Application of three-dimensional flower-like nanomaterials in the fabrication of sandwich-type electrochemical immunosensors

Abstract

A novel and ultrasensitive sandwich-type electrochemical immunosensor was developed for the quantitative detection of carcinoembryonic antigen (CEA) in this work. Three-dimensional (3D) ZnO nanoflowers (NFs) were 3-aminopropyl-functionalized as the matrix to immobilize the primary antibodies (Ab₁). The unique 3D flower-like nanostructure of ZnO provided a higher surface area for the capture of proteins. 3D AuPd NFs were employed as the label to immobilize the secondary antibodies (Ab₂). The unique 3D flower-like nanostructure of AuPd produced a better electrocatalytic performance towards the reduction of hydrogen peroxide (H₂O₂). Due to the special electrochemical performance of 3D flower-like nanomaterials, the facile and sensitive fabrication of the designed immunosensor was achieved. Under optimal conditions, the designed immunosensor exhibited a wide linear range from 10⁻⁵ ng mL⁻¹ to 10² ng mL⁻¹ with a low detection limit of 3.2 fg mL⁻¹ for CEA. It also displayed an electrochemical performance with good reproducibility, selectivity and stability, which would provide potential applications in the clinical diagnosis of other tumor markers.

---

1. Introduction

In recent years, the fast, accurate and sensitive detection of tumor markers has resulted in a wide range of research,^1–5 which is beneficial to the sensing and diagnosis of cancers.^6,7 Therefore, a number of methods have been proposed for the quantitative detection of tumor markers, such as radioimmunoassay, chemiluminescence immunoassay, electrochromatography, latex particle immunoassay, time-resolved fluorometry and enzyme-linked immunosorbent assay (ELISA), etc.^8–16 Nevertheless, these methods usually entail complicated label processing and time-consuming separations, and cannot meet the increasing clinical demands for the rapid detection.¹⁷ Taking advantage of easy-to-operate, economical, sensitive, portable and simple-to-operate,^18–20 considerable attention has been devoted to the development of electrochemical immunosensors.^21–24

As the most used and classic analytical model, the sandwich-type structure has been widely employed for the study of electrochemical immunosensor.^25–31 For the sandwich-type electrochemical immunosensor, the matrix and the label are the two key factors to influence the sensitivity. At the present time, all kinds of nanomaterials have been multifunctionalized as the matrix or the label to achieve the high sensitivity of the sandwich-type electrochemical immunosensor.^32–36 However, the multifunctionalized procedure of the nanomaterials, including the complex operation steps and strict experimental conditions, makes the immunosensor become complicated and not easy to be repeated. Therefore, close attention should be paid to the nanomaterials, which possess special nanostructures and simple preparation methods, in the application of the fabrication of sandwich-type electrochemical immunosensor. Special nanostructures will bring some special chemical properties, such as the flower-like nanostructure has a larger specific surface area. Simple preparation methods will simplify operation steps and make the immunosensor become easy to be repeated.

In this work, three-dimensional (3D) flower-like nanomaterials were first employed as the matrix and the label simultaneously in the fabrication of sandwich-type electrochemical immunosensor. The special 3D flower-like structure can provide a higher surface area, which will bring some special electrochemical performance for the nanomaterials. As the matrix, 3D flower-like nanomaterials with the higher surface area can capture more proteins.^37,38 As the label, 3D flower-like nanomaterials with the higher surface area can produce better electrocatalytic performance.^35,39 In this sense, 3D ZnO nanoflowers (NFs) were synthesized by a simple hydrothermal synthesis method⁴⁰ and employed as the matrix in the designed immunosensor. In order to immobilize the primary antibodies (Ab₁), the amino-functionalized 3D ZnO NFs (NH₂–ZnO) was obtained by the 3-aminopropyl-functionalized procedure. 3D AuPd NFs with good electrocatalytic performance towards the reduction of hydrogen peroxide (H₂O₂) were synthesized by a simple coreduction method⁴¹ and employed as the label to immobilize the secondary antibodies (Ab₂). With the increase of concentration of antigen, the specific recognized conjugate (AuPd@Ab₂) will produce an increasing electrocatalytic current signal to achieve the electrochemical detection of antigen. Using carcinoembryonic antigen (CEA) as the target analyte, the facile and sensitive fabrication of the sandwich-type electrochemical immunosensor can be achieved.

2. Materials and methods

2.1. Apparatus and reagents

All electrochemical measurements were performed on a CHI760D electrochemical workstation (Chenhua Instrument Shanghai Co., Ltd, China). A conventional three-electrode system was used for all electrochemical measurements: a GCE (4 mm in diameter) as the working electrode, a saturated calomel electrode (SCE) as the reference electrode, and the platinum wire electrode as the counter electrode. Scanning electron microscope (SEM) images were obtained by using Quanta FEG250 field emission environmental SEM (FEI, United States) operated at 4 kV. Transmission electron microscope (TEM) images were obtained from a JEOL-1400 microscope (Japan).

Human CEA and antibody to human CEA (anti-CEA) were purchased from Shanghai Linc-Bio Science Co., Ltd, China. Zinc nitrate hexahydrate (Zn(NO₃)₂·6H₂O), hexamethylenetetramine (HMT, C₆H₁₂N₄), trisodium citrate, 1-(3-(dimethylamino)-propyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 3-aminopropyltriethoxysilane (APTES), chloroauric acid (HAuCl₄·4H₂O) and potassium hexachloropalladate(IV) (K₂PdCl₆) were purchased from Shanghai Aladdin Chemistry Co., Ltd, China. Phosphate buffered saline (PBS 1/15 M Na₂HPO₄ and KH₂PO₄) was used as an electrolyte for all electrochemistry measurements, which was purged with nitrogen gas for 20 min to remove the dissolved oxygen. All other reagents were of analytical grade and ultrapure water was used throughout the study.

2.2. Synthesis of the NH₂–ZnO

The 3D ZnO NFs were synthesized by a simple method.⁴⁰ 15 mL of 0.01 M Zn(NO₃)₂ aqueous solution was mixed with 15 mL of 0.01 M HMT aqueous solution, and the resulting mixture was stirred thoroughly. Then, 1 mL of 0.005 M trisodium citrate aqueous solution was added into the mixture under stirring. This final mixture was transferred into a Teflon-lined autoclave for hydrothermal synthesis at 95 °C in an oven for 1 h. After that, the autoclave was removed and cooled to room temperature. The resulting white precipitate was washed with water several times and dried in an oven at 65 °C.

The NH₂–ZnO was synthesized by a modified method according to the literature.⁴² Briefly, 0.1 g of 3D ZnO NFs were dispersed in a solution of 10 mL of ethanol solution containing 0.1 mL of APTES, which was placed in a vial and preheated to 70 °C in an oil bath under magnetic stirring for 1.5 h. After mild centrifugation, the resulting white precipitate was dried at 35 °C under high vacuum overnight.

2.3. Synthesis of the AuPd@Ab₂

The 3D AuPd NFs were prepared by the coreduction of Au and Pd ions using trisodium citrate.⁴¹ Typically, 64.8 μL of 0.04 g mL⁻¹ HAuCl₄ and 2.5 mg of K₂PdCl₆ were mixed in a 50 mL of aqueous solution. After the mixture was heated to boiling under vigorous stirring, 3 mL of 2 wt% trisodium citrate aqueous solution was rapidly added. The solution was kept on boiling for 10 min. The mixture was finally cooled down to room temperature.

100 μL of 100 μg mL⁻¹ Ab₂ dispersion was added into 1 mL of 2 mg mL⁻¹ the prepared 3D AuPd NFs solution under stirring for 12 h at 4 °C, followed by centrifugation. The resulting AuPd@Ab₂ was redispersed in 1 mL of PBS and stored at 4 °C until use.

2.4. Fabrication of the immunosensor

Fig. 1 shows the schematic diagram of the designed sandwich-type electrochemical immunosensor. A GCE was polished to a mirror-like finish with alumina powder (1.0, 0.3 and 0.05 μm), and then it was thoroughly cleaned before use. First, an aqueous solution of NH₂–ZnO (2 mg mL⁻¹, 6 μL), which was dispersed in 10 mM per 2 mM EDC/NHS, was added onto the surface of bare GCE and then dried. After washed, Ab₁ dispersion (10 μg mL⁻¹, 6 μL) was added onto the electrode. After incubated at 4 °C for 1 h and washed, BSA solution (10 mg mL⁻¹, 3 μL) was added onto the electrode to eliminate nonspecific binding sites. After incubated for another 1 h at 4 °C, the electrode was washed and incubated with a varying concentration of CEA (10⁻⁵ to 10² ng mL⁻¹, 6 μL) for 1 h at room temperature, and then the electrode was washed extensively to remove unbounded CEA molecules. Finally, the prepared AuPd@Ab₂ solution (2 mg mL⁻¹, 6 μL) was added onto the modified electrode surface for 1 h at room temperature, and the electrode was washed thoroughly for measurement. For amperometric i–t curve to record the amperometric response, a detection potential of −0.4 V was selected. 5 mM H₂O₂ was added into the PBS after the back ground current was stabilized.

Fig. 1 The schematic diagram of the sandwich-type electrochemical immunosensor.

3. Results and discussion

3.1. Design and preparation of 3D flower-like nanomaterials for SEM and TEM characterization

A well-defined 3D flower-like spherical nanostructure can be observed in the SEM image of the ZnO nanoparticles (Fig. 2A). The 3D ZnO NFs are relatively monodisperse in size and are around 2–4 μm in diameter. A high-magnification SEM image of one ZnO nanoflower is shown in Fig. 2B. The ZnO nanoflower is actually composed of a random growth of nanosheets that can be bent and connected with each other. Fig. 2C gives a TEM image of 3D ZnO NFs. It reveals that the nanosheets can indeed extend from outward far into the interior. This unique 3D flower-like nanostructure provides a higher surface area than similar-sized ZnO nanomaterials, which is beneficial for the capture of protein. Fig. 2D displays a TEM image of 3D AuPd NFs. The synthesized AuPd nanoparticles have a flower-like shape with the average diameter around 30 nm. The special 3D flower-like nanostructure has a higher surface area than spherical AuPd nanoparticles, which is beneficial for the electrocatalysis of H₂O₂.

Fig. 2 SEM images of 3D ZnO NFs (A and B); TEM images of 3D ZnO NFs (C) and 3D AuPd NFs (D).

3.2. Electrochemical characterization

In order to investigate the signal amplification strategy of the 3D AuPd NFs, amperometric i–t curve method was employed to characterize the electrocatalytic performances of different nanomaterials towards the reduction of H₂O₂. It can be seen in the Fig. 3A that either NH₂–ZnO (curve a) or Au nanoparticles (curve b) modified electrode has no obvious electrocatalytic performance. Therefore, the electrocatalytic current of NH₂–ZnO could be ignored in the detection process of the designed immunosensor. However, the Pd nanoparticles (curve c) modified electrode exhibits an electrocatalytic current of about 30 μA. As expected, the electrocatalytic current of the 3D AuPd NFs (curve e) modified electrode increases to about 120 μA. The high electrocatalytic performance can mainly be ascribed to the special nanostructure of the 3D AuPd NFs. It also indicates that a good synergic effect between the Au and Pd has a positive effect on the electrocatalytic performance of the 3D AuPd NFs. In conclusion, the signal amplification strategy is based on the good electrocatalytic performance of the 3D AuPd NFs.

Fig. 3 (A) Electrocatalytic current responses towards the reduction of 5 mM H₂O₂ of NH₂–ZnO (curve a), Au nanoparticles (curve b), Pd nanoparticles (curve c) and 3D AuPd NFs (curve d) in PBS at pH 6.8; (B) CVs of the 2 mg mL⁻¹ 3D AuPd NFs modified electrode before (curve a) and after (curve b) the addition of 5 mM H₂O₂; (C) effect of pH on the electrocatalytic current responses towards the reduction of 5 mM H₂O₂; (D) effect of concentration of 3D AuPd NFs on the electrocatalytic current responses towards the reduction of 5 mM H₂O₂. Error bar = RSD (n = 5).

In order to investigate the electrocatalytic mechanism of 3D AuPd NFs towards the reduction of H₂O₂, cyclic voltammetry was employed to further characterize the electrocatalytic performance. Fig. 3B displays cyclic voltammograms (CVs) of the 2 mg mL⁻¹ 3D AuPd NFs modified electrode before and after the addition of 5 mM H₂O₂ in PBS at pH = 6.8. Before the addition of H₂O₂ (curve a), CV of the 2 mg mL⁻¹ 3D AuPd NFs modified electrode exhibits no apparent redox activity. After the addition of H₂O₂ (curve b), a dramatic increase of the reduction current is observed, demonstrating the good electrocatalytic performance of 3D AuPd NFs towards the reduction of H₂O₂. According to the literature,^43,44 the mechanism for H₂O₂ electroreduction can be expressed as following:

H₂O₂ + e⁻ → OH_ad + OH⁻ (1)

OH_ad + e⁻ → OH⁻ (2)

2OH⁻ + 2H⁺ → 2H₂O (3)

In order to achieve an optimal electrochemical signal, optimizations of experimental conditions are necessary. The pH value of PBS and the concentration of 3D AuPd NFs influence the electrocatalytic process of 3D AuPd NFs towards the reduction of H₂O₂. Fig. 3C shows the different electrocatalytic current responses of the electrode modified with 2 mg mL⁻¹ 3D AuPd NFs in different pH values of PBS. Fig. 3D shows the different electrocatalytic current responses of the electrode modified with different concentrations of 3D AuPd NFs in PBS at pH = 6.8. As shown in these figures, the optimal amperometric response was achieved at a pH of 6.8 and at a concentration of 2 mg mL⁻¹. Therefore, PBS at pH = 6.8 and 2 mg mL⁻¹ 3D AuPd NFs were selected for the test throughout this study.

3.3. Characterization of the immunosensor

A.C. impedance method was employed to characterize the fabrication process of the sandwich-type electrochemical immunosensor. Nyquist plots of the A.C. impedance method was recorded from 1 to 10⁵ Hz at 0.19 V in a solution containing 0.1 M KCl and 2.5 mM Fe(CN)₆³⁻/Fe(CN)₆⁴⁻. Nyquist plots are consist of two portions. The linear portion at low frequencies is associated with electrochemical behavior limited by diffusion. The semicircle portion at high frequencies is associated with the electrochemical process subject to electron transfer, where the diameter corresponds to the resistance. Simply, resistance change could be judged by observing the diameter change of semicircle portion. Thus, A.C. impedance is a suitable method for monitoring the changes in the surface features during the fabrication process.⁴⁵ As shown in the Fig. 4, it can be observed that the bare GCE exhibits a very small resistance (curve a), which is characteristic of a diffusion-limiting step in the electrochemical process. After the modification of NH₂–ZnO, the electrode shows an increasing resistance (curve b), implying that the successful immobilization of NH₂–ZnO on the surface of bare GCE. The gradually increasing resistance of electrodes further modified with Ab₁ (curve c), BSA (curve d) and CEA (curve e) indicates the successful immobilization of the non-conductive bioactive substances. After the modification of AuPd@Ab₂, the electrode shows a decreasing resistance (curve f), implying that the successful specific recognition between AuPd@Ab₂ and CEA. It also indicates that 3D AuPd NFs can not only offer a biocompatible surface for the capture of protein but also provide a sensitive interface to accelerate the electron transfer.

Fig. 4 Nyquist plots of the A.C. impedance method: bare GCE (a), NH₂–ZnO/GCE (b), Ab₁/NH₂–ZnO/GCE (c), BSA/Ab₁/NH₂–ZnO/GCE (d), CEA/BSA/Ab₁/NH₂–ZnO/GCE (e) and AuPd@Ab₂/CEA/BSA/Ab₁/NH₂–ZnO/GCE (f).

Under optimal conditions, the sandwich-type electrochemical immunosensor was employed to detect different concentrations of CEA. Fig. 5A shows the electrocatalytic current responses of the designed immunosensor for the detection of CEA covering the concentration range from 10⁻⁵ ng mL⁻¹ (curve a) to 10² ng mL⁻¹ (curve h). Fig. 5B shows a linear relationship between electrocatalytic current responses and the logarithmic values of CEA concentration. When the concentration of CEA increased at high concentration range, the increase rate of electrocatalytic current response slowed down, due to steric hindrance or saturation of couple antigen molecules.^46,47 Therefore, the electrocatalytic current responses have a linear relationship with the logarithmic values of CEA concentration. And the linear regression equation of the calibration curve was I = 6.04 log C + 36.36 with correlation coefficient of 0.99. The low detection limit of 3.2 fg mL⁻¹ was obtained, which was ascribed to the novel signal amplification strategy based on 3D flower-like nanomaterials in the fabrication of the designed immunosensor.

Fig. 5 (A) Electrocatalytic current responses of the immunosensor for the detection of different concentrations of CEA: 10⁻⁵ ng mL⁻¹ (a), 10⁻⁴ ng mL⁻¹ (b), 10⁻³ ng mL⁻¹ (c), 10⁻² ng mL⁻¹ (d), 0.1 ng mL⁻¹ (e), 1 ng mL⁻¹ (f), 10 ng mL⁻¹ (g) and 100 ng mL⁻¹ (h); (B) calibration curve of the immunosensor for the detection of different concentrations of CEA. Error bar = RSD (n = 5).

3.4. Reproducibility, selectivity and stability

To evaluate the reproducibility of the sandwich-type electrochemical immunosensor, a series of five electrodes were prepared for the detection of 1 ng mL⁻¹ CEA. The relative standard deviation (RSD) of the measurements for the five electrodes was less than 5% (Fig. 6A), suggesting the precision and reproducibility of the designed immunosensor was quite good.

Fig. 6 (A) Electrochemical signal responses of the immunosensor fabricated on five different electrodes for the detection of 1 ng mL⁻¹ CEA; (B) electrocatalytic current responses of the immunosensor for the detection of 1 ng mL⁻¹ CEA + 100 ng mL⁻¹ AFP, 100 ng mL⁻¹ AFP, 1 ng mL⁻¹ CEA + 100 ng mL⁻¹ PSA, 100 ng mL⁻¹ PSA, 1 ng mL⁻¹ CEA + 100 ng mL⁻¹ IgG, 100 ng mL⁻¹ IgG, 1 ng mL⁻¹ CEA + 100 ng mL⁻¹ BSA, 100 ng mL⁻¹ BSA and 1 ng mL⁻¹ CEA; (C) electrochemical signal responses of the immunosensor for the detection of 10 ng mL⁻¹ CEA after different weeks. Error bar = RSD (n = 5).

To investigate the specificity of the sandwich-type electrochemical immunosensor, interference study was performed by using alpha fetoprotein (AFP), prostate specific antigen (PSA), human immunoglobulin (IgG) and BSA. 100 ng mL⁻¹ interfering substances with and without 1 ng mL⁻¹ CEA solution was measured by the immunosensor. As indicated from Fig. 6B, samples with the CEA had significantly higher electrocatalytic current responses than those without CEA. The electrocatalytic current response variation of samples with interference and CEA was less than 5% of that only CEA, indicating the selectivity of the designed immunosensor was good.

To test the stability of the sandwich-type electrochemical immunosensor, it was stored at 4 °C when not in use. After one month, the electrocatalytic current response change was less than 5% for the detection of 10 ng mL⁻¹ CEA (Fig. 6C). The good stability of the designed immunosensor can be ascribed to the good biocompatibility of the 3D ZnO NFs and 3D AuPd NFs. The reproducibility, selectivity and stability of the designed immunosensor were all acceptable, thus it was suitable for quantitative detection of CEA in real human samples.

3.5. Real sample analysis

In order to validate the sandwich-type electrochemical immunosensor, a comparative experiment with the commercialized available ELISA method for the detection of CEA in human serum sample was conducted (Table 1). The relative error between the two methods was in the range from −4.0% to 4.7%. These data revealed a good agreement between the two analytical methods, indicating the feasibility of the designed immunosensor for clinical application.

Table 1 Human serum sample analysis using the designed method and the ELISA method

Sample	This method^a (ng mL^−1)	ELISA^a (ng mL^−1)	Relative error (%)
a Each value is the average of five measurements.
1	0.51	0.49	4.1
2	2.4	2.5	−4.0
3	4.5	4.3	4.7

---

4. Conclusions

This work has developed a novel and ultrasensitive sandwich-type electrochemical immunosensor for the quantitative detection of CEA. 3D flower-like nanomaterials was employed as the matrix and the label simultaneously to achieve the facile and sensitive fabrication of the immunosensor. The designed immunosensor can also be applied in clinical detection of other tumor markers. Compared with traditional immunosensors, immunosensors based on nanomaterials with special nanostructures and simple preparation methods would occupy more advantages in clinical applications in future. Different kinds of 3D nanomaterials with simple preparation methods may be developed and employed in the fabrication of immunosensor.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (No. 21175057, 21375047, 21377046, 21575050 and 21505051), the Science and Technology Plan Project of Jinan (No. 201307010), the Science and Technology Development Plan of Shandong Province (No. 2014GSF120004), the Special Project for Independent Innovation and Achievements Transformation of Shandong Province (No. 2014ZZCX05101), and QW thanks the Special Foundation for Taishan Scholar Professorship of Shandong Province (No. ts20130937) and UJN.

References

1. P. Li, B. Zhang and T. Cui, Biosens. Bioelectron., 2015, 72, 168–174.
2. X. Jia, Z. Liu, N. Liu and Z. Ma, Biosens. Bioelectron., 2014, 53, 160–166.
3. S. Du, Z. Guo, B. Chen, Y. Sha, X. Jiang, X. Li, N. Gan and S. Wang, Biosens. Bioelectron., 2014, 53, 135–141.
4. Z. Guo, T. Hao, S. Du, B. Chen, Z. Wang, X. Li and S. Wang, Biosens. Bioelectron., 2013, 44, 101–107.
5. C. Wang, F. Hou and Y. Ma, Biosens. Bioelectron., 2015, 68, 156–162.
6. S. Cheng, S. Hideshima, S. Kuroiwa, T. Nakanishi and T. Osaka, Sens. Actuators, B, 2015, 212, 329–334.
7. B. Rapp, F. Gruhl and K. Länge, Anal. Bioanal. Chem., 2010, 398, 2403–2412.
8. Z.-M. Zhou, Z. Feng, J. Zhou, B.-Y. Fang, X.-X. Qi, Z.-Y. Ma, B. Liu, Y.-D. Zhao and X.-B. Hu, Biosens. Bioelectron., 2015, 64, 493–498.
9. B. Passelecq, M. de Bo, C. Huber, J. P. Gennart, A. Bernard and R. Lauwerys, J. Immunol. Methods, 1988, 109, 69–74.
10. S. H. Chan, S. H. Heng and M. J. Simons, J. Immunol. Methods, 1979, 29, 191–196.
11. E. P. Diamandis, T. K. Christopoulos and C. C. Bean, J. Immunol. Methods, 1992, 147, 251–259.
12. G. I. Abelev, E. R. Karamova, N. L. Lazarevich, V. I. Kiseleva and A. M. Poverenny, Immunol. Lett., 1994, 40, 133–138.
13. W. Brummund, D. A. Aryan, M. T. Mennuti and N. A. Starkovsky, Clin. Chim. Acta, 1980, 105, 25–40.
14. S. Aoyagi, M. Kusumi, A. Matsuyuki, M. Maeda and A. Tsuji, J. Immunol. Methods, 1991, 137, 73–78.
15. I. A. Darwish, T. A. Wani, A. M. Alanazi, M. A. Hamidaddin and S. Zargar, Talanta, 2013, 111, 13–19.
16. X. Yang, Y. Zhuo, S. Zhu, Y. Luo, Y. Feng and Y. Xu, Biosens. Bioelectron., 2015, 64, 345–351.
17. H. Li, J. He, S. Li and A. P. F. Turner, Biosens. Bioelectron., 2013, 43, 25–29.
18. J. Narayanan, M. K. Sharma, S. Ponmariappan, Sarita, M. Shaik and S. Upadhyay, Biosens. Bioelectron., 2015, 69, 249–256.
19. J. Liu, G. Lin, C. Xiao, Y. Xue, A. Yang, H. Ren, W. Lu, H. Zhao, X. Li and Z. Yuan, Biosens. Bioelectron., 2015, 71, 82–87.
20. Z.-H. Yang, Y. Zhuo, R. Yuan and Y.-Q. Chai, Biosens. Bioelectron., 2015, 69, 321–327.
21. M. K. Sharma, J. Narayanan, S. Upadhyay and A. K. Goel, Biosens. Bioelectron., 2015, 74, 299–304.
22. S. Zhang, H. Ma, L. Yan, W. Cao, T. Yan, Q. Wei and B. Du, Biosens. Bioelectron., 2014, 59, 335–341.
23. H. Ilkhani, M. Sarparast, A. Noori, S. Zahra Bathaie and M. F. Mousavi, Biosens. Bioelectron., 2015, 74, 491–497.
24. M. Pandiaraj, N. K. Sethy, K. Bhargava, V. Kameswararao and C. Karunakaran, Biosens. Bioelectron., 2014, 54, 115–121.
25. N. Xia, D. Deng, L. Zhang, B. Yuan, M. Jing, J. Du and L. Liu, Biosens. Bioelectron., 2013, 43, 155–159.
26. J. Zhou, J. Tang, G. Chen and D. Tang, Biosens. Bioelectron., 2014, 54, 323–328.
27. G.-H. Yang, J.-J. Shi, S. Wang, W.-W. Xiong, L.-P. Jiang, C. Burda and J.-J. Zhu, Chem. Commun., 2013, 49, 10757–10759.
28. J. Liu, C.-Y. Lu, H. Zhou, J.-J. Xu, Z.-H. Wang and H.-Y. Chen, Chem. Commun., 2013, 49, 6602–6604.
29. Q. Wang, Y. Song, Y. Chai, G. Pan, T. Li, Y. Yuan and R. Yuan, Biosens. Bioelectron., 2014, 60, 118–123.
30. J. Zhang, B. P. Ting, M. Khan, M. C. Pearce, Y. Yang, Z. Gao and J. Y. Ying, Biosens. Bioelectron., 2010, 26, 418–423.
31. F. Li, J. Han, L. Jiang, Y. Wang, Y. Li, Y. Dong and Q. Wei, Biosens. Bioelectron., 2015, 68, 626–632.
32. J. Han, Y. Zhuo, Y. Chai, Y. Xiang, R. Yuan, Y. Yuan and N. Liao, Biosens. Bioelectron., 2013, 41, 116–122.
33. Q. Wang, Y. Song, Y. Chai, G. Pan, T. Li, Y. Yuan and R. Yuan, Biosens. Bioelectron., 2014, 60, 118–123.
34. N. Li, H. Ma, W. Cao, D. Wu, T. Yan, B. Du and Q. Wei, Biosens. Bioelectron., 2015, 74, 786–791.
35. Z. He, S. Zang, Y. Liu, Y. He and H. Lei, Biosens. Bioelectron., 2015, 73, 85–92.
36. B. Kavosi, A. Salimi, R. Hallaj and F. Moradi, Biosens. Bioelectron., 2015, 74, 915–923.
37. G. Sun, Y.-N. Ding, C. Ma, Y. Zhang, S. Ge, J. Yu and X. Song, Electrochim. Acta, 2014, 147, 650–656.
38. C. Mouli Pandey, G. Sumana and I. Tiwari, Biosens. Bioelectron., 2014, 61, 328–335.
39. P. V. Suneesh, V. Sara Vargis, T. Ramachandran, B. G. Nair and T. G. Satheesh Babu, Sens. Actuators, B, 2015, 215, 337–344.
40. C.-L. Kuo, T.-J. Kuo and M. H. Huang, J. Phys. Chem. B, 2005, 109, 20115–20121.
41. J. Han, Z. Zhou, Y. Yin, X. Luo, J. Li, H. Zhang and B. Yang, CrystEngComm, 2012, 14, 7036–7042.
42. A. Cauvel, G. Renard and D. Brunel, J. Org. Chem., 1997, 62, 749–751.
43. Y. Zhou, G. Yu, F. Chang, B. Hu and C.-J. Zhong, Anal. Chim. Acta, 2012, 757, 56–62.
44. F. Meng, X. Yan, J. Liu, J. Gu and Z. Zou, Electrochim. Acta, 2011, 56, 4657–4662.
45. W. Lu, X. Cao, L. Tao, J. Ge, J. Dong and W. Qian, Biosens. Bioelectron., 2014, 57, 219–225.
46. K. Wang, J. Liao, X. Yang, M. Zhao, M. Chen, W. Yao, W. Tan and X. Lan, Biosens. Bioelectron., 2015, 63, 172–177.
47. J. Huang, G. Yang, W. Meng, L. Wu, A. Zhu and X. A. Jiao, Biosens. Bioelectron., 2010, 25, 1204–1211.

---