An aptamer electrochemical assay for sensitive detection of immunoglobulin E based on tungsten disulfide–graphene composites and gold nanoparticles

Abstract

Two-dimensional transition metal dichalcogenides are attracting increasing attention in electrochemical sensing due to their unique electronic properties. In this work, a label-free electrochemical assay for sensitive determination of IgE was developed by assembling aptamers on a glassy carbon electrode modified with WS₂–graphene nanosheets and gold nanoparticles. The WS₂–graphene nanosheet was prepared by a simple one-step hydrothermal process and its properties were characterized by X-ray powder diffraction, scanning electron microscopy and transmission electron microscopy. The WS₂–graphene nanosheet acted as a relatively good electrical conductor for accelerating electron transfer, while the modification of gold nanoparticles provided signal amplification for electrochemical detection. The detection of IgE was performed by measuring the electrochemical signal response of [Fe(CN)₆]^3−/4− using differential pulse voltammetry. A good linear relationship between the peak current and the logarithm of IgE concentration from 1.0 × 10⁻¹² to 1.0 × 10⁻⁸ M was obtained with a detection limit of 1.2 × 10⁻¹³ M (3σ/S). The developed assay exhibited high sensitivity, selectivity and long-term stability, and it was successfully applied for the determination of IgE in serum samples. This work indicated that WS₂–graphene nanosheets were promising in electrochemical detection applications.

---

1. Introduction

The human immune system is very important and protects the body from exterior invaders. The over-reactions of the human immune system can cause allergies, anaphylactic shock, and even death. Immunoglobulin E (IgE) is an antibody subclass that is found only in mammals.¹ It is capable of triggering the most powerful immune reactions and its rapid and sensitive detection is of great interest in dealing with patients afflicted with allergy-mediated disorders.²

Electrochemical methods have become the preferred choice for protein detection due to their inherent advantages such as low cost of instrumentation and operation, simplicity for operators and on-site monitoring.^3,4 But, an obstacle is that the ordinary electrode can not offer enough sensitivity to determine protein at ultra-trace level. The selectivity is another challenge for an electrochemical approach. Therefore, there were some works focused on biomolecules, such as antibodies^5,6 modified on the electrode to detect protein selectively. Aptamers are artificial single-stranded DNA or RNA oligonucleotides which can recognize various targets including small species, sugars, proteins, and even whole cells with high specificity.^7,8 Compared with antibody, aptamers exhibit several considerable advantages, such as high specificity, good stability, desirable biocompatibility and significant chemical simplicity. Therefore, they offer a powerful alternative to antibody as recognition molecules. Recently, several aptamer-based biosensors have been developed for IgE determination.^1,9,10

Graphene (Gr)-based assays have stimulated intense research interest because of the unique properties, such as high surface area, excellent electrical conductivity and strong mechanical strength.^11,12 However, Gr tend to form agglomerates through the van der Waals interactions, which greatly restricts its further application in electrochemical sensing. Therefore, the modification of Gr to prevent the aggregation is very important for enlarging its application. WS₂, as one of two-dimensional (2D) transition-metal dichalcogenides, is composed of W layer sandwiched between two sulfur layers and stacked together by weak van der Waals interactions.¹³ It was reported to possess good biocompatibility, large electroactive surface area and dispersibility in water.^14,15 Moreover, WS₂ nanosheets were reported to allow self-assembly of thiolated compounds on its surfaces, indicating another advantage of WS₂ nanosheets over Gr in facile surface modification.¹⁶ However, it is not favorable for applications in electrode materials because WS₂ is a semiconductor and has relatively low conductivity. Thus, it is highly desirable to prepare WS₂ nanosheets on electronically conductive support to facilitate the charge transfer for electrochemical biosensing applications. Therefore, the combination of Gr and WS₂ nanosheets to prepare the nanocomposite would be a good way to overcome the low conductivity of WS₂ and the poor dispersibility of Gr nanosheets.

In this work, a 2D WS₂–Gr nanosheet was prepared by a simple hydrothermal method, and then a novel electrochemical sensing platform was fabricated for IgE detection based on WS₂–Gr nanosheets coupled with gold nanoparticles (AuNPs) as sensing platform and aptamer as recognition group. WS₂–Gr nanosheets and AuNPs may provide signal amplification for electrochemical detection due to their good conductivity and large electroactive surface area. Under optimal experimental conditions, the developed aptamer assay showed excellent sensitivity and good selectivity to IgE, and has been applied for serum samples analysis.

2. Experimental

2.1 Apparatus

Electrochemical measurements were performed on a CHI 660E Electrochemical Workstation (Shanghai CH Instruments, China) with a conventional three-electrode system composed of platinum wire as auxiliary, saturated calomel electrode (SCE) as reference and modified GCE as working electrode. A JEM 2100 transmission electron microscope (TEM) with an accelerating voltage of 200 kV and a Hitachi S-4800 scanning electron microscope (SEM) were used to record the morphologies of the nanocomposite. X-ray powder diffraction (XRD) pattern was operated on a Japan RigakuD/Maxr-A X-ray diffractometer equipped with graphite monochromatized high-intensity Cu Kα radiation (λ = 1.54178 Å) and operated at 40 kV and 20 mA. Raman spectra were recorded at ambient temperature on a Renishaw Raman system model 1000 spectrometer with a 200 mW argon-ion laser at an excitation wavelength of 514.5 nm and an integration time of 30 s were used during the test.

2.2 Reagents

Graphite powder, Na₂WO₄·2H₂O, hydrazine solution (50 wt%) and ammonia solution (28 wt%) were purchased from Shanghai Chemical Reagent Corporation (Shanghai, China). 6-Mercapto-1-hexanol (MCH), chloroauric acid (HAuCl₄·4H₂O) and trisodium citrate were purchased from Sigma-Aldrich (St. Louis, MO). 0.1 M phosphate buffer solution (PBS, pH 7.0) was prepared with 0.1 M Na₂HPO₄ and NaH₂PO₄ and adjusted by 0.1 M H₃PO₄ or 0.1 M NaOH solutions. IgE and its aptamer were obtained from Sangon Biological Engineering Technology & Co. Ltd (Shanghai, China). The sequences of the aptamer of IgE was: 5′-SH–(CH₂)₆–TTT TTT TTT T GGG GCA CGT TTA TCC GTC CCT CCT AGT GGC GTG CCC C-3′, and it was dissolved in 10 mM PBS (7.4) containing 1.0 M NaCl. All reagents were of analytical grade and used without further purification.

2.3 Preparation of Au nanoparticles

First, the colloidal AuNPs with about 3–4 nm diameter were prepared according to the previous protocol.¹⁷ Briefly, 20 mL of 2.5 × 10⁻⁴ mol L⁻¹ HAuCl₄ containing 2.5 × 10⁻⁴ mol L⁻¹ Na₃C₆H₅O₇ was prepared in a conical flask. 0.6 mL of 0.1 M NaBH₄ solution (ice cold) was quickly added to the solution with vigorous stirring. The solution turned pink immediately after adding NaBH₄, indicating the formation of AuNPs.

2.4 Preparation of tungsten disulfide–graphene nanosheets

Graphite oxide (GO) was synthesized by oxidizing natural graphite powder using the modified Hummers method.¹⁸ The GO prepared as such was dispersed in deionized water and then exfoliated by ultrasonication to get graphene oxide sheets.

WS₂–Gr composites were prepared by a modified L-cysteine-assisted solution-phase method.¹⁹ In short, 40 mL deionized water was added in the GO suspension prepared above and then 0.5 g of Na₂WO₄·2H₂O was added with stirring. After 30 min, the pH value of the mixture was adjusted to 6.5 with 0.1 M NaOH. 0.8 g L-cysteine was then added to the mixture followed by 80 mL deionized water. After ultrasonication and stirring for 10 min, the mixture was transferred to a 100 mL Teflon-lined stainless steel autoclave, sealed tightly, and heated at 240 °C for 24 h. After cooling to room temperature naturally, a black precipitate was collected by centrifugation, washed with deionized water and ethanol, and then dried in a vacuum oven at 80 °C for 24 h.

2.5 Preparation of modified electrode

The procedure for the modified electrode fabrication is illustrated in Scheme 1.

Scheme 1 Schematic diagram of the modified electrode.

The WS₂–Gr nanocomposites were firstly dispersed in water with ultrasonication for 30 min to get a homogenous suspension (5 mg mL⁻¹). A GCE was polished to a mirror-like smoothness with 1, 0.3, and 0.05 μm alumina slurry sequentially, and then cleaned ultrasonically in absolute ethanol and water, respectively. The cleaned GCE was dried with nitrogen gas. After that, the WS₂–Gr/GCE was prepared by casting 6 μL WS₂–Gr suspension onto the pretreated GCE and dried in the air. Dried electrode was then exposed to AuNPs for 6 h to obtain AuNPs/WS₂–Gr/GCE. Subsequently, 8 μL 1.0 × 10⁻⁶ M IgE aptamer was covalently immobilized onto the AuNPs/WS₂–Gr/GCE by the Au–thiol chemistry for 12 h at room temperature. To block the uncovered gold surface, 20 μL of 1 mM MCH was dropped on the surface of as-prepared aptamer/AuNPs/WS₂–Gr/GCE for 1 h, followed by washing with 0.1 M PBS for three times.

For detection of IgE, the modified electrode was first incubated with 10 μL of different concentration of IgE for 120 min at 37 °C. Then the electrochemical measurement was performed in 0.1 M PBS (pH 7.0) containing 1.0 mmol L⁻¹ [Fe(CN)₆]^3−/4− and 0.1 mol L⁻¹ KCl by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) with instrumental parameters as: 0.005 V of pulse amplitude, 0.05 s of pulse width and 0.2 s of pulse period. Electrochemical impedance spectroscopy (EIS) experiment was performed in a 10.0 mL aqueous solution containing 1.0 mmol L⁻¹ [Fe(CN)₆]^3−/4− and 0.1 mol L⁻¹ KCl at a potential of 0.2 V over the frequency range from 0.1 Hz to 100 kHz, using an amplitude of 5 mV.

3. Results and discussion

3.1 Tungsten sulfide–graphene composites characterization

The morphologies of the as-prepared Gr and WS₂–Gr composite were characterized with SEM and TEM. Fig. 1A shows the SEM image of as-synthesized Gr nanosheets. It can be seen that most of Gr nanosheets are curled and entangled together. Fig. 1B draws the SEM image of WS₂–Gr composites. It is clearly observed that the Gr nanosheets are covered by densely WS₂ layers. Fig. 1C shows the corrugated and scrolled sheets resemble crumpled silk veil waves. Gr layers interact with each other to form an open pore system, through which electrolyte ions easily access the surface of Gr to form electric double layers.^20,21 The TEM images of WS₂–Gr hybrid material are shown in Fig. 1D, where WS₂ nanosheets distribute well on the Gr 2D skeleton, evidencing the well-behaved assembly process. In the WS₂–Gr hybrid material, the overlapping or coalescing of the Gr and WS₂ nanosheets would form the interconnected conducting network, which facilitated rapid electronic transport in electrode reactions. Furthermore, the interconnected conducting network and layered structure also enhanced the stability of the WS₂–Gr composites due to superstrength of Gr. The high-resolution TEM (HRTEM) image of WS₂–Gr in Fig. 1E shows that the interlayer spacing between the WS₂ sheets in the composite is estimated to be 0.69 nm.

Fig. 1 SEM images of Gr (A) and WS₂–Gr composites (B); TEM images of Gr (C) and WS₂–Gr composites (D); HRTEM image of WS₂–Gr composites (E).

It is reported that W(VI) can be easily reduced in solution by reductants such as thiourea and H₂S, etc.^22–24 The sulfurization reagent L-cysteine will easily decompose and form H₂S during hydrothermal procedure.²⁵ Thus, WS₂ might be obtained by the reaction of Na₂WO₄ and sulfurization reagent without using additional reductant.

Fig. 2B shows the XRD patterns of the Gr and WS₂–Gr composites. A broad diffraction peak with low intensity centers at about 24.5° corresponding to Gr is observed. The reflections observed for WS₂ (2θ = 13.75, 33.82, 59.87, 69.79) can be indexed on the hexagonal WS₂ structure (JCPDS no. 84-1398). The presence of (002), (100) and (110) reflections strongly suggests a few-layered structure for WS₂ in the WS₂–Gr composites.²⁶ The diffraction peaks display very weak, indicating the crystallinity of WS₂ is very poor. The poor crystallinity of WS₂ is attributed to the incorporation of the Gr inhibiting the growth of the layered WS₂ crystal during the hydrothermal process.

Fig. 2 (A) XRD patterns of WS₂–Gr composites; (B) Raman spectra of Gr and WS₂–Gr composites.

Raman spectroscopy was carried out to further confirm the formation of the WS₂–Gr composites. As shown in Fig. 2B, the peaks observed at 343.5 cm⁻¹ and 422.6 cm⁻¹ correspond to the E_2g and A_1g modes of WS₂, respectively.²⁷ The D-band (1346.7 cm⁻¹) and G-band (1592.1 cm⁻¹) associated with the defects in graphitic carbons and the E_2g phonon of sp² carbon atoms, respectively.²⁸ The I_D/I_G ratio is generally related to the density of defects in graphene-based materials. The results also confirmed the presence of the Gr and WS₂ in the composite and complete correspondence with the findings from the XRD diffraction studies.

The elemental composition of the as-prepared WS₂–Gr composite was identified with energy-dispersive spectroscopy (EDS). The obtained results showed that the products contain C (6.13), W (58.6), S (32.3), and a small quantity of O (2.93). The calculated atomic ratio of S to W element was 1.81, approaching the theoretical value of WS₂. These values indicated that the products were stoichiometric WS₂. C was mainly provided by Gr, while a small quantity of O came from a few parts of the Gr that were not completely reduced during the hydrothermal process.

3.2 Electrochemical characterization

Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were employed to validate the fabrication of the electrode. Fig. 3A shows the CVs of the sequentially modification processes in a 1.0 mM [Fe(CN)₆]^3−/4− containing 0.1 M KCl. The probe [Fe(CN)₆]^3−/4− reveals a nearly reversible CV at the bare GCE (curve a). After WS₂–Gr composites are applied on the GCE (curve c), the peak currents are much higher than that on the bare GCE because of excellent electro-conductibility of the WS₂–Gr. When the AuNPs are further coated on WS₂–Gr/GCE, the peak currents improve greatly (curve c) due to the synergistic effect of WS₂–Gr nanosheets and AuNPs, which improve the effective active area of electrode and accelerate the electron transfer. The assembly of IgE aptamer on AuNPs/WS₂–Gr/GCE surface induces an obviously decrease in peak currents (curve d). One reason can explain this phenomenon: the electrostatic repulsion between the negatively charged phosphate backbone of oligonucleotides in aptamer and [Fe(CN)₆]^3−/4− controlled by negative charge. After IgE was immobilized on the aptamer/AuNPs/WS₂–Gr/GCE surface, the peak currents decrease obviously (curve e), which suggest that the protein IgE severely reduced effective area and active sites for electron transfer.

Fig. 3 CVs (A) and EIS (B) of GCE (a), WS₂–Gr/GCE (b), AuNPs/WS₂–Gr/GCE (c), aptamer/AuNPs/WS₂–GR/GCE (d), and IgE/aptamer/AuNPs/WS₂–GR/GCE (e) in 1 mM [Fe(CN)₆]^3−/4− containing 0.1 M KCl.

EIS is an effective method for studying the interface properties of surface-modified electrodes and electron-transfer resistance at the electrode surface. The semicircle diameter in EIS equals to the electron transfer resistance (R_et) and the linear part at lower frequencies represents the diffusion process. As shown in Fig. 3B, the EIS of GCE displays a large semicircle part (curve a), indicating that GCE had a relatively low conductivity. Then the R_et value decreases greatly when WS₂–Gr is coated on GCE (curve b), indicating that WS₂–Gr film promotes the electron transfer and enhanced the conductivity of the electrode. After modification of AuNPs, a further decrease of R_et is observed (curve c) because of the formation of the conductive AuNPs monolayer. Subsequently, when the IgE aptamer is adsorbed on the surface of AuNPs, a high interfacial R_et (curve d) is obtained in the impedance spectrum because aptamer insulates the conductive support and perturbs the interfacial electron transfer between the electrode and the redox indicator in the solution. When IgE is assembled on the electrode, the result is consistent with the fact that the hydrophobic layer of protein further hinders the interfacial electron transfer (curve e).

3.3 Optimization of the experiment conditions

The effect of IgE aptamer concentration was studied by varying its concentration on AuNPs/WS₂–Gr/GCE and then evaluated the DPV response of [Fe(CN)₆]^3−/4−. The results showed that the peak current of [Fe(CN)₆]^3−/4− decreased with increase of the probe concentration in the range of 0.5–1.0 μM. The current response reached a platform when the probe concentration exceeded 1.0 μM, indicating the most aptamer has been immobilized on the electrode. Thus the aptamer concentration of 1.0 μM was used in the further experiments.

The influence of the reaction time of aptamer and IgE on the DPV response of [Fe(CN)₆]^3−/4− was also evaluated. Fig. 4A shows the peak current of [Fe(CN)₆]^3−/4− obviously increases with increasing the reaction time from 20 to 100 min, and almost keeps stable after 120 min, suggesting the reaction is completed. Thus 100 min of reaction time was used.

Fig. 4 Effect of the incubation time (A) and the temperature (B) of IgE on the amperometric responses.

The effects of the reaction temperature of aptamer and IgE on the DPV response of [Fe(CN)₆]^3−/4− were also studied in range of 30–45 °C. As shown in Fig. 4B, the DPV response of [Fe(CN)₆]^3−/4− increases with the increase of the temperature in the range of 30–37 °C, and then decreased when exceeded 37 °C. Therefore, 37 °C was employed in the subsequent experiments for capture of IgE with the aptamer.

3.4 Analytical performance

Under the optimal experiment conditions, we examined the analytical performances of the developed method upon the addition of different concentrations of IgE and then measured the DPV response of [Fe(CN)₆]^3−/4−. As shown in Fig. 5A, the DPV peak currents decrease with the increasing of IgE concentrations in the range of 1.0 × 10⁻¹² to 1.0 × 10⁻⁸ M, and a linear relationship between the DPV peak currents and the logarithm of concentrations is observed (inset of Fig. 5A). The regression equation was i (μA) = −6.20 log(−c/M) − 2.37 (i is the peak current and c is the concentration of IgE) with a correlation coefficient (R) of 0.991. The detection limit was calculated to be 1.2 × 10⁻¹³ M based on three times the standard deviation of the blank sample measurement. The analytical performance of this fabricated electrode was compared with previously reported different modified electrodes based aptamer and nanomaterials for the determination of protein. The comparison results are presented in Table 1. It can be seen that the developed biosensor displays better analytical performance toward protein than previously reported other methods.^29–32 The reason might be as follows: firstly, the excellent electrical conductivity of layered WS₂–Gr composites enhanced the charge transport; secondly, the formation of the AuNPs film on the WS₂–Gr/GCE not only promoted the electron transfer but also increased the immobilization amount of aptamer, which resulted in the lower detection limit.

Fig. 5 (A) DPVs of aptamer/AuNPs/WS₂–Gr/GCE after the reaction with different concentrations of IgE (from a to h: 0; 1.0 × 10⁻¹²; 1.0 × 10⁻¹¹; 1.0 × 10⁻¹⁰; 8.0 × 10⁻¹⁰; 1.0 × 10⁻⁹; 5.0 × 10⁻⁹; 1.0 × 10⁻⁸ M). Inset: calibration curve for I_pa vs. −log c; (B) DPVs of aptamer/AuNPs/WS₂–Gr/GCE (a) and after reaction with 1.0 × 10⁻⁷ M thrombin (b), 1.0 × 10⁻⁷ M BSA (c), and 1.0 × 10⁻⁹ M IgE (d); (C) six parallel-made aptamer/AuNPs/WS₂–Gr/GCEs were applied to detect a 1.0 × 10⁻¹¹ IgE.

Table 1 Comparison between the proposed assay and other reported method for IgE detection

Electrodes	Analytes	Analytical technique	Linear range (nM)	LOD (pM)	Ref.
Multiwalled carbon nanotubes/ionic liquid/chitosan/GCE	IgE	DPV	0.5–30	37	1
Streptavidin–Ag/Gr/screen printed electrode	IgE	Square wave anodic stripping voltammetry	0.053–53	0.19	9
AuNPs/graphite-based screen-printed electrode with cDNA amplification	IgE	DPV	0.001–100	0.16	29
Fe3O4/Au	Thrombin	DPV	1.0–75	100	30
AuNPs/1,6-hexanedithiol/Au	Thrombin	EIS	0.1–30	13	31
Poly(pyrrole-nitrilotriacetic acid)/glassy carbon disk electrodes	Thrombin	EIS	0.047–0.5	4.4	32
AuNPs/WS2–Gr/GCE	IgE	DPV	0.001–10	0.12	This work

---

3.5 The specificity and repeatability

The selectivity of the developed assay was evaluated and two proteins (thrombin and BSA) were chosen as control samples. As shown in Fig. 5B, only IgE can cause an obvious decrease in peak current even when the two control samples were presented at 100-fold concentrations. It is clear that the aptamer probe can bind its target molecule with high affinity and selectivity. These data obtained provided direct evidence that the current signal is generated from the specific interaction between the aptamer and IgE.

To evaluate the repeatability of the assay, six parallel-made aptamer/AuNPs/WS₂–Gr/GCEs were applied to detect a 1.0 × 10⁻¹¹ IgE and the relative standard deviation (RSD) of 4.5% was achieved (Fig. 5C), suggesting good reproducibility of the developed sensing platform.

3.6 Practical application

In order to estimate the applicability of the methodology, the proposed assay was tested by applying it to the analysis of IgE concentration in human serum sample by the DPV technique. The standard addition method was adopted for this purpose and the obtained result was compared with the determined value through the standard ELISA method. The results and the relative deviations between the two methods are shown in Table 2. It describes the correlation between the results obtained by the proposed assay and ELISA method. It could be seen that there is a good agreement between results obtained by two methods and thus, the aptamer/AuNPs/WS₂–Gr/GCE might be a promising system for detecting IgE in real samples.

Table 2 Comparison of IgE levels determined using proposed method and ELISA (n = 3)

Serum samples	1	2	3	4	5
Aptamer assay (ng mL^−1)	36.9	44.7	68.2	72.3	124.3
ELISA (ng mL^−1)	35.6	43.4	65.4	75.6	130.4
Relative deviation (%)	3.7	3.0	4.3	−4.4	−4.7

---

4. Conclusion

This work reported a novel electrochemical assay for IgE by assembling an aptamer on AuNPs/WS₂–Gr film modified GCE using [Fe(CN)₆]^3−/4− as indicator. The introduction of WS₂–Gr nanosheets and AuNPs efficiently accelerated the electron transfer and enhanced the detection signal, which led to a high sensitivity with a detection limit of 0.12 pM. The assay also displayed excellent precision and accuracy, wide linear range, high selectivity and good repeatability, and was applied successfully for IgE detection in real samples. The proposed approach would be attractive for protein analysis in bioanalytical and clinic biomedical application.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (U1304214) and the State Key Laboratory of Chemo/Biosensing and Chemometrics (no. 2013013, 2012011).

References

1. S. Khezrian, A. Salimi, H. Teymourian and R. Hallaj, Biosens. Bioelectron., 2013, 43, 218–225.
2. K. Maehashi, T. Katsura, K. Kerman, Y. Takamura, K. Matsumoto and E. Tamiya, Anal. Chem., 2007, 79, 782–787.
3. A. Erdem and G. Congur, Sens. Actuators, B, 2014, 196, 168–174.
4. H. Li, W. Zhang and H. H. Zhou, Anal. Biochem., 2014, 449, 26–31.
5. C. M. Pandey, G. Sumana and I. Tiwari, Biosens. Bioelectron., 2014, 61, 328–335.
6. J. Zhou, L. P. Du, L. Zou, Y. C. Zou, N. Hu and P. Wang, Sens. Actuators, B, 2014, 197, 220–227.
7. A. K. Sharma, A. D. Kent and J. M. Heemstra, Anal. Chem., 2012, 84, 6104–6109.
8. P. Wu, K. Hwang, T. Lan and Y. A. Lu, J. Am. Chem. Soc., 2013, 135, 5254–5257.
9. W. Song, H. Li, H. P. Liu, Z. S. Wu, W. B. Qiang and D. K. Xu, Electrochem. Commun., 2013, 31, 16–19.
10. Y. C. Chen, W. T. Chang, C. C. Cheng, J. Y. Shen and K. S. Kao, Curr. Appl. Phys., 2014, 14, 608–613.
11. S. Mao, K. Yu, J. Chang, D. A. Steeber, L. E. Ocola and J. Chen, Sci. Rep., 2013, 3(1–6), 1696.
12. S. Mao, K. Yu, G. Lu and J. Chen, Nano Res., 2011, 4, 921–930.
13. G. F. Ma, H. Peng, J. J. Mu, H. H. Huang, X. Z. Zhou and Z. Q. Lei, J. Power Sources, 2013, 229, 72–78.
14. Q. Xi, D. M. Zhou, Y. Y. Kan, J. Ge, Z. K. Wu, R. Q. Yu and J. H. Jiang, Anal. Chem., 2014, 86, 1361–1365.
15. Y. X. Yuan, R. Q. Li and Z. H. Liu, Anal. Chem., 2014, 86, 3610–3615.
16. S. S. Chou, M. De, J. Kim, S. Byun, C. Dykstra, J. Yu, J. Huang and V. P. Dravid, J. Am. Chem. Soc., 2013, 135, 4584–4587.
17. N. R. Jana, L. Gearheart and C. J. Murphy, J. Phys. Chem. B, 2001, 105, 4065–4067.
18. K. J. Huang, L. Wang, Y. J. Liu, Y. M. Liu, H. B. Wang, T. Gan and L. L. Wang, Int. J. Hydrogen Energy, 2013, 38, 14027–14034.
19. K. J. Huang, L. Wang, Y. J. Liu, T. Gan, Y. M. Liu, L. L. Wang and Y. Fan, Electrochim. Acta, 2013, 107, 379–387.
20. H. B. Li, T. Lu, L. K. Pan, Y. P. Zhang and Z. Sun, J. Mater. Chem., 2009, 19, 677–681.
21. W. Lv, D. M. Tang, Y. B. He, C. H. You, Z. Q. Shi, X. C. Chen, C. M. Chen, P. X. Hou, C. Liu and Q. H. Yang, ACS Nano, 2009, 3, 3730–3736.
22. S. X. Cao, T. M. Liu, S. Hussain, W. Zeng, X. H. Peng and F. S. Pan, Mater. Lett., 2014, 129, 205–208.
23. K. Shiva, H. S. S. R. Matte, H. B. Rajendra, A. J. Bhattacharyya and C. N. R. Rao, Nano Energy, 2013, 2, 787–793.
24. Z. Z. Wu, B. Z. Fang, A. Bonakdarpour, A. Sun, D. P. Wilkinson and D. Wang, Appl. Catal., B, 2012, 125, 59–66.
25. K. Chang and W. X. Chen, ACS Nano, 2011, 5, 4720–4728.
26. P. Joensen, R. F. Frindt and S. R. Morrison, Mater. Res. Bull., 1986, 21, 457–461.
27. C. S. Reddy, A. Zak and E. Zussman, J. Mater. Chem., 2011, 21, 16086–16093.
28. R. H. Wang, C. H. Xu, J. Sun, L. Gao and C. C. Lin, J. Mater. Chem. A, 2013, 1, 1794–1800.
29. C. Y. Lee, K. Y. Wu, H. L. Su, H. Y. Hung and Y. Z. Hsieh, Biosens. Bioelectron., 2013, 39, 133–138.
30. S. Zhang, G. L. Zhou, X. L. Xu, L. L. Cao, G. H. Liang, H. Chen, B. H. Liu and J. L. Kong, Electrochem. Commun., 2011, 13, 928–931.
31. L. D. Li, H. T. Zhao, Z. B. Chen, X. J. Mu and L. Guo, Sens. Actuators, B, 2011, 157, 189–194.
32. H. Xu, K. Gorgy, C. Gondran, A. Goff, N. Spinelli, C. Lopez, E. Defrancq and S. Cosnier, Biosens. Bioelectron., 2013, 41, 90–95.

---