Xu, Y. N. Jiang and Q. Lin, J. Colloid Interface Sci., 2015, 456, 1.
12. S. B. Ruan, J. Qian, S. Shen, J. H. Zhu, X. G. Jiang, Q. He and H. L. Gao, Nanoscale, 2014, 6, 10040.
13. Y. Q. Dong, R. X. Wang, G. L. Li, C. Q. Chen, Y. W. Chi and G. N. Chen, Anal. Chem., 2012, 84, 6220.
14. K. P. Gong, F. Du, Z. H. Xia, M. Durstock and L. M. Dai, Science, 2009, 323, 760.
15. J. P. Paraknowitsch, Y. J. Zhang, B. Wienert and A. Thomas, Chem. Commun., 2013, 49, 1208.
16. X. Wang, J. Wang, D. L. Wang, S. Dou, Z. L. Ma, J. H. Wu, L. Tao, A. L. Shen, C. B. Ouyang, Q. H. Liu and S. Y. Wang, Chem. Commun., 2014, 50, 4839.
17. D. Qu, M. Zheng, P. Du, Y. Zhou, L. G. Zhang, D. Li, H. Q. Tan, Z. Zhao, Z. G. Xie and Z. C. Sun, Nanoscale, 2013, 5, 12272.
18. S. P. Wu, R. Y. Huang and K. J. Du, Dalton Trans., 2009, 24, 4735.
19. F. Li, J. Wang, Y. M. Lai, C. Wu, S. Q. Sun, Y. H. He and H. Ma, Biosens. Bioelectron., 2013, 39, 82.
20. H. S. Jung, P. S. Kwon, J. W. Lee, J. Il Kim, C. S. Hong, J. W. Kim, S. H. Yan, J. Y. Lee, J. H. Lee, T. Joo and J. S. Kim, J. Am. Chem. Soc., 2009, 131, 2008.
21. E. Gaggelli, H. Kozlowski, D. Valensin and G. Valensin, Chem. Rev., 2006, 106, 1995.
22. S. Anbu, R. Ravishankaran, M. F. C. Guedes da Silva, A. A. Karande and A. J. L. Pombeiro, Inorg. Chem., 2014, 53, 6655.
23. L. Järup, Br. Med. Bull., 2003, 68, 167.
24. I. Y. Goon, C. C. Zhang, M. Lim, J. J. Gooding and R. Amal, Langmuir, 2010, 26, 12247.
25. J. S. Becker, A. Matusch, C. Depboylu, J. Dobrowolska and M. V. Zoriy, Anal. Chem., 2007, 79, 6074.
26. W. B. Lu, X. Y. Qin, S. Liu, G. H. Chang, Y. W. Zhang, Y. L. Luo, A. M. Asiri, A. O. Al-Youbi and X. P. Sun, Anal. Chem., 2012, 84, 5351.
27. J. Zong, X. L. Yang, A. Trinchi, S. Hardin, I. Cole, Y. H. Zhu, C. Z. Li, T. Muster and G. Wei, Biosens. Bioelectron., 2014, 51, 330.
28. X. M. Yang, Y. Zhuo, S. S. Zhu, Y. W. Luo, Y. J. Feng and Y. Dou, Biosens. Bioelectron., 2014, 60, 292.
29. Q. Q. Shi, Y. H. Li, Y. Xu, Y. Wang, X. B. Yin, X. W. He and Y. K. Zhang, RSC Adv., 2014, 4, 1563.
30. R. Z. Zhang and W. Chen, Biosens. Bioelectron., 2014, 55, 83.
31. J. Ju and W. Chen, Biosens. Bioelectron., 2014, 58, 219.
32. D. A. Skoog, F. J. Holler and T. A. Nieman, Principles of Instrumental Analysis, Hartcourt Brace & Company, Philadelphia, 1998.
33. H. J. Zhang, Y. L. Chen, M. J. Liang, L. F. Xu, S. D. Qi, H. L. Chen and X. G. Chen, Anal. Chem., 2014, 86, 9846.
34. J. I. Paredes, S. Villar-Rodil, A. Martıínez-Alonso and J. M. D. Tascón, Langmuir, 2008, 24, 10560.
35. L. Wang and H. Susan Zhou, Anal. Chem., 2014, 86, 8902.
36. R. L. Liu, J. Zhang, M. P. Gao, Z. L. Li, J. Y. Chen, D. Q. Wu and P. Liu, RSC Adv., 2015, 5, 4428.
37. B. de and N. Karak, RSC Adv., 2013, 3, 8286.
38. Y. S. Liu, Y. N. Zhao and Y. Y. Zhang, Sens. Actuators, B, 2014, 196, 647.
39. H. Huang, C. G. Li, S. J. Zhu, H. L. Wang, C. L. Chen, Z. R. Wang, T. Y. Bai, Z. Shi and S. H. Feng, Langmuir, 2014, 30, 13542.
40. S. Liu, J. Q. Tian, L. Wang, Y. W. Zhang, X. Y. Qin, Y. L. Luo, A. M. Asir, A. O. Al-Youbi and X. P. Sun, Adv. Mater., 2012, 24, 2037.
41. Q. Qu, A. W. Zhu, X. L. Shao, G. Y. Shi and Y. Tian, Chem. Commun., 2012, 48, 5473.

---