Fluorographene nanosheets: a new carbon-based matrix for the detection of small molecules by MALDI-TOF MS

Abstract

Fluorographene nanosheets (FG) were synthesized via one-pot chloroform-mediated sonochemical exfoliation under ambient conditions. For the first time, this material served as a novel matrix for the detection of small molecules by negative ion MALDI-TOF MS. The structural characterization and ionization mechanism of FG were illustrated. The unique properties of FG endow it with properties such as low background, excellent chemical compatibility and homogeneity over conventional carbon based matrices. Most significantly, application of FG as matrix resolved the contamination problem of conventional carbon based matrices to ion source. With the association of FG, a variety of small molecules containing amino acids, peptides, small metabolites, drugs and organonitrogen compounds were analyzed. The developed method was found to have excellent feasibility with biosystems. Based on this, qualitative analysis of sialic acid in human saliva, melatonin in human urine and in situ analysis of MCF-7 cells were done, besides, quantification of anti-cancer drug in human serum and uric acid in human urine were displayed. This work extends the application fields of FG and provides a good candidate of matrix for MALDI-TOF MS detection of small molecules.

---

1. Introduction

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has become an effective tool for the analysis of large molecules such as proteins, peptides, carbohydrates and synthetic polymers since the late 1980s.¹ Despite its outstanding performance, MALDI MS still underperforms for small molecules because of the background interference caused by conventional organic matrices in the low-mass range (MW < 500 Da). To solve this problem, many efforts have been made to minimize the background in the low-mass range. Several organic matrixes with low background were developed.^2–4 Furthermore, various nanomaterials such as metal nanoparticles,^5–7 metal oxides nanoparticles⁸ and non-metallic nanoparticles⁹ have been applied as alternative matrices due to their low background interference. Among the numerous candidates, the type of carbon-based nanomaterials like graphene,^10–13 fullerene,¹⁴ carbon nanotubes¹⁵ and carbon nanodots^16–18 displayed advantageous efficacy in laser desorption/ionization owing to their specific structures and unique properties of remarkable charge mobility and universal optical absorption. However, there are still lots of deficiencies of carbon based matrices. For one thing, the poor dispersibility of carbon-based materials in solvents makes it hard to deposit carbon-based matrix onto the sample target and to form a homogeneous layer, leading to the time-consuming searching for “sweet spots”, poor reproducibility and low peak resolution. To solve this, methods including the combination¹⁹ and chemical functionalization^20–22 of carbon materials have been established. For the other thing, carbon matrices often cause contamination of ion source for that they could not adhere to the sample target tightly. To minimize the contamination, means like material immobilization,²³ film construction,^19,24 and hydrophilic modification²⁰ were adopted to decrease the tendency to fly off when laser pulse was applied. But, all these ways suffered from fussy operation, contemporary compositions, and did not solve the discharge problem of carbon matrices from the nature of themselves. Besides, the abundant small carbon cluster signals produced by carbon nanomaterials upon laser ablation would restrict the detection mass range and compromise the sensitivity.²⁵ Therefore, designing a new kind of carbon based matrix to overcome the above-mentioned challenges facilely will be of great significance.

Fluorographene (FG),²⁶ whose basal plane is decorated with fluorine atoms in terms of C–F bonds together with structural transformation of the C–C bonds from sp² to sp³ configuration, has gradually received particular attention owing to its wide bandgap, high transparency and fascinating insulation.^27,28 Inheriting from graphene, FG is born with continuous π-conjugated network, which is beneficial to efficient laser absorption and energy transfer. In this study, we prepared FG via one-pot chloroform-mediated sonochemical exfoliation under ambient conditions and introduced FG as a new matrix for analysis of small molecules for the first time. With FG as matrix, bio-molecules, pollutants and explosives (amino acids, peptides, small metabolites, drugs, organonitrogen compounds) was detected. The mechanism and advantages of FG as matrix were also discussed. To investigate the compatibility of FG with complex substrates, analysis of melatonin in human serum, sialic acid in human saliva and in situ analysis of MCF-7 cells were carried out. In addition, quantification of anti-cancer drug in human serum and uric acid in human urine was performed to illustrate the homogeneity of FG matrix.

2. Experimental

2.1. Synthesis and characterization of fluorographene (FG)

FG was synthesized following the reported method:²⁷ 20 mg graphite fluoride powders were added into 40 mL chloroform solvent and the system was ultrasonic treated for 5 h in an ice-bath under ambient conditions. Then the suspension was centrifuged to remove impurities and a transparent yellow supernatant (ca. 0.1 mg mL⁻¹) was collected for further use. UV-vis adsorption spectrum was measured by double-beam UV-visible light spectrometer (TU-1900, Beijing's general instrument co., LTD). The morphology of FG was characterized by transmission electron microscopy (TEM, JEM-2010, JEO, Japan) with an accelerating voltage of 120 kV. The chemical composition of FG was investigated using XPS spectral measurements (ESCALab220i-XL).

2.2. Preparation of FG as matrix and processing of analytes

The chemical and reagents information can be found in the ESI.† FG was dissolved in various solvents accordingly, as a concentration of 0.1 mg mL⁻¹. For assist of analytes' dissociation and ionization, 1 μL of FG solution was mixed with 1 μL of analyte solution and then 1 μL of the mixture was spotted onto the target plate for MALDI-TOF MS detection after air-dried. The preparation of analytes solution was described specifically in the ESI.† For detection of MT in human serum, SA in human saliva, DOX in human serum and uric acid in human urine, the detailed sample treatments were all presented in the ESI.† For in situ analysis of MCF-7 cells, matrix solution was spotted on the cells cultured on ITO glass directly. The details of cell culture and processing was shown in the ESI.† All experiments were performed in compliance with the relevant laws and institutional guidelines, and were approved by the ethics committee of Chinese Academy of Sciences. Informed consent was obtained for any experimentation with human subjects.

2.3. MALDI-TOF MS analysis or MALDI-TOF MS imaging

MALDI-TOF MS analysis was performed on an Ultraflextreme MALDI-TOF/TOF MS (Bruker Daltonics, Billerica, MA) in negative reflection mode. The mass spectrometer was equipped with a smartbeam Nd:YAG pulsed laser operated at 355 nm. MS imaging and other detailed instrumental parameters, mass calibration and data handling process can be found in the ESI.†

3. Results and discussion

3.1. Characterization of FG

The morphology of synthesized FG was characterized using transmission electron microscope (see Fig. 1B). The transparent lamellar multilayer or monolayer nanosheets with diameters of a few hundred nanometres could be observed. XPS spectra (see Fig. 1C) showed two main peaks located at 284.8 and 288.8 eV, which were attributed to the binding energy of the core levels of C 1s in the form of C=C and C–F bonds according to previous work,^27,29–32 indicating that the synthesized FG are decorated with F atoms in terms of C–F bonds and the π-conjugated network of graphene was reserved to a certain extent. It is well known that π-conjugated network plays a crucial role in assisting D/I process because the laser absorption and energy transfer is highly dependent on it. The basic requirement of being a MALDI matrix is the strong absorption of laser energy, to investigate the light absorption of FG, UV-vis absorption spectrum was measured (see Fig. 1D). FG has similar absorption with GO (graphene oxide), and has high absorption at the wavelength of laser used by MALDI (355 nm). Scheme 1 shows the possible mechanism of FG as matrix: F atom bonds to carbon atom in two forms (Scheme 1a and b), since F atom possesses three pairs of sp² electrons, it can act as Lewis base and trap protons from analytes. Moreover, the extremely strong electronegativity of fluorine leads to high polarity of C–F bonds, where the F atoms have excessive negative charges, thus the strong electrostatic attraction between negative charged F atoms and positive charged protons makes it toilless to realize the ionization process, forming [M − H]⁻ ions.

Fig. 1 (A) Photograph of FG dissolved in acetonitrile; (B) TEM image of FG; (C) XPS spectra of the C 1s of FG; (D) UV-vis spectrum of FG and GO.

Scheme 1 Schematic distribution and electron configuration of F species in FG network, (a) and (b) represented the bonding pattern between F and C atoms.

3.2. Analysis of amino acids and peptides

Firstly, we investigated the background of FG compared with GO and CNT–COOH by negative ion mode MALDI-TOF MS (see Fig. 2A). Compared with conventional carbon-based matrix, whose spectra were dominated by carbon cluster related peaks in the low mass range, FG possesses an extremely low background with limited carbon cluster related peaks. In the initial test, Gln (MW 146.07) were analyzed by MALDI-TOF MS using FG and GO as matrices in negative ion mode (see Fig. 2B). The characteristic [M − H]⁻ ion peak at m/z 145.07 were observed with both matrices. For GO matrix, carbon cluster related peaks, located at m/z 60, 72, 84 and 96, were strong. Meanwhile, during the experimental process, the ion source was contaminated seriously, along with the high voltage disappearance, partially due to the electric conductivity of GO. For FG matrix, however, the carbon clusters related peaks significantly decreased and higher signal intensity as well as signal to noise ratio (S/N) were obtained. Owing to the inert property, FG-based spectrum exhibits a cleaner background than that of other carbon based matrix in m/z 0–500 and cause no contamination to ion source. Based on above, analysis of amino acids mixture including Leu (MW 131.09), Asp (MW 133.04), Arg (MW 174.11), Tyr (MW 181.07) and Trp (MW 204.09) was performed with FG matrix (see Fig. 3A(a)). The [M − H]⁻ ions were all observed at m/z 130.09, 132.04, 173.12, 180.08 and 203.10, respectively. Furthermore, other amino acids containing Asn, His, Phe, Ser, Thr and Val were all detected by FG matrix (see Fig. S2, ESI†).

Fig. 2 MALDI-TOF mass spectra of (A) FG, GO and CNT–COOH in negative ion mode, laser intensity 70%; (B) glutamine (m/z 145.07) by using FG and GO matrix in negative ion mode. Carbon cluster anions were marked at m/z ★60, ○72, ◇84 and ☆96, laser intensity 70%.

Fig. 3 Negative ion mode MALDI-TOF mass spectra of (A) (a) amino acids mixture. (b) Peptides mixture. (B) (a) Hypoxanthine; (b) glutathione; (c) and (d) mixture of small metabolites with FG as matrix. The amount of each analyte was 500 pmol.

To date, for low-MW peptides less than 5 amino acids, α-cyano-4-hydroxycinnamic acid (CHCA) is one of the most common used matrix, however, their signals are often overlapped and suppressed by CHCA signals. Encouraged by the outstanding performance of FG in the low-MW range, a standard solution containing three dipeptides of Gly-Ala (MW 146.07), Gly-His (MW 190.06) and Gly-Asp (MW 212.09) were analysed using FG matrix (Fig. S3A†). Result shows that [M − H]⁻ ions of short-chain peptides yield strong signals at m/z 145.07, 189.06 and 211.09 with low background. In addition to low-MW peptides, medium-MW peptides up to octapeptide were also analyzed by FG matrix (Fig. 3A(b)). The four peptides: Gly-Tyr (MW 237.95), Val-Tyr-Val (MW 378.71), Tyr-Gly-Gly-Phe-Leu (MW 555.16) and Asp-Arg-Val-Tyr-Ile-His-Pro-Phe (MW 573.08) were detected with deprotonated signals at m/z 236.96, 377.73, 554.16 and 572.08, respectively. In comparison, GO was also used to analyzed the four peptides (Fig. S3B†).

3.3. Analysis of small metabolites

Metabolites are intermediates and products formed during metabolism, analysis of which is generally used to discover biomarkers of diseases, identify candidate signalling pathways, and understand disease mechanisms. For small metabolites detected by MALDI-TOF MS, one of the major challenges is that the background signals of matrix do not interfere with the analytes signals. To verify whether FG is suitable for analysis of small metabolites, hypoxanthine (MW 136.05) and glutathione (MW 307.15) were analyzed using FG matrix (Fig. 3B(a) and (b)), the two metabolites were detected effectively with deprotonated ions without interference from m/z 0 to 400. Then mixture of ten metabolites containing creatinine (MW 113.09), creatine (MW 131.47), aspartic acid (MW 133.04), hypoxanthine riboside (MW 136.04), glutamine (MW 146.08), glutamic acid (MW 147.06), taurine (MW 125.01), N-acetyl aspartic acid (MW 175.07), vitamin C (MW 176.07), and citric acid (MW 192.06) were measured under the same situations with FG matrix (Fig. 3B(c) and (d)). All were observed with [M − H]⁻ ions, the results validated that FG as matrix for negative ion MALDI MS could detect a variety of small metabolites simultaneously and effectively. To investigate the feasibility of FG in bio-samples, ectogenic melatonin (MT) spiked in human urine and endogenous sialic acid (SA) in human saliva were measured (Fig. S4 and S5†). Fig. S4a and b† represented mass spectrum of 1 mM MT in ethanol with [M − H]⁻ ions and 1 mM MT spiked in human serum with [M − CH₃ − H]⁻ ions. Fig. S5a† showed mass spectrum of 1 mM SA with [M − H]⁻ and [M + Cl]⁻ ions. Fig. S5b† showed mass spectrum of saliva from a female volunteer. The saliva collection and treating process was described in ESI.† With FG as matrix, endogenic SA in saliva was detected. Sialic acid is an important biomarker of cancer and the malignant transformation of cells is often accompanied with over expression of it. In this paper, in situ detection of SA in MCF-7 cells (from the Cell Resource Centre of Peking Union Medical College Hospital) was performed and the result indicated that using FG as matrix, SA can be directly probed without any purification or preparation (Fig. S6†).

3.4. Detection and monitor of drugs

Detection and monitor of drugs and their derivatives during metabolic process plays an important role in pharmacodynamics, pharmacokinetic studies and disease-related biomedical research. MALDI-TOF MS as a powerful tool for fast and high-throughput analysis, however, was unfortunately limited in drug molecules due to the lack of proper matrix. In this study, with FG matrix, female hormone drugs (estradiol (MW 272.28), 17α-ethynylestradiol (MW 296.40)), senile dementia treating drugs huperzine-A (MW 242.20) and anticancer drugs doxorubicin hydrochloride (MW 579.99) were analyzed (Fig. 4A). All were detected as [M − H]⁻ ions at m/z 271.28, 295.30, 241.20 and [M − X − H]⁻ ion at m/z 396.41 (for fragment process see Scheme S1†). Therapeutic drug monitoring (TMD), such as measuring concentration of drugs and metabolites in serum, is regarded as an essential workflow to optimizing dosage and evaluating adherence. Doxorubicin is a medication used in cancer chemotherapy which works by intercalating DNA, and is commonly used in the treatment of a wide range of cancers. In this paper, we monitored DOX in human serum (from Beijing Jiufengrunda biological technology co., LTD.) with FG matrix (Fig. 4B). At concentration of 0.5 mM, the signal intensity of DOX in spiked serum was comparable to that in standard test solution (Fig. 4A), displaying good signal reproducibility and resistance to complex substrate when using FG as matrix. After step-by-step concentration decreased from 600 μM to 1 μM, the characteristic peaks keep being visible clearly from the background at m/z 396.41. The insert is the semi-quantitative results. A linear range of 300 pmol to 500 fmol was obtained which satisfy the needs of detection of clinical medicine. Stimulated by the results in real sample, we believe that FG-based MS tool can be further broadened to monitor and quantify other drugs and small molecules in biological cases.

Fig. 4 (A) Negative ion mode FG-assisted mass spectra of drugs (estradiol, [M − H]⁻, m/z 271.28; 17-ethynelestradiol, [M − H]⁻, m/z 295.30; huperzine-A, [M − H]⁻, m/z 241.20; DOX, [M − X − H]⁻, m/z 396.41). (B) Semi-quantification of DOX in human serum.

3.5. Environmental pollutants and explosives analysis

According to report, FG synthesized via this method has excellent dispersibility in many kinds of solvents²⁷ (Table S1†) and long term stability, which could improve the chemical compatibility of FG matrix. Organonitrogen compounds exist widely in nature and are very valuable and essential, among which nitro compounds, nitroso compounds, amides and imines are the representative species. Based on the fact that FG co-dissolve and co-crystallize finely with a variety of analytes and a good co-crystallization is conducive to energy transfer from matrix to analytes in the D/I process, we measured four kinds of organonitrogen substances by FG matrix. Nitro polycyclic aromatic hydrocarbons (nitro-PAHs), a class of genotoxic environmental pollutants that often been found in air and aquatic systems are potentially mutagenic and carcinogenic. With FG as matrix, nitro-PAHs were analysed (Fig. 5). For 1-nitropyrene, [M + O − H]⁻ at m/z 247.11 and [M − NO]⁻ at m/z 217.10 (Fig. 5A) and for 2-nitrofluorene, [M − NO]⁻ at m/z 181.09, [M − H]⁻ at m/z 210.09, [M + O − H]⁻ at m/z 226.08 and [M + O − H + NO − H]⁻ at m/z 255.08 were observed (Fig. 5B). For 6-nitrochrysene, [M − NO]⁻ at m/z 243.12, [M − H]⁻ at m/z 273.13 and [M + O − H]⁻ at m/z 288.75 (Fig. 5C) and for 9-nitroanthracene, [M − NO]⁻ at m/z 193.11 and [M − H]⁻ at m/z 223.10 were generated (Fig. 5D). Nitroso compounds are proven to be poisonous and carcinogenic to human beings, however, they are hard to be detected by MALDI due to their instability. Here, 5-nitroso-2,4,6-triaminopyrimidine, an intermediate for drugs triamterene (potassium-sparing diuretic) and aminopterin (antineoplastic drugs) were analyzed (Fig. S7†) with [M − H]⁻ ions. Imide compounds are monomers of polyimide (PI) which is frequently used in aerospace, machinery and electronics. In this chapter, three imide monomers: N-hydroxysuccinimide, 1-methylpyrrolidine-2,5-dione and 1-phenylpyrrolidine-2,5-dione were explored (Fig. S7b–d†). [M + H₂O − H]⁻ at m/z 132.05, [M − H]⁻ at m/z 115.02 and [M + H₂O − H]⁻ at m/z 190.08 were all observed with high S/N ratios.

Fig. 5 FG-assisted MALDI-TOF MS spectra of four kinds of nitro-PAHs in negative ion mode. The amount of each analyte is 500 pmol. Laser intensity: 70%.

Although many nitroaromatic compounds naturally occur in the environment, a variety of them come from anthropogenic sources. Indeed, nitroaromatic compounds like 2,4,6-trinitrotoluene (TNT) are commonly used as chemicals and intermediates industrial manufacturing of explosives. Considering the significance of them in forensic sciences, we applied high-throughput FG-assisted MALDI-TOF MS to analyse explosives. The qualitative results of two kinds of explosives (2,4,6-trinitrotoluene (TNT) and pentaerythritol tetranitrate (PETN)) were shown in Fig. S8.†

3.6. Investigation of spot homogeneity of FG

“Dried droplet” standard MALDI sample preparation procedure is the most used method over the past years. However, surface tension leads to a non-homogeneous distribution of the individual crystals near the rim of the sample spot and the best MALDI signals are usually achieved only at the certain locations of the crystals, which often requires manual control and that is why it is difficult for MALDI MS to realize absolute quantitative analysis. With FG as matrix, the “sweet spot” and “coffee-ring” effect have been greatly reduced (Fig. S9†). Uric acid (UA) is a vital metabolite and the increase of its contents will lead to gout. So the quantitative determination of UA proves medically important. With FG matrix, both UA and isotope internal standard UA − 1,3-¹⁵N₂ can be directly detected in the diluted urine sample with [M − H]⁻ (Fig. 6A). A calibration curve was plotted according to the ion intensity ratio of [UA − H]⁻ and [UA − 1,3-¹⁵N₂]⁻ in the range of 0.2–5.0 mM (R² = 0.9978) (Fig. 6B). With the calibration curve, UA in untreated human urine sample was directly determined (Fig. S10,† Table 1).

Fig. 6 (A) MALDI-TOF mass spectra of UA in uric acid-free artificial urine for quantitative curve with FG as matrix. m/z 167.03, [UA − H]⁻; m/z 169.05, [UA − 1,3-¹⁵N₂ − H]⁻. The concentrations of uric acid are 0.2, 0.5, 1.0, 3.0, and 5.0 mM, and the internal standard added is 2 mM. Laser intensity: 70%. (B) The calibration curves used for quantitative determination of uric acid according to the ion intensity ratio of [UA − H]⁻ and [UA − 1,3-¹⁵N₂ − H]⁻. The regression equation is y = 0.3888x + 0.0517 with an R-square of 0.998.

Table 1 Determination results of uric acid (UA) in human urine^a

Experiments	Exp 1	Exp 2	Exp 3	Main
a The typical concentration of UA in human urine is 2 mM.^33 Exp 1, exp 2 and exp 3 represent three parallel measurements of the same human urine sample.
I167.03/169.03	136/1082 = 0.1257	371/2858 = 0.1298	146/1047 = 0.1394	0.1316
Concentration (mM)	3.81	4.01	4.51	4.11

---

4. Conclusions

In conclusion, FG was successfully synthesized via a simple one-pot ambient method and applied as a versatile matrix for MALDI-TOF MS analysis of small molecules. A variety of small molecules as well as practical samples were analyzed. Results showed that compared to conventional carbon based matrices, FG own more superior properties: (i) clean background, high signal intensity and S/N ratio; (ii) no discharge or contamination to ion source; (iii) excellent dispersibility which makes for the homogeneity of matrix spots and improved chemical compatibility and provide possibility of quantification. This study provides a novel carbon-based matrix which solves the problems of usual carbon-based matrices, expanding the application range of FG. Further investigations such as sensitivity of FG matrix would be conducted.

Acknowledgements

This work was supported by the National Natural Sciences Foundation of China (Grants 21127901, 21321003, 21305144, 21475139, 21505140 and 21675160) and Chinese Academy of Sciences.

Notes and references

1. M. Karas and F. Hillenkamp, Anal. Chem., 1988, 60, 2299–2301.
2. R. L. Vermillion-Salsbury and D. M. Hercules, Rapid Commun. Mass Spectrom., 2002, 16, 1575–1581.
3. A. R. Korte and Y. J. Lee, J. Mass Spectrom., 2014, 49, 737–741.
4. R. Chen, W. Xu, C. Xiong, X. Zhou, S. Xiong, Z. Nie, L. Mao, Y. Chen and H. C. Chang, Anal. Chem., 2012, 84, 465–469.
5. C. L. Su and W. L. Tseng, Anal. Chem., 2007, 79, 1626–1633.
6. Z. Lin, W. Bian, J. Zheng and Z. Cai, Chem. Commun., 2015, 51, 8785–8788.
7. Y. H. Shih, C. H. Chien, B. Singco, C. L. Hsu, C. H. Lin and H. Y. Huang, Chem. Commun., 2013, 49, 4929–4931.
8. Z. Li, Y. W. Zhang, Y. L. Xin, Y. Bai, H. H. Zhou and H. W. Liu, Chem. Commun., 2014, 50, 15397–15399.
9. Z. Lin, J. Zheng, G. Lin, Z. Tang, X. Yang and Z. Cai, Anal. Chem., 2015, 87, 8005–8012.
10. J. Zhang, L. Zhang, R. Li, D. Hu, N. Ma, S. Shuang, Z. Cai and C. Dong, Analyst, 2015, 140, 1711–1716.
11. X. L. Dong, J. S. Cheng, J. H. Li and Y. S. Wang, Anal. Chem., 2010, 82, 6208–6214.
12. M. Lu, Y. Lai, G. Chen and Z. Cai, Anal. Chem., 2011, 83, 3161–3169.
13. Q. Min, X. Zhang, X. Chen, S. Li and J. J. Zhu, Anal. Chem., 2014, 86, 9122–9130.
14. J. Shiea, J.-P. Huang, C.-F. Teng, J. Jeng, L. Y. Wang and L. Y. Chiang, Anal. Chem., 2003, 75, 3587–3595.
15. S. Xu, Y. Li, H. Zou, J. Qiu, Z. Guo and B. Guo, Anal. Chem., 2003, 75, 6191–6195.
16. W. Lu, Y. Li, R. Li, S. Shuang, C. Dong and Z. Cai, ACS Appl. Mater. Interfaces, 2016, 8, 12976–12984.
17. S. Chen, H. Zheng, J. Wang, J. Hou, Q. He, H. Liu, C. Xiong, X. Kong and Z. Nie, Anal. Chem., 2013, 85, 6646–6652.
18. Y. Wang, D. Gao, Y. Chen, G. Hu, H. Liu and Y. Jiang, RSC Adv., 2016, 6, 79043–79049.
19. Y. K. Kim, H. K. Na, S. J. Kwack, S. R. Ryoo, Y. Lee, S. Hong, S. Hong, Y. Jeong and D. H. Min, ACS Nano, 2011, 5, 4550–4561.
20. C. Pan, S. Xu, L. Hu, X. Su, J. Ou, H. Zou, Z. Guo, Y. Zhang and B. Guo, J. Am. Soc. Mass Spectrom., 2005, 16, 883–892.
21. J. Meng, C. Shi and C. Deng, Chem. Commun., 2011, 47, 11017–11019.
22. C. Shi, C. Deng, X. Zhang and P. Yang, ACS Appl. Mater. Interfaces, 2013, 5, 7770–7776.
23. S. F. Ren, L. Zhang, Z. H. Cheng and Y. L. Guo, J. Am. Soc. Mass Spectrom., 2005, 16, 333–339.
24. X. M. He, G. T. Zhu, J. Yin, Q. Zhao, B. F. Yuan and Y. Q. Feng, J. Chromatogr. A, 2014, 1351, 29–36.
25. S. Chen, C. Xiong, H. Liu, Q. Wan, J. Hou, Q. He, A. Badu-Tawiah and Z. Nie, Nat. Nanotechnol., 2015, 10, 176–182.
26. R. R. Nair, W. Ren, R. Jalil, I. Riaz, V. G. Kravets, L. Britnell, P. Blake, F. Schedin, A. S. Mayorov, S. Yuan, M. I. Katsnelson, H. M. Cheng, W. Strupinski, L. G. Bulusheva, A. V. Okotrub, I. V. Grigorieva, A. N. Grigorenko, K. S. Novoselov and A. K. Geim, Small, 2010, 6, 2877–2884.
27. M. Zhu, X. Xie, Y. Guo, P. Chen, X. Ou, G. Yu and M. Liu, Phys. Chem. Chem. Phys., 2013, 15, 20992–21000.
28. L. P. Wang, X. D. Xie, W. F. Zhang, J. Zhang, M. S. Zhu, Y. L. Guo, P. L. Chen, M. H. Liu and G. Yu, J. Mater. Chem. C, 2014, 2, 6484–6490.
29. H. Chang, J. Cheng, X. Liu, J. Gao, M. Li, J. Li, X. Tao, F. Ding and Z. Zheng, Chemistry, 2011, 17, 8896–8903.
30. P. W. Gong, Z. F. Wang, J. Q. Wang, H. G. Wang, Z. P. Li, Z. J. Fan, Y. Xu, X. X. Han and S. R. Yang, J. Mater. Chem., 2012, 22, 16950–16956.
31. S. Kwon, J. H. Ko, K. J. Jeon, Y. H. Kim and J. Y. Park, Nano Lett., 2012, 12, 6043–6048.
32. Z. F. Wang, J. Q. Wang, Z. P. Li, P. W. Gong, J. F. Ren, H. G. Wang, X. X. Han and S. R. Yang, RSC Adv., 2012, 2, 11681–11686.
33. E. Popa, Y. Kubota, D. A. Tryk and A. Fujishima, Anal. Chem., 2000, 72, 1724–1727.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra21083j

---