Efficient electrochemiluminescence quenching of carbon-coated petalous CdS nanoparticles for an ultrasensitive tumor marker assay through coreactant consumption by G-quadruplex-hemin decorated Au nanorods

Abstract

A novel competitive electrochemiluminescence (ECL) aptasensor was designed for the detection of carcinoembryonic antigen (CEA) using carbon-coated petalous CdS nanopaticles (CdS–C petalous nanoparticles) as an ECL emitter and DNAzyme/Au nanorods–complementary DNA as a quenching probe. The quenching probe was firstly prepared by assembling guanine (G)-rich ssDNA and cDNA on Au nanorods and then reacting with hemin to form hemin/G-quadruplex DNAzyme units. CdS–C nanoparticles were synthesized and employed as the matrix for the construction of the CdS–C/Chit/aptamer platform. In the absence of CEA, the DNAzyme/Au nanorods as the quenching probe can be introduced by hybridization with aptamer on the surface of the sensing platform. In this state, DNAzyme immobilized on the probe catalyzes the reduction of H₂O₂, producing a decreased ECL emission. Upon both CEA and quenching probe addition, competitive reaction of the quenching probe and CEA with capture aptamer immobilized on the electrode occurred and thus resulted in the decreased amount of quenching probe on the electrode, which decreased the consumption of H₂O₂, producing an increased ECL signal. Based on this strategy, the aptasensor enables the sensitive detection of CEA in a range of 0.1 pg mL⁻¹ to 0.5 ng mL⁻¹ with a detection limit of 0.036 pg mL⁻¹. The limit of quantification in human serum samples was experimentally found to be 0.21 pg mL⁻¹. Moreover, the application of the aptasensor was demonstrated in the analysis of CEA in human serum samples with recoveries of 88.2–106%. The proposed method holds great promise in the highly sensitive and selective detection of CEA in biological samples.

---

Introduction

Specific and sensitive detection of biomarkers holds great promise for early disease diagnosis, for guidance for personalized therapy, and for drug development. Various analytical technologies including Raman spectroscopy,¹ radioimmunoassay,² enzyme-linked immunosorbent assay,³ fluorescence,⁴ and electrochemiluminescence (ECL)⁵ techniques have been developed for the detection of biomarkers, especially proteins. Owing to its intrinsic sensitivity, simplified setup, excellent temporal and spatial controllability, the electrochemiluminescence technique (ECL) has attracted considerable attention in biomarker analysis. Up to now, three types of ECL luminophores have been exploited including inorganic and organic molecules and semiconductor nanocrystals (NCs).^6,7

Compared with molecular luminophores, NCs have been widely used as ECL emitters for the construction of ECL biosensors due to their unique features size or surface-trap controlled luminescence and stability. The NCs-based ECL emission is usually cathodic emission in the presence coreactants such as hydrogen peroxide, oxygen, or peroxydisulfate.^8,9 Cadmium sulfide (CdS), one of the most technologically important semiconductor materials, has been extensively investigated in the construction of ECL sensors.^10–12 However, it has been reported that cadmium-based NCs pose risks to human health and the environment associated with the toxicity of leached Cd²⁺ ions from them, which will limit their biological applications. Coating CdS NCs with cadmium free materials such as ZnS and polymer coatings has been prepared to repress the Cd²⁺ release.^13–17 Recently, Hu and coworkers¹⁸ prepared the carbon layer coated CdS petalous nanostructures coating with facile one-pot method. The carbon layer not only reduces the amount of surface traps of CdS NCs, but also inhibits the leaching of free Cd²⁺ ions via degradation. To the best of our knowledge, the ECL behavior of the CdS–C composites has not been explored so far and the low toxic CdS–C might provide an ingenious alternative for the fabrication of ECL biosensors.

Although great advantages of the ECL methods have been manifested, the increasing demand for early, specific, and ultrasensitive detection of biomarker is further pushing the enhancement of the detection sensitivity. Recently, some interesting ECL biosensors have been proposed with the use of different enzyme labels, such as horseradish peroxidase (HRP) and glucose oxidase (GOD).^19,20 In such analytical systems, the enzyme-labeling could enhance the sensitivity by using nanocarriers. However, the conformation and specific substrate of these natural enzymes greatly influence their catalytic activity. In addition, the loading capacity of these enzymes on the nanocarriers is limited by the relatively large occupied volume of them. Enzyme like mimics as small molecule complex provide effective alternatives for the enzyeme-labeling. DNAzymes as a kind of artificial enzyme have gradually achieved applications in biosensing due to their high catalytic activity and good stability. G-quadruplex-hemin DNAzyme with HRP-like activity, shows excellent catalytic activity to H₂O₂ and has been used as for ultrasensitive DNA biosensing.²¹ The G-quadruplex-hemin DNAzyme shows great promising in the fabrication of sensitive ECL biosensor as a novel biocatalyst.

Herein, a novel competitive ECL strategy was designed for protein detection by using carbon-coated petalous CdS nanopaticles (CdS–C petalous nanoparticles) as ECL emitter and DNAzyme decorated Au nanorod as quenching probe. CdS–C petalous nanoparticles were prepared and coated on the electrode as a platform both for strong and stable ECL emitting and subsequent aptamer immobilization. The quenching probe showed sensitive ECL quenching efficiency of CdS–C due to the consumption of H₂O₂, the ECL coreactant, by its electrocatalytic reduction. Carcinoembryonic antigen (CEA), which has been identified as a critical biomarker in the clinical diagnosis for breast tumors, colon tumors, lung cancer and other cancers, was chosen as a model analyte. In the presence of CEA, a competitive interaction of the CEA and the quenching probe with the aptamer on the electrode occurred and thus an ECL increase appeared compared to the electrode only with quenching probe incubation. Under the optimum conditions, the as-prepared aptasensor exhibits excellent performance for CEA detection. Finally the aptasensor was utilized in the determination of CEA in human serum samples.

Experimental

Materials and reagents

Hemin was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Polyvinylpyrrolidone (PVP), Chitosan (CS), human serum albumin (HSA), human IgG (hIgG), and bovine serum albumin (BSA) were from Shanghai Solarbio Bioscience & Technology Co., Ltd (see bio Biotechnology). CEA was donated as gift by Ms Yan-Li Zheng from Zhengzhou Immuno Biotech Co., Ltd (Zhengzhou, China). Hydrogen tetrachloroaurate (III) trihydrate (HAuCl₄·3H₂O) and ascorbic acid (AA) were from Alfa Aesar (Ward Hill, MA). 0.1 mol L⁻¹ phosphate buffer saline (PBS) as the detection solution was prepared with 0.1 mol L⁻¹ Na₂HPO₄, 0.1 mol L⁻¹ NaH₂PO₄ and 0.1 mol L⁻¹ KNO₃. The DNA oligonucleotides were synthesized by Shanghai Sangon Biotechnology Co. Ltd (China). The sequences are listed as follows:

CEA binding aptamer (NH₂-APT): 5′-NH₂–(CH₂)₆-TTT TAT ACC AGC TTA TTC AAT T-3′, complementary DNA (cDNA): 5′-SH–(CH₂)₆-TTT TAA TTG AAT A-3′, ssDNA: 5′-SH-TTT TTT TTT GGG TTG GGC GGG ATG GG-3′.

Apparatus

The ECL measurement was carried out on a model BPCL ultraweak luminescence analyzer (Institute of Biophysics, Chinese Academy of Science, Beijing, China). A RST5200 electrochemical workstation (Zhengzhou Shiruisi Technology Co., Ltd) was used to record cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). A conventional three-electrode system was used with a glass carbon electrode (GCE, Φ = 3 mm) as the working electrode, an Ag/AgCl (sat. KCl) as the reference electrode and a platinum electrode as the counter electrode. Transmission electron microscopy (TEM) images were obtained with a Tecnai G2 F20 transmission electron microscope (FEI Co., USA). The scanning electron micrographs were taken with scanning electron microscope (SEM, S-4800, Hitachi). X-ray powder diffraction (XRD) patterns were performed on a RigakuD/Maxr-A X-ray diffractometer (Tokyo, Japan).

Preparation of DNAzyme/Au nanorods quenching probe

Au nanorods were synthesized based on the silver ion-assisted seed-mediated protocol according to the previous literature.²² Firstly, the seed solution was prepared by mixing 0.25 mL 0.01 mol L⁻¹ HAuCl₄ and 10 mL 0.1 mol L⁻¹ CTAB. 0.6 mL 0.01 mol L⁻¹ fresh, ice-cold NaBH₄ was then injected to the mixture under vigorous stirring for 2 min. The seed solution was kept at room temperature for 2 h. Secondly, 2 mL 0.01 mol L⁻¹ HAuCl₄ and 0.1 mL 0.01 mol L⁻¹ AgNO₃ were added into 40 mL 0.1 mol L⁻¹ CTAB, followed by the addition of 0.8 mL 1.0 mol L⁻¹ HCl. After gentle mixing of the solution, 0.32 mL 0.1 mol L⁻¹ AA was added to the mixture to form a growth solution. Thirdly, 0.15 mL of the seed solution was added to the growth solution, and the solution was gently mixed for 5 min and left at 30 °C for 2 h. Finally, the Au nanorods were obtained by removing the excess CTAB by centrifugation.

The DNAzyme/Au nanorods quenching probe was prepared following a previous report.²³ As shown in Scheme 1A, cDNA and ssDNA were added into 1 mL Au nanorods solution and incubated for 16 h with stirring at room temperature, followed by a salt-stabilization in 0.1 mol L⁻¹ NaCl. Then, 0.01 mol L⁻¹ PBS containing 0.1 mol L⁻¹ KCl was dropped into the solution. After stirring for 2 h, the excess of DNA was removed by centrifugation and re-dispersed in PBS. Excess amount of hemin was added to the resulting solution, and incubated for 1.5 h in dark at 4 °C. After centrifuged to remove the unreacted hemin, the obtained probe was re-dispersed in 0.01 mol L⁻¹ pH 7.4 PBS containing 0.1 mol L⁻¹ KCl and stored at 4 °C.

Scheme 1 Schematic illustration of the ECL aptasensor fabrication process.

Preparation of ECL aptasensor and detection procedure

The flower-like CdS–C composites were synthesized by means of a one-pot solvothermal method described elsewhere.¹⁸ 3.5 mmol L⁻¹ CdCl₂·2.5H₂O, 3.5 mmol L⁻¹ thiourea, 0.389 g PVP and 0.75 g glucose were dissolved in ethylene glycol with the assistance of sonication. Then, the mixture was transferred into a 50 mL Teflon and kept at 160 °C for 12 h. After cooling, the carbon-coated CdS particles were collected by centrifugation, washed with ethanol and water, and finally dried in air at 80 °C overnight.

CdS–C/chit composites were prepared by mixing CdS–C and chitosan in acetic acid solution and then 5 μL CdS–C/chit composites were dropped onto the surface of GCE electrode. After drying in air, 5 μL glutaraldehyde was pipetted on the electrode. Thereafter, CEA aptamer was coated on the electrode by an overnight incubation at 4 °C. The resulting electrode was washed with 0.01 mol L⁻¹ PBS for removing the unbound aptamer and then blocked with BSA to form the ECL aptasensor.

To carry out the ECL measurements, the sensor was dipped into a mixture solution containing CEA and probe at 37 °C for 1 h followed by washing with 0.01 mol L⁻¹ PBS. ECL signals were acquired in CV mode with continuous potential scanning from −1.7 V to 0 V at a scanning rate of 100 mV s⁻¹ applied to achieve ECL signals in 0.1 mol L⁻¹ PBS containing H₂O₂.

Results and discussion

Characterization of CdS–C nanocomposites

SEM image of the as-synthesized CdS–C product is shown in Fig. 1A. The nanospheres are made up of petal-like units with the size of about 300 nm. The morphology and structure are further elucidated by TEM. From Fig. 1B, the flower-like nanostructures can be observed, which are in good agreement with the SEM observation. Fig. 1C shows a high-resolution TEM image of an individual particle. It can be seen that the CdS particle is coated with carbon layer of about 5.6 nm in thickness. The phase and structure of the CdS–C nanoparticles were examined by XRD. As shown in Fig. 1D, all of the diffraction peaks of the CdS–C nanoparticles match well with those of the hexagonal CdS phase. The morphology of Au nanorods was shown in Fig. 1E. It can be seen that the average aspects ratio of Au nanorods was 2.4 : 1.

Fig. 1 SEM (A), TEM (B), HRTEM (C) images, and XRD pattern (D) of CdS–C petalous nanoparticles. (E) TEM image of Au nanorods.

Characterization of the aptasensor

The fabrication process for the ECL aptasensor was monitored by CV and EIS. The sensitive redox couple, [Fe(CN)₆]³⁻/⁴⁻, was employed for electrochemical measurements toward the sensing electrodes at different stages. As shown in Fig. 2A, a couple of reversible redox peaks of [Fe(CN)₆]³⁻/⁴⁻ was observed on the bare GCE (curve a). When CdS–C/chit nanocomposites were modified onto the electrode, the peak current (curve b) responses suffer sharply decreased due to low conductivity of CdS–C/chit. After the assembly of the aptamer, the current further decreased (curve c) due to the repellence of negatively charged aptamer on the electrode to the [Fe(CN)₆]³⁻/⁴⁻, suggesting the successful immobilization of the aptamer layer on the electrode surface. Subsequently, the modified electrode was treated with BSA to block nonspecific sites, a further decrease of current (curve d) was observed. In case of quenching probe (curve e) was immobilized in the electrode, the CV responses continuously declined. The EIS data in Fig. 2B coincide with the results of the CV test, which shows the successful fabrication of the aptasensor.

Fig. 2 CV (A) and EIS (B) of the bare GCE (a), CdS–C/chit/GCE (b), aptamer/CdS–C/chit/GCE (c), BSA/aptamer/CdS–C/chit/GCE (d), probe/BSA/aptamer/CdS–C/chit/GCE (e). (C) ECL behaviors of the bare GCE (a), CdS–C/chit/GCE (b), aptamer/CdS–C/chit/GCE (c), probe/BSA/aptamer/CdS–C/chit/GCE (d), CEA + probe/BSA/aptamer/CdS–C/chit/GCE (e).

ECL behaviors at the self-assembly process were also investigated. As shown in Fig. 2C, the bare GCE has weak ECL signal (curve a). The CdS–C/chit/GCE showed a strong ECL emission in the presence of coreactant H₂O₂, thanks to the excellent ECL property of the CdS–C nanocomposites, which could be attributed to the large surface area of the petalous nanostructures and the perfect chemical protection of the carbon layer to the inner CdS nanoparticles.¹⁸ Compared with other many Cd semiconductor materials such as CdS, CdSe, CdTe, the prepared CdS–C petalous nanoparticles could inhibit the leaching of free Cd²⁺ ions. This environmentally friendly and nontoxic nanoparticle-based ECL luminophores is beneficial to bioanalysis. Then the ECL intensity decreased with the immobilization of aptamer (curve c). In the presence of DNAzyme/Au nanorods quenching probe (curve d), the electrode showed an obvious decrease in ECL intensity, owing to the fact that the introduction of the DNAzyme could deplete the coreactant, i.e. H₂O₂, in the detection solution. While the electrode was incubated with the mixture solution containing quenching probe and target CEA, compared with the electrode only with quenching probe incubation, the electrode incubation with the mixture containing quenching probe and target CEA produced an increased ECL signal, which could be ascribed to the decreased consumption of H₂O₂ by quenching probe owing to the occupancy of some aptamer binding sites by CEA (curve e). The principle of ECL emission and quenching mechanism could be described as follows:

CdS–C + e⁻ → CdS–C⁻˙ (1)

H₂O₂ + e⁻ → OH⁻ + ˙OH (2)

CdS–C⁻˙ + ˙OH → CdS–C* + OH⁻ or (3)

2CdS–C⁻˙ + H₂O₂ → 2CdS–C* + 2OH⁻ (4)

2DNAzyme + H₂O₂ → 2OH⁻ + 2DNAzyme⁺ (5)

CdS–C* → CdS–C* + hν (6)

Analytical performance

Under the optimum conditions, the ECL intensity of the aptasensor in response to CEA of various concentrations was tested (Fig. 3A). As can be seen, a good linear relationship between the ECL intensity and the logarithmic value of CEA concentration ranged from 0.1 pg mL⁻¹ to 0.5 ng mL⁻¹ was observed (Fig. 3B). The regression equation was I = 15 857.3 + 2986.4 log C_CEA (ng mL⁻¹), with the correlation coefficient of 0.996. The limit of detection was experimentally found to be 0.036 pg mL⁻¹. Compared with other CEA assays reported in the literatures^24–28 (Table 1), the proposed ECL aptasensor had a relative large linear range and low detection limit. The high sensitivity could be attributed to the following factors: (i) the petalous CdS–C nanomaterials possess excellent ECL performance and large surface area; (ii) the DNAzyme/Au nanorod complex as effective ECL quenching probe efficiently magnifies the detection signal. In addition, the limits of quantitation (LOQs) of CEA in human serum have also been determined and the LOQ for CEA was experimentally found to be 0.21 pg mL⁻¹.

Fig. 3 (A) ECL-time curves of the aptasensor for CEA detection at different concentrations (0.1, 0.5, 1, 5, 10 and 50 pg mL⁻¹ and 0.1, 0.5 ng mL⁻¹) in 0.1 M pH 8.0 PBS containing 30 mM H₂O₂. (B) The linear calibration for CEA detection.

Table 1 Comparison of the different methods for CEA detection

Analytical methods	Linear range (ng mL^−1)	Detection limit (ng mL^−1)	Ref.
Electrochemical immunosensor	0.001–50	0.0003	24
Flow-injection ECL	0.01–50	0.006	25
Photoelectrochemical immunosensor	0.05–20	0.01	26
Light scattering assay	0.1–60	0.03	27
Time resolved fluorescence immunoassay	1–1000	0.5	28
ECL aptasensor	0.0001–0.5	0.000036	This work

---

Specificity and stability of the ECL aptasensor

To test the specificity, the proposed aptasensor was incubated with some possible interfering proteins. Fig. 4A shows the ECL signal of the electrode after incubation with blank, 0.6 mmol L⁻¹ HSA, 50 μmol L⁻¹ IgG, 0.01 ng mL⁻¹ CEA and the mixture containing HSA, IgG and CEA, respectively. As can be seen, the ECL values for interfering proteins were much smaller than those for CEA. Furthermore, the presence of IgG and high concentration of HSA did not interfere with the assay for CEA. The results clearly suggested that the proposed aptasensor could be utilized as a selective platform for the detection of the target molecule. The stability of the aptasensor is shown in Fig. 4B under continuous cyclic potential scans for 21 cycles. The RSD was less than 1.0%, showing that the aptasensor exhibits good stability.

Fig. 4 (A) The specificity of the ECL aptasensor towards different targets. (B) The stability of the ECL aptasensor under continuous cyclic scans for 21 cycles. The concentration of the CEA for stability test is 50 pg mL⁻¹. Working solution: 0.1 mol L⁻¹ PBS containing H₂O₂, scan rate: 100 mV s⁻¹, scan range: −1.7 to 0 V.

Analytical applications

To demonstrate the applicability of the proposed method, the contents of CEA in human serum samples collected from Xinyang Central Hospital were determined using the aptasensor and the ROCHE ECL method adopted in Xinyang Central Hospital as the reference method. When the level of CEA was over the calibration range, the serum sample was appropriately diluted with 0.1 M pH 7.4 PBS, prior to assay. The analytical results were listed in Table 2. The agreement of the present protocol with ROCHE ECL assay results implies that the present method is feasible for biological sample analysis. In addition, the recovery experiments were also implemented in human serum sample. Three different concentrations of CEA (0.001, 0.01 and 0.1 ng mL⁻¹) were added to the serum sample with the recoveries of 88.2–106%, indicative of the acceptable accuracy.

Table 2 Analytical results of CEA content in human serum samples

No. of samples	Content of CEA (ng mL^−1)
	This method	Reference method	Relative errors (%)
1	0.14	0.15	−6.7
2	0.37	0.40	−7.5
3	0.42	0.38	10.5
4	1.05	0.98	7.1
5	3.04	2.87	5.9

---

Conclusions

In conclusion, a new and competitive ECL aptasensor for CEA assay has been developed based on the efficient ECL quenching of carbon-coated petalous CdS nanoparticles by G-quadruplex-hemin decorated Au nanorods. The petalous CdS–C composite exhibits high ECL efficiency. G-quadruplex-hemin decorated Au nanorods acted as ECL quenching probe possesses high electrocatalytic activity toward the reduction of H₂O₂ and thus quench the ECL emission efficiently. On the basis of competitive reaction between CEA and the quenching probe, the aptasensor demonstrates an excellent identification and quantitative detection of CEA. The presented strategy shows high sensitivity, wide linear range and low detection limit. The good performance indicates that this method is a new promise for detecting CEA and could be applied to other biological assays.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (21675136, 21405129, 21375114), Plan for Scientific Innovation Talent of Henan Province, State Key Laboratory of Analytical Chemistry for Life Science (SKLACLS1419), and Nan Hu Young Scholar Supporting Program of XYNU.

References

1. H. Chon, S. Lee, S. W. Son, C. H. Oh and J. Choo, Anal. Chem., 2009, 81, 3029–3034.
2. M. A. Virji, D. W. Mercer and R. B. Herberman, Ca-Cancer J. Clin., 1988, 38, 104–126.
3. T. C. Pina, I. T. Zapata, F. C. Hernández, J. B. López, P. P. Paricio and P. M. Hernández, Clin. Chim. Acta, 2001, 305, 27–34.
4. J. Kim, S. Kwon, J.-K. Park and I. Park, Biosens. Bioelectron., 2014, 55, 209–215.
5. Y. Shan, J. J. Xu and H. Y. Chen, Chem. Commun., 2010, 46, 4187–4189.
6. W. J. Miao, Chem. Rev., 2008, 108, 2506–2553.
7. L. Z. Hu and G. B. Xu, Chem. Soc. Rev., 2010, 39, 3275–3304.
8. Z. Liu, W. Qi and G. Xu, Chem. Soc. Rev., 2015, 44, 3117–3142.
9. W. W. Zhao, J. Wang, Y. C. Zhu, J. J. Xu and H. Y. Chen, Anal. Chem., 2015, 87, 9520–9531.
10. Y. Y. Zhang, Q. M. Feng, J. J. Xu and H. Y. Chen, ACS Appl. Mater. Interfaces, 2015, 7, 26307–26314.
11. H. Zhou, T. Han, Q. Wei and S. Zhang, Anal. Chem., 2016, 88, 2976–2983.
12. J. J. Zhang, J. T. Cao, G. F. Shi, Y. M. Liu, Y. H. Chen and S. W. Ren, Talanta, 2015, 141, 158–163.
13. L. Ye, K. T. Yong, L. Liu, I. Roy, R. Hu, J. Zhu, H. Cai, W. C. Law, J. Liu, K. Wang, J. Liu, Y. Liu, Y. Hu, X. Zhang, M. T. Swihart and P. N. Prasad, Nat. Nanotechnol., 2012, 7, 453–458.
14. A. M. Derfus, W. C. W. Chan and S. N. Bhatia, Nano Lett., 2003, 4, 11–18.
15. W. E. Smith, J. Brownell, C. C. White, Z. Afsharinejad, J. Tsai, X. Hu, S. J. Polyak, X. Gao, T. J. Kavanagh and D. L. Eaton, ACS Nano, 2012, 6, 9475–9484.
16. G. F. Shi, J. T. Cao, J. J. Zhang, K. J. Huang, Y. M. Liu, Y. H. Chen and S. W. Ren, Analyst, 2014, 139, 5827–5834.
17. G. F. Shi, J. T. Cao, J. J. Zhang, Y. M. Liu, Y. H. Chen and S. W. Ren, Sens. Actuators, B, 2015, 220, 340–346.
18. Y. Hu, X. H. Gao, L. Yu, Y. R. Wang, J. Q. Ning, S. J. Xu and X. W. Lou, Angew. Chem., Int. Ed., 2013, 52, 5636–5639.
19. H. Wang, R. Yuan, Y. Chai, H. Niu, Y. Cao and H. Liu, Biosens. Bioelectron., 2012, 37, 6–10.
20. X. Jiang, Y. Chai, H. Wang and R. Yuan, Biosens. Bioelectron., 2014, 54, 20–26.
21. H. Zhou, Y. Y. Zhang, J. Liu, J. J. Xu and H. Y. Chen, Chem. Commun., 2013, 49, 2246–2248.
22. Y. Liu, K. Ai, J. Liu, M. Deng, Y. He and L. Lu, Adv. Mater., 2013, 25, 1353–1359.
23. C. Zong, J. Wu, J. Xu, H. Ju and F. Yan, Biosens. Bioelectron., 2013, 43, 372–378.
24. G. Sun, Y. Ding, C. Ma, Y. Zhang, S. Ge, J. Yu and X. Song, Electrochim. Acta, 2014, 147, 650–656.
25. Y. Zhang, W. Y. Liu, S. G. Ge, M. Yan, S. W. Wang, J. H. Yu, N. Q. Li and X. R. Song, Biosens. Bioelectron., 2013, 41, 684–690.
26. W. W. Tu, W. J. Wang, J. P. Li, S. Y. Deng and H. X. Ju, Chem. Commun., 2012, 48, 6535–6537.
27. Z. Chen, Y. Lei, Z. Liang, F. Li, L. Liu, C. Li and F. Chen, Anal. Chim. Acta, 2012, 747, 99–105.
28. J. Y. Hou, T. C. Liu, G. Lin, Z. X. Li, L. P. Zou, M. Li and Y. S. Wu, Anal. Chim. Acta, 2012, 734, 93–98.

---