An amperometric glucose enzyme biosensor based on porous hexagonal boron nitride whiskers decorated with Pt nanoparticles

Abstract

A novel amperometric electrode is fabricated using platinum nanoparticle (Pt NP) decorated porous hexagonal boron nitride (h-BN) whiskers. The BN–Pt composite with a mass ratio of BN whiskers to H₂PtCl₆ of 1 : 2 exhibits a good amperometric response towards the reduction and oxidation of H₂O₂. This feature allows us to apply it as a bioplatform on which glucose oxidase (GOD) is immobilized through a simple adsorption mechanism for the construction of the glucose biosensor. The developed glucose biosensor showed a fast response time (within 5 s), high sensitivity (6.37 mA M⁻¹ cm⁻²) and a linear range of 0.1–2.7 mM with a detection limit of 14.1 μM (S/N = 3). The biosensor also exhibits good anti-interference ability.

---

1. Introduction

Diabetes is a worldwide public health problem, which is one of the leading causes of death and disability in the world. Since patients with diabetes have high blood glucose, accurate determination and regular monitoring of blood glucose concentration is very important in the diagnosis and treatment of diabetes or metabolic disorders.^1–3 Among the various detection techniques, electrochemical methods have attracted considerable attention by virtue of their fast response, simple operation, high sensitivity and excellent selectivity.⁴ Usually two kinds of amperometric biosensors have been developed. One is glucose enzyme sensors based on the immobilization of glucose oxidase. In this model, glucose determination is implemented by the measurement of the current response originated from the oxidation of hydrogen peroxide, a side product during the course of the enzymatic reaction. For instance, Zhang Y. J. et al. fabricated the CNTs–GOD-based biosensor to monitor the glucose levels.⁵ The other is a direct electrocatalysis nonenzymatic glucose sensor. The amperometric response of nonenzymatic glucose sensor is based on the direct electrochemical oxidation of glucose. Baghayeri. M. et al. fabricated the nonenzymatic glucose sensor based on efficient loading Ag nanoparticles on functionalized carbon nanotubes.⁶ Among them, glucose enzyme sensors usually suffer from drawbacks, such as easily affected by temperature, pH value and toxic chemicals.⁷ By comparison, nonenzymatic glucose sensor is free from the environmental conditions mentioned above. Whatever type, electrode materials are considered to be the determinant factor affecting the analytical properties of a biosensor.

Recently semiconductor materials have received extensive attentions. Due to low intrinsic carrier concentration, semiconductor materials tend to form an electric double layer on the surface, which is important for electrocatalysis,⁸ particularly, semiconductor materials with a wide band gap such as SiC,⁹ ZnO¹⁰ and TiO₂,¹¹ could lower intrinsic carrier concentration and have been applied in the electrochemical field. Hexagonal boron nitride (h-BN) is a promising wide band gap semiconductor and have been widely applied as composite materials, luminescent nanomaterials, thermal conductive polymer due to its super elasticity and strength, high thermal conductivity, high resistance to oxidation, unique luminescence and high surface-to-volume ratio etc.^11–15 Great efforts have been made to fabricate various h-BN nanomaterials including BN nanocarpets,¹⁶ BN nanonet,¹⁷ BN nanosheet,¹⁸ BN fibers¹⁹ and BN hollow sphere²⁰ and so on. From the viewpoint of structure, h-BN, so-called ‘white graphite’, is isostructural to graphite which has been widely used in the field of biosensors.²¹ There is every reason to believe that BN whiskers can be a kind of candidate in biosensor applications. Yin L. W. et al. fabricated a novel amperometric biosensor based on the BN nanotubes–polyaniline–Pt hybrids electrode. The developed biosensor exhibited good performance.²² Besides this, no other biosensor based on BN is reported.

Herein, on the basis of large scale synthesis of h-BN whiskers in our work, the BN whiskers are successfully decorated with Pt nanoparticles (NPs) using solvothermal method to improve the electrocatalytic behavior. By adjusting the mass ratio of BN whiskers to H₂PtCl₆, moderate Pt NPs are uniformly dispersed on the surface of BN whiskers. The BN–Pt composites are explored to apply as an electrochemical glucose biosensor. The performance of the prepared glucose biosensor with respect to sensitivity, detection limit, linear range and response time is presented and discussed.

2. Experimental

2.1 Materials

Glucose oxidase (GOD, E.C. 1.1.3.4, from Aspergillus niger, 200 U per mg), glucose, H₂PtCl₆, N,N-dimethylformamide (DMF), ethylene glycol, glutaraldehyde, acetone, p-acetamidophenol (PA), ascorbic acid (AA) and uric acid (UA) were used as received. Phosphate buffer solution (PBS, 0.2 M, pH = 6.5) was made up of Na₂HPO₄, NaH₂PO₄ and the pH was adjusted by NaOH and HCl. Deionized water was used throughout the experiments.

2.2 Material preparation

Porous h-BN whiskers were synthesized using wet chemical method. H₃BO₃ and C₃N₆H₆ with the molar ration of 3 : 1 were used as raw materials to get white precursor. The white precursor was then slowly heated in a tube furnace to the require temperature, i.e. 900 °C for 3 h in flowing nitrogen/hydrogen (5% hydrogen) and then taken out from the furnace at 700 °C to cool to room temperature in nitrogen. Finally the white BN whiskers in large scale were produced.

The BN–Pt composite was prepared by dispersing 50 mg BN whiskers into 25 ml ethylene glycol under ultrasonic treatment for 30 min. Then H₂PtCl₆ was dropped into above solution under vigorously stirring and the pH of the solution was adjusted to 10 using 0.04 M NaOH. The mass ratio of BN whiskers to H₂PtCl₆ ranging from 1 : 1 to 1 : 3 was adopted. The reaction was carried out under magnetic stirring at 75 °C for 5 h. Finally, the precipitate was separated and rinsed with acetone several times and was then dried in a vacuum oven at 80 °C overnight.

2.3 Fabrication of the modified electrode

Glass carbon (GC, 3.0 mm in diameter) electrode was polished with 1.0, 0.3 and 0.05 μm alumina slurry sequentially and then washed ultrasonically in water and ethanol for a few minutes, respectively. The cleaned GC electrode was dried in nitrogen steam for next modification. 2 mg BN–Pt composite was dispersed in 1 ml of DMF by strong sonication over 1 h to from a stable suspension. Then this suspension was spread evenly on GC electrode. Following 5 μl GOD and 5 μl glutaraldehyde (2.5 wt%) were dissolved in 5 mg l⁻¹ PBS (pH = 6.5) under ultrasonic treatment to form a homogeneous suspension. Then the suspension was also cast on the GC electrode. The GC electrode was dried at room temperature and the electrode was called BN–Pt–GOD/GC. Finally, 5 μl 1 wt% Nafion was cast on the modified electrode to avoid the enzyme leakage. The enzyme modified electrode was stored in refrigerator (4 °C) when not in use. For comparison, BN/GC and BN–Pt/GC electrode were prepared using a similar procedure.

2.4 Phase and microstructure characterization

The phase of the samples was examined by powder X-ray diffraction (XRD, M21XVHF22, MAC Science, Yokohama, Japan) with a TTRIII diffractometer equipped with CuKα radiation over a 2θ range from 10 to 90°. Morphology of the samples was examined using cold field emission scanning electron microscopy (FESEM, ZEISS SUPRATM 55, Germany). Further structural characterization was observed using transmission electron microscope (TEM, JEM 2010, Joel Ltd Japan) with high resolution transmission electron microscopy (HRTEM) and selected area electron diffraction (SAED) pattern. The electrochemical tests were carried out on a CHI660D electrochemical working station consisting of an Ag/AgCl reference electrode, a platinum wire counter electrode, and a modified GC working electrode.

3. Results and discussion

3.1 Phase and microstructure characterization

Fig. 1 shows XRD patterns of the obtained BN–Pt. BN whiskers show broad peak at 2θ of 23° corresponding to (002) plane of BN.²³ The diffraction peaks at 40°, 46°, 68° and 82° can be indexed to the (111), (200), (220) and (311) planes of the Pt (JCPDS card 4-802) respectively. What's more, the characteristic peaks of the Pt are obviously broadened, indicating Pt NPs are in nanometer size.²⁴ No impurities were observed in the XRD diffraction pattern.

Fig. 1 XRD pattern of the BN–Pt sample.

The morphology of BN before and after decorated with Pt NPs is investigated using SEM. Fig. 2b–d present the SEM images of the BN–Pt obtained with 1 : 2 mass ratio of BN whiskers to H₂PtCl₆. Compared with pristine BN whiskers as shown in Fig. 2a, a certain amount of Pt NPs with average particle size of 10–20 nm are uniformly covered on the surface of BN whiskers (Fig. 2b–d). As for the morphology of Pt NPs, it can be seen that most of the Pt NPs have nearly spherical shapes and the phenomenon of aggregation of Pt NPs are rarely found (Fig. 2d).

Fig. 2 SEM micrographs of BN before and after decorated with Pt NPs. (a) SEM image of the BN whiskers. (b) Low-magnification SEM image of BN–Pt. (c and d) High-magnification SEM images of BN–Pt.

The effect of different ratios of BN whiskers to H₂PtCl₆ on the microstructure was also investigated. Fig. 3 show SEM images of BN–Pt composite synthesized with different mass ratios of BN whiskers to H₂PtCl₆ at 1 : 1, 1 : 2 and 1 : 3. It can be seen that Pt NPs gradually transformed from random shape into spherical shape with increasing mass ratio of H₂PtCl₆. Meanwhile the loading density of Pt NPs increased and uniformity declined. When the mass ratio of BN to H₂PtCl₆ is 1 : 2, the BN whiskers were almost uniformly covered by Pt NPs and there is almost no aggregation. Since the aggregation or excessive loading of the Pt NPs can not only reduce their surface activity, which is very important for the applications in sensors, but also affects the synergy of the components in the hybrids.^25,26 On the other hand, H₂PtCl₆ is expensive. Therefore the loading density of Pt should be controlled within a moderate range.

Fig. 3 SEM micrographs of the BN–Pt synthesized with different mass ratios of BN whiskers to H₂PtCl₆. (a and b) 1 : 1. (c and d) 1 : 2. (e and f) 1 : 3.

In order to further observe the microstructure, the sample was also characterized by TEM. Fig. 4a–c are the typical TEM images of BN whiskers and BN–Pt at a low and a higher magnification, respectively. In contrast to the as-prepared BN whiskers with smooth surfaces (Fig. 4a), the surface of BN–Pt becomes relatively rough, further suggesting Pt NPs have been immobilized on the whisker surfaces. HRTEM image (Fig. 4d) shows the BN whiskers have a randomly stacked layer structure and poor crystallization. In addition, the interlayer distance is 0.341 nm, which corresponds to the (002) planes of defective h-BN.²⁷ As for Pt NPs, they are single crystalline and the lattice spacing is measured to be 0.198 nm (Fig. 4e). The corresponding SAED pattern (Fig. 4f) shows five diffraction rings. The (112) plane corresponding to h-BN, while the (111), (200), (220) and (420) planes correspond with Pt.

Fig. 4 SEM micrographs of the BN whiskers and BN–Pt. (a) TEM image of the BN whiskers. (b) Low-magnification TEM image of BN–Pt. (c) High-magnification TEM image of BN–Pt. (d) HRTEM image. (e) HRTEM image of the area M in (d). (f) SAED.

3.2 Electrochemical performance of the as-prepared electrodes

The electrochemical behaviour of the BN–Pt/GC electrode was investigated by cyclic voltammetry (CV) using Fe(CN)₆^3−/4− as the redox marker. Fig. 5a shows the CVs of the BN–Pt/GC electrode recorded at different scan rates in 0.1 M KCl solution containing 5 mM of Fe(CN)₆^3−/4−. The Fe(CN)₆^3−/4− redox process is observed with an peak separation (ΔE_p) of about 110 mV at a specific scan rate of 0.05 V s⁻¹. Although the delta E_p of the Fe(CN)₆]^3−/4− couple is large, the ratio of the cathodic current over the anodic one was close to 1. Therefore it should be a quasi-reversible electron transfer process. The peak currents of the Fe(CN)₆^3−/4− redox process (anodic and cathodic) at the BN–Pt/GC electrode are dependent on the scan rate (Fig. 5b). The peak currents are linearly correlated to the square root of the scan rate in a range from 0.05 to 0.19 V s⁻¹ (Fig. 5b), implying that the electrochemical kinetics is a diffusion-controlled process.²⁸

Fig. 5 (a) CVs of the BN–Pt/GC electrode at scan rates of 0.05, 0.07, 0.09, 0.11, 0.13, 0.15, 0.17, 0.19 V s⁻¹ in 0.10 M KCl containing [Fe(CN)₆]^3−/4−. (b) The plot of anodic/cathodic peak currents versus the square root of scan rate.

3.3 Detection of hydrogen peroxide

Before using BN–Pt/GC electrode to fabricate the glucose biosensor, the performance of BN–Pt/GC electrode toward the detection of H₂O₂ was examined because unless BN–Pt show high sensitivity toward H₂O₂, the glucose biosensor based on these nanomaterials will not show high sensitivity toward the detection of glucose. Fig. 6 shows the CVs obtained from the modified BN–Pt/GC electrode in 0.2 M PBS containing 1 mM H₂O₂ in comparison with a bare GC electrode and BN/GC electrode. As shown in Fig. 6, the oxidation and reduction currents from H₂O₂ at the bare GC electrode are quite small, and the oxidation of H₂O₂ starts at 1.4 V. However, the electrochemical responses obtained at the BN/GC electrode and the BN–Pt/GC electrode is much larger than that obtained at the bare GC electrode. Moreover, the oxidation of H₂O₂ starts at 1.2 V. The obvious increase of anodic and cathodic current at BN–Pt/GC electrode indicated the BN–Pt composites exhibited excellent electrocatalytic activity toward H₂O₂.

Fig. 6 CVs obtained at (a) bare GC electrode; (b) BN/GC electrode and (c) BN–Pt/GC electrode in 0.2 M PBS buffer solution containing 1 mM H₂O₂.

Fig. 7a shows the typical amperometric response of BN–Pt/GC electrode to successive additions of 4 μl H₂O₂ with an applied potential at 0.4 V. The BN–Pt/GC electrode exhibits a fast and sensitive response to the addition of H₂O₂ with steady-state current reached within 5 s (95% of steady-state current). Fig. 7b displays the plot of the electrochemical response vs. the H₂O₂ concentration with a correlation coefficient of 0.994. It can be seen from Fig. 7b that this biosensor shows a linear range from 0.1 mM to 1.2 mM H₂O₂. The limit of detection (LOD) is 0.11 μM based on signal/noise (S/N) = 3. These could be attributed to the synergistic effects of Pt NPs and porous h-BN whiskers, which include the high catalytic activity of Pt NPs and a large surface area of h-BN whiskers.

Fig. 7 (a) Amperometric response for BN–Pt/GC electrode upon successive addition of H₂O₂. (b) The linear regression analysis of the H₂O₂ concentration–current curves.

3.4 Detection of glucose

Due to the outstanding electrocatalytic activities of the BN–Pt towards H₂O₂, an amperometric glucose biosensor was constructed by immobilizing GOD onto BN–Pt. As shown in Fig. 8a, the amperometric response of the BN–Pt–GOD biosensor to the successive addition of glucose at a constant applied potential of 0.55 V at room temperature. A response time of about 5 s is obtained. The response time is shorter than that obtained by decorating Pt NPs on mesoporous silica nanoparticles (10 s),²⁹ suggesting that the electrode responds rapidly to the change of glucose concentration. Fig. 8b displays a linear response range from 0.1 to 2.7 mM with a correlation coefficient of 0.994. LOD is estimated to be 14.1 μM (S/N = 3). Table 1 shows the comparative performance of our biosensor with other reported Pt-based enzyme glucose biosensors. It can be concluded that the BN–Pt–GOD/GC electrode possesses comparable sensitivity, linear range and short response time toward glucose detection. The main reason is that the BN possesses good adsorption capacity due to high surface area, which is beneficial to electron transfer and substance diffusion.

Fig. 8 (a) Amperometric response for BN–Pt–GOD/GC electrode upon successive addition of glucose in 0.2 M PBS (pH = 6.5) at an applied potential of 0.55 V. (b) The linear regression analysis of the glucose concentration–current curves.

Table 1 Comparison of the analytical performance of different Pt-based enzyme glucose biosensors

Electrode	Linearity (mM)	Sensitivity (mA M^−1 cm^−2)	Detection limit (μM)	Response time (s)	Reference
GOx–Pt–MWCNT	1–28	52.72	30	30	30
BNNTS–Pani–Pt–GOD	0.01–505	19.02	6	3	22
GOD/Pt/OMC/Au	0.05–3.7	12.10	50	7	31
(GOx)/BSA/PtNP–SWCNT	0.04–0.87	4.54	40	20	32
MSN–PtNP–GOx	0.001–26	4.38	0.8	10	33
BN–Pt–GOD	0.1–2.7	6.37	14.1	Less than 5 s	This work

---

3.5 Selectivity and reproducibility of the BN–Pt–GOD/GC electrode

Considering various interferents typically exist randomly in practice, the selectivity of BN–Pt–GOD/GC electrode was evaluated by amperometic method in 1 mM glucose concentration at a constant applied potential of 0.55 V at room temperature, with the additions of 0.5 mM PA, UA and AA. As shown in Fig. 9, no noteworthy response was observed, indicating the BN–Pt–GOD/GC electrode displays outstanding anti-interference behavior.

Fig. 9 Amperometric response of the BN–Pt GOD/GC electrode upon successive additions of 1 mM glucose (a), 0.5 mM AA (b), UA (c) and PA (d) at an applied potential of 0.55 V.

The reproducibility of the sensor was carefully investigated. Four different BN–Pt–GOD/GC electrodes were prepared under the same conditions. The relative standard deviation (RSD) of these sensor responses was 4.91%, indicating better reproducibility.

4. Conclusions

BN–Pt composite is prepared by a simple solvothermal method to construct glucose biosensor. The effect of the mass ratio of BN whiskers to H₂PtCl₆ ranging from 1 : 1 to 1 : 3 on the microstructure is also discussed. It shows that spherical Pt NPs with the size of 10–20 nm are uniformly covered on the surface of BN whiskers when the mass ratio of BN whiskers to H₂PtCl₆ is 1 : 2. The developed glucose biosensor exhibits a fast response time (within 5 s), a linear range of 0.1–2.7 mM with the detection limit of 14.1 μM (S/N = 3). It also shows an excellent anti-interference ability. The good electrochemical properties attribute to the large surface area of composite providing large surface-to-volume ratio and enhancing active sites for the oxidation of glucose. Therefore, the novel BN–Pt composite is a potential candidate for the development of biosensor devices.

Acknowledgements

The authors express their appreciation to the National Science Foundation of China (No. 51572019), the National Science Foundation for Excellent Young Scholars of China (No. 51522402) and the Central Universities of No. FRF-TP-15-006C1 and FRF-SD-13-006A for financial support.

Notes and references

1. T. Yang, J. Xu, L. Lu, X. Zhu, Y. Gao, H. Xing, Y. Yu, W. Ding and Z. Liu, J. Electroanal. Chem., 2016, 761, 118–124.
2. T. D. Thanh, J. Balamurugan, S. H. Lee, N. H. Kim and J. H. Lee, Biosens. Bioelectron., 2016, 81, 259–267.
3. R. Prasad, N. Gorjizadeh, R. Rajarao, V. Sahajwalla and B. R. Bhat, RSC Adv., 2015, 5, 44792–44799.
4. L. Fang, B. Liu, L. Liu, Y. Li, K. Huang and Q. Zhang, Sens. Actuators, B, 2016, 222, 1096–1102.
5. Y. Zhang, Y. Shen, D. Han, Z. Wang, J. Song, F. Li and L. Niu, Biosens. Bioelectron., 2007, 23, 438–443.
6. M. Baghayeri, A. Amiri and S. Farhadi, Sens. Actuators B, 2016, 225, 354–362.
7. Y. Yang, Y. Wang, X. Bao and H. Li, J. Electroanal. Chem., 2016, 775, 163–170.
8. C. A. Koval and J. N. Howard, Chem. Rev., 1992, 92, 411–433.
9. T. Yang, L. Zhang, X. Hou, J. Chen and K. C. Chou, Sci. Rep., 2016, 6, 24872.
10. Y. Yang, Y. Wang, X. Bao and H. Li, J. Electroanal. Chem., 2016, 775, 163–170.
11. D. Wen, S. Guo, J. Zhai, L. Deng, W. Ren and S. Dong, J. Phys. Chem. C, 2009, 113, 13023–13028.
12. Q. Li, T. Yang, Q. Yang, F. Wang, K. C. Chou and X. Hou, Ceram. Int., 2016, 42, 8754–8762.
13. Q. Weng, X. Wang, C. Zhi, Y. Bando and D. Golberg, ACS Nano, 2013, 7, 1558–1565.
14. H. Zhao, X. Song and H. Zeng, NPG Asia Mater., 2015, 7, 1–8.
15. W. Luo, T. Yang, L. Su, K. C. Chou and X. Hou, RSC Adv., 2016, 6, 27767–27774.
16. X. Zhang, G. Lian, S. Zhang, D. Cui and Q. Wang, CrystEngComm, 2012, 14, 4670–4676.
17. G. Lian, X. Zhang, H. Si, J. Wang, D. Cui and Q. Wang, ACS Appl. Mater. Interfaces, 2013, 5, 12773–12778.
18. W. Lei, D. Portehault, D. Liu, S. Qin and Y. Chen, Nat. Commun., 2013, 4, 1777.
19. L. Xue, B. Lu, Z. S. Wu, C. Ge, P. Wang, R. Zhang and X. D. Zhang, Chem. Eng. J., 2014, 243, 494–499.
20. G. Lian, X. Zhang, S. Zhang, D. Liu, D. Cui and Q. Wang, Energy Environ. Sci., 2012, 5, 7072–7080.
21. C. Shan, H. Yang, J. Song, D. Han, A. Ivaska and L. Niu, Anal. Chem., 2009, 81, 2378–2382.
22. J. Wu and L. Yin, ACS Appl. Mater. Interfaces, 2011, 3, 4354–4362.
23. Q. Weng, X. Wang, C. Zhi, Y. Bando and D. Golberg, ACS Nano, 2013, 7, 1558–1565.
24. J. Bai, C. L. Fang, Z. H. Liu and Y. Chen, Nanoscale, 2016, 2875–2880.
25. G. Lu, L. E. Ocola and J. Chen, Adv. Mater., 2009, 21, 2487–2491.
26. R. P. Bagwe, L. R. Hilliard and W. Tan, Langmuir, 2006, 22, 4357–4362.
27. J. Li, X. Xiao, X. Xu, J. Lin, Y. Huang, Y. Xue, P. Jin, J. Zou and C. Tang, Sci. Rep., 2013, 3, 0328.
28. D. Wan, S. Yuan, G. L. Li, K. G. Neoh and E. T. Kang, ACS Appl. Mater. Interfaces, 2010, 2, 3083–3091.
29. H. Li, J. He, Y. Zhao, D. Wu, Y. Cai, Q. Wei and M. Yang, Electrochim. Acta, 2011, 56, 2960–2965.
30. J. Xie, S. Wang, L. Aryasomayajula and V. K. Varadan, Nanotechnology, 2007, 18, 1–9.
31. X. Jiang, Y. Wu, X. Mao, X. Cui and L. Zhu, Sens. Actuators B, 2011, 153, 158–163.
32. Z. Zeng, X. Zhou, X. Huang, Z. Wang, Y. Yang, Q. Zhang, F. Boey and H. Zhang, Analyst, 2010, 135, 1726–1730.
33. H. Li, J. He, Y. Zhao, D. Wu, Y. Cai, Q. Wei and M. Yang, Electrochim. Acta, 2011, 56, 2960–2965.

---