Study on an improved bio-electrode made with glucose oxidase immobilized mesoporous carbon in biofuel cells

Xuewei Yangab, Wenqiao Yuan*b, Dawei Lic and Xiangwu Zhangc
aCollege of Life Science, Shenzhen University, Shenzhen, Guangdong 518055, China
bDepartment of Biological and Agricultural Engineering, North Carolina State University, Raleigh, NC State 27695, USA. E-mail: wyuan2@ncsu.edu; Tel: +1-919-515-6742
cDepartment of Textile Engineering, Chemistry and Science, North Carolina State University, Raleigh, NC State 27695, USA

Received 18th December 2015 , Accepted 25th February 2016

First published on 26th February 2016


Abstract

Response surface methodology (RSM) was used for process optimization to immobilize glucose oxidase (GOx) on ordered mesoporous carbon (OMC). Results showed that the maximum GOx adsorption on OMC was 37.38 mg g−1 OMC with the activity of 8531.59 U g−1 GOx-immobilized OMC under the optimized conditions. The interaction effect between GOx, OMC concentration and NaCl and pH were observed. By immobilizing GOx on OMC, we can improve the retained catalyst activity at high temperature (37.3 °C) and shift the optimum reaction pH towards a high value (pH 7.2). The electrochemical characteristics of immobilized GOx can also be enhanced with higher current peak (85.12 μA cm−2) and lower reduction potential.


1. Introduction

Biofuel cells (BFCs) as a renewable energy system employ enzymes to harvest electrical energy from biofuels. Distinct from conventional H2/O2 fuel cells, BFCs can use simple biomolecules (such as xylose1) while working under moderate conditions, such as in mild medium and at ambient temperature/pressure,2 thus becoming a very attractive green energy system.3 Moreover, since fuels consumed by BFCs, such as glucose and oxygen, are generally rich in biological systems, BFCs can be used as the stand-alone power source for implantable medical devices (IMDs, e.g., pacemakers,4 insulin pumps,5 cardiac defibrillators,6 neurological stimulators7) and/or an autonomous health/disease monitoring or management system (e.g., for body glucose/lactose/uric acid level monitoring,8 and diabetes or gout management9 for humans or animals). These unique characteristics and important potential applications have attracted great interest in the fundamental study and development of BFCs.10,11

The performance of BFCs is strongly dependent on several factors: the enzymes used and how they are immobilized on the electrodes, and electrode miniaturization. Research showed that the theoretical open-circuit electric potential of glucose oxidase (vs. Ag/AgCl) on a direct electron transfer (DET) anode is between −0.4 V and 0.25 V.12 However, if a mediator electron transfer (MET) anode is used, the power density will be significantly lower than DET.13 Immobilizing enzymes on the electrode surface, can not only realize the DET of electrode, but also increase the lifetime of the enzyme activity. Liu et al. have performed biofuel cells by using microbial surface displayed enzymes with acceptable stability and energy conversion efficiency.14,15 Research by Kim et al. has shown that by encapsulating enzyme, the active lifetime can be extended beyond to 7–20 days, compared to free enzyme short lifetime (8 h to 2 days).16

Many attempts have been made to immobilize enzymes using various nanomaterials such as multi-walled carbon nanotubes (MWNT),17 single-walled nanotubes (SWNT),18 graphene,19 carbon black and hollow carbon spheres.20 For efficient biocatalytic processes, it is highly desirable to develop nanostructured materials that enable high loading and long-term stability of the biocatalysts, as well as low mass-transfer resistance. In this regard, mesoporous materials with well-controlled pore structures have gained much attention as appropriate, high-capacity hosts for enzymes. Ordered mesoporous carbons (OMCs) have been recently developed as a new class of carbon nanomaterials with high specific surface area, uniform pore distribution, high thermal/mechanical stability and flexible framework composition. OMCs could also be a good candidate to achieve fast direct surface and hydrophobic property due to lack of oxygen-containing functional groups caused by the high carbonization temperature during synthesis.

Recently, researches about GOx immobilization on OMCs are mainly focused on the electrode modification for biofuel cell or biosensor. Yu et al.21 immobilized glucose oxidase (GOx) on the Pt nanoparticles/mesoporous carbon (Pt/CMK-2) matrix, and then cast the mixture on a glassy carbon electrode (GCE) with the maximum current density of 908 μA cm−2. Wang et al. immobilized GOx on glassy carbon electrode modified with mesoporous carbon FDU-15 (MC-FDU-15) and Nafion, and found that MC-FDU-15 and Nafion matrices possessed an excellent bioelectrocatalytic activity and facilitated the electron exchange between the active center of GOx and the electrode.22 However, due to the involvement of electrode material (such as GCE), the size of the electrode modified with OMC is hard to be micro-scale, which can prohibit its future application in the micro-scale biofuel cell or biosensor implanted in human body. By fabricating the electrode with enzyme immobilized-OMC particle, it is possible to construct a micro-scale biofuel cell or biosensor with efficient direct electron transference. Moreover, it was also reported that the adsorption capacity of mesoporous carbon CMK-3-150 with a pore size (6.5 nm) for GOx was determined to be 3.8% (w/w carbon), which was about five times higher than that of CMK-3-100 with a smaller pore size (3.9 nm).23,24 Nevertheless, the factors that affect the glucose oxidase immobilization on OMC have not been thoroughly studied.

In order to explore the possibility of electrode fabrication by GOx immobilized OMC, the GOx adsorption condition (time, pH, GOx concentration, OMC concentration), enzymatic (catalyst activity stability), chemical (Fourier transform infrared spectroscopy) and electrochemical (cyclic voltammetry) characteristics analysis of GOx immobilized OMC have been studied in this paper.

2. Materials and method

2.1. Chemicals

Glucose oxidase (GOx) (type X-S from Aspergillus niger) and ordered mesoporous carbon (OMC) (average pore diameter 100 ± 10 Å, 0.5 cm3 g−1 pore volume) were purchased from Sigma. All other chemicals used were of analytical grade and were used without further purification. Water used was doubly distilled and deionized.

2.2. Optimization of enzyme immobilization

To obtain the highest enzyme immobilization efficiency on OMC, response surface methodology (RSM) based on full factorial central composite design (CCD) for four factors with replicates at the centre point and star point was used and analyzed by Design Expert 8.0.6 software (Stat-Ease, Inc., MN, USA). The variables used were buffer pH values, sodium chloride, enzyme and OMC concentrations each at five coded levels (−α, −1, 0, +1, +α) as shown in Table 1. The actual levels of the variables for the CCD experiments were selected based on our previous results of single factor effect experiments. The CCD contains a total of 30 experimental trials that include 16 trials for factorial design, 8 trials for axial points (2 for each variable) and 6 trials for replications of the central points. The response value (Y) in each trial was average of duplicates (shown in Table 2).
Table 1 The variables and their levels for the central composite experimental design
Variables Symbols Code levels
α −1 0 +1 +α
a Glucose oxidase.b Ordered mesoporous carbon.c Sodium chloride.
GOxa concentration (mg ml−1) A 3.00 4.00 5.00 6.00 7.00
OMCb concentration (mg ml−1) B 20.00 30.00 40.00 50.00 60.00
NaClc concentration (mol l−1) C 0.75 1.00 1.25 1.50 1.75
pH value D 6.00 6.50 7.00 7.50 9.00


Table 2 Experimental design and results of the central composite design
Run Factor 1 Factor 2 Factor 3 Factor 4 GOx activity (U g−1 OMC)
GOx (mg ml−1) OMC (mg ml−1) NaCl (mol l−1) pH value
1 6 40 1.25 7 6484.57
2 3 50 1 7.5 5766.95
3 3 30 1.5 7.5 6223.08
4 5 30 1.5 7.5 5180.23
5 5 50 1.5 6.5 6130.86
6 5 50 1 6.5 6981.24
7 2 40 1.25 7 4595.36
8 3 50 1 6.5 5839.36
9 4 40 1.25 6 6932.50
10 4 40 1.75 7 6201.42
11 4 40 1.25 7 5957.72
12 4 40 1.25 8 6083.44
13 3 30 1 7.5 6291.16
14 4 40 1.25 7 6083.44
15 4 40 1.25 7 6083.44
16 3 30 1.5 6.5 6780.09
17 5 30 1 6.5 8531.59
18 5 30 1.5 6.5 6665.59
19 3 50 1.5 7.5 5675.97
20 4 40 1.25 7 6083.44
21 4 40 0.75 7 6470.64
22 5 30 1 7.5 6832.70
23 3 30 1 6.5 5891.97
24 5 50 1.5 7.5 5826.36
25 4 20 1.25 7 8197.38
26 4 60 1.25 7 6093.11
27 4 40 1.25 7 6083.44
28 4 40 1.25 7 6083.44
29 5 50 1 7.5 6180.99
30 3 50 1.5 6.5 6500.35


2.3. Glucose oxidase activity and stability analysis

After glucose oxidase immobilization, the samples were centrifuged at 8000 rpm for 3 min, and solid part which was the GOx-immobilized OMC (GOx-OMC), was repeatedly washed by the PBS for 3 times and freeze dried for 72 h. The effect of pH on the activity of immobilized glucose oxidase was determined in 50 mM phosphate buffer (pH 5.8–8.0). The effect of temperature (25 °C, 30 °C, 37.3 °C) on the activity of immobilized glucose oxidase was determined in 50 mM phosphate buffer pH 7.0. The glucose oxidase activity of GOx-OMC was analyzed by using Amplex Red Glucose/Glucose Oxidase Assay Kit (Life technologies, Inc., NY, USA).

2.4. Bio-electrode fabrication

The bio-electrode was fabricated by coating GOx immobilized-OMC (GOx-OMC) and high electricity conductive gel PEDOT:PSS (poly(3,4-ethylenedioxythiophene)–poly(styrenesulfonate), 1.1% in H2O, Sigma Inc., USA), to polyethylene terephthalate (PET) film (0.5 mm, Goodfellow, Inc., WI, USA). PET was served as separator for the multi-layer electrode. The dispersion of GOx immobilized-OMC/PEDOT–PSS was made with a ratio of 100 mg[thin space (1/6-em)]:[thin space (1/6-em)]1.5 ml. The dispersions were put into an ultrasonic bath to prevent agglomeration of the nanoparticles. Polyethylene terephthalate (PET) (2 cm × 3 cm) substrates were washed successively by 1[thin space (1/6-em)]:[thin space (1/6-em)]1 ethanol/acetone mixture, 5% NaOH solution and de-ionized water in sequence in an ultrasonic bath for 15 minutes, and then dried under vacuum in a clean chamber under 60 °C for 24 h. 250 μl of the dispersion was sprayed on the PET substrate and dried under vacuum at 30 °C for 30 min to eliminate the residues of water in the PEDOT:PSS/GOx-OMC layer. The resulting film will be formed as bio-electrode. The same protocol was also applied to the fabrication of OMC/PEDOT–PSS electrode with a ratio of 100 mg (OMC)[thin space (1/6-em)]:[thin space (1/6-em)]1.5 ml (PEDOT–PSS), GOx/PEDOT–PSS electrode with a ratio of 23.67 mg (OMC)[thin space (1/6-em)]:[thin space (1/6-em)]1.5 ml (PEDOT–PSS), GOx-OMC/PEDOT–PSS electrode with a ratio of 23.67 mg (GOx)[thin space (1/6-em)]:[thin space (1/6-em)]100 mg (OMC)[thin space (1/6-em)]:[thin space (1/6-em)]1.5 ml (PEDOT–PSS).

2.5. Electrochemical characteristics analysis of the bio-electrode

The electrochemical experiments were carried out with a CHI 660A electrochemical workstation with a conventional three-electrode cell. The Pt wire electrode (MW1032, Basi Inc., West Lafayette, IN, USA) was used as the counter electrode. The Ag/AgCl electrode (Aldrich glass, Sigma Inc., St. Louis, MO, USA) was used as the reference electrode and all the potentials were quoted with respect to the Ag/AgCl electrode. All experiments were performed at a temperature of 25 °C. 10 mM glucose in 0.1 M phosphate buffer (PBS) (pH 7.0) saturated with air and N2 were used as the electrolyte. The purpose of this manuscript is to investigate the possibility of making GOx/OMC/PEDOT–PSS as micro-scaled electrode that can be performed in human physiological environment, such as blood. We expected that by consuming glucose in the blood, this kind of FCs can be used as the stand-alone power source for implantable medical devices and/or an autonomous health/disease monitoring or management system. Thus we studied the electrochemical characteristics and responses of the electrode with the glucose addition. The cyclic voltammogram (CV) test of the bio-electrode were performed with the scan rate at 100 mV s−1.

3. Results

3.1. Response surface methodology for GOx immobilization in OMC

The experimental results of GOx immobilization by a complete four-factor-two-level factorial experiment design with six replications of the central point and eight axial points are shown in Table 2. The responses of the CCD design were fitted with a second-order polynomial equation:
Glucose oxidase activity = 5300.72 + 472.30A − 526.07B − 67.31C − 96.90D − 42.95AB − 382.10AC − 202.15 + 128.96BC + 83.79BD − 62.43CD + 41.67A2 + 442.99B2 + 240.69C2 + 341.36D2

As shown in Table 3, the model F-value of 102.45 implies the model is significant. There is only a 0.01% chance that an F-value this large could occur due to noise. Values of “Prob > F” less than 0.05 indicate model terms are significant. In this case A, B, D, AC, AD, BC, BD, CD, B2, C2, D2 are significant model items. The coefficient of determination (R2) was calculated to be 0.9963, indicating that the model could explain 99% of the variability. The “lack of fit test” compares the residual error to the “Pure Error” from replicated design points. The “lack of fit F-value” of 26[thin space (1/6-em)]111.80 implies the lack of fit is significant. Thus, the estimated models fit the experimental data adequately. “Adeq. Precision” measures the signal (response) to noise (deviation) ratio. A ratio greater than 4 is desirable. The ratio of 48.152 indicates an adequate signal. This model can be used to navigate the design space.

Table 3 Results of the regression analysis of the central composite rotatory design
Source Mean square F-Value Prob > F
Model 1.003 × 106 102.45 <0.0001
A-GOx concentration 1.785 × 106 182.24 <0.0001
B-OMC concentration 2.214 × 106 226.11 <0.0001
C-NaCl concentration 36[thin space (1/6-em)]240.89 3.70 0.0906
D-pH 75[thin space (1/6-em)]115.49 7.67 0.0243
AB 29[thin space (1/6-em)]499.67 3.01 0.1208
AC 2.336 × 106 238.56 <0.0001
AD 6.538 × 105 66.77 <0.0001
BC 2.661 × 105 27.18 0.0008
BD 1.123 × 105 11.47 0.0095
CD 62[thin space (1/6-em)]368.73 6.37 0.0356
A2 47[thin space (1/6-em)]633.89 4.86 0.0585
B2 5.383 × 106 549.70 <0.0001
C2 1.589 × 106 162.27 <0.0001
D2 3.196 × 106 326.40 <0.0001
Lack of fit 26[thin space (1/6-em)]111.80    


Three-dimensional response surfaces were plotted on the basis of the model equation, to investigate the interaction among the variables and to determine the optimum concentration of each factor for maximum GOx immobilization in OMC. The effects of varying concentrations of GOx, OMC, NaCl and pH were shown in Fig. 1, which showed that the response surfaces for AC and AD were similar to each other, while that for BD and CD were similar to each other. It suggested that the interaction mechanism for GOx, OMC concentration with NaCl and pH were similar,25 which was probably due to the effect of charge state on enzyme.26 The simultaneous effect of all the variations on glucose oxidase immobilization can be seen in the perturbation graph (Fig. 2). The plot reveals that the immobilization process is most sensitive to OMC concentration while least influenced by NaCl concentration. The maximum GOx adsorption on OMC was 37.38 mg g−1 OMC with the activity of 8531.59 U g−1 GOx-immobilized OMC under the optimized condition: GOx (5 mg ml−1), OMC concentration (30 mg ml−1), NaCl (1 mol l−1) and pH (6.5), which is 2-fold higher than the control medium.


image file: c5ra27111h-f1.tif
Fig. 1 Response surface plot for glucose oxidase (GOx) activity of GOx immobilized OMC. The interaction between (A) GOx concentration (mg ml−1), (B) OMC concentration (mg ml−1), (C) NaCl concentration (mol l−1), (D) pH value.

image file: c5ra27111h-f2.tif
Fig. 2 Perturbation graph showing the effect of variables on immobilization (A = GOx concentration; B = OMC concentration; C = NaCl concentration; D = pH value).

3.2. Enzyme activity stability analysis of immobilized GOx

Fig. 3a shows the activity of the free and immobilized glucose oxidase at different temperatures. There is no significant difference between immobilized and free GOx in its optimum reaction temperature (30 °C). However, for lower temperature (25 °C) and higher temperature (37.3 °C), the retaining activities of immobilized enzyme were 15.7–16.3% higher than that of the free enzyme. Enzyme immobilization on OMC can provide a large surface area for substrate contact within a small total volume of enzyme and minimize barriers to mass transport of substrate and product.27 It has been reported that in the glucose oxidation, GOx undergoes conformational changes, groups on the GOx change their charge state and an oxygen molecule is used to form a hydrogen peroxide molecule. Thus the conductivity of the immobilization support is very important for the glucose catalytic reaction of GOx.26 Carbon nano materials such as carbon nanotube (CNT), mesoporous carbon (OMC), have been found to have the ability to promote electron transfer reactions when they were used to fabricate electrodes for the oxidation of biomolecules such as glucose.28 Above all, the promotion of retaining activities of immobilized GOx might be due to the facilitation of substrate contact and electron transportation. Previous research has reported that the immobilization processes can protect the enzyme substantially against heat inactivation.29 It indicates that immobilized GOx showed the potential to work well in human body temperature (36.3–37.2 °C),30 which can be used as biocatalyst in the implanted device as power supply.31
image file: c5ra27111h-f3.tif
Fig. 3 Temperature and pH stability analysis of free and immobilized glucose oxidase (free enzyme = free glucose oxidase; immobilized enzyme = glucose oxidase immobilized on OMC).

Fig. 3b shows the activity of the free and immobilized glucose oxidase at different pH values. The optimum pH values of the free and immobilized glucose oxidase were pH 6.6 and pH 7.2, respectively. A forward shift in pH optima of immobilized enzyme may be the result of change in support chemistry by pH which further influenced the protein-support interaction and activity.32 A marginal effect on the enzyme optimum pH due to the interaction of support material with the enzymes charged amino acids has been observed during immobilization.33 A forward shift of pH also suggested that immobilized GOx might be more suitable to be used in the human body, whose environment pH is around 7.4.34 It has been reported that enzyme stability appeared to be the main bottle neck for utilization of biofuel cell as the power supply in implanted medical device.10 Results of enzyme stability showed that GOx immobilized in OMC had advantages as the biocatalyst of bio-electrode in the human body, with free GOx.

3.3. Electrochemistry characteristics of electrode fabricated by immobilized GOx

Cyclic voltammetry (CV) is a valuable and convenient tool to monitor the electrochemical performance of the electrode. As shown in Fig. 4, a pair of well-defined and quasi-reversible redox peaks was observed at the GOx/OMC/PEDOT–PSS (curve 3), and GOx immobilized-OMC/PEDOT–PSS (curve 4). These redox peaks are characteristics of reversible electron transfer process of redox active center in the GOx (GOx(FADH2) ↔ GOx(FAD) + 2e + 2H+) proving a fast electron transfer process.35 The electrode OMC/PEDOT–PSS (curve 1), that has been observed with a pair of redox peaks, can be considered as a non-enzymatic electro-catalyst. It has been reported that non-enzymatic electrocatalysts come in a number of forms, specifically; metals (e.g. Pt, Au), metal oxides/semiconductors (e.g. Ni(OH)2, RuO2), alloys (e.g. PtPb, PtRu), complexes (e.g. cobalt phthalocyanine) and carbon based (e.g. carbon nanotubes, boron doped diamond).36 Non-enzymatic and unmodified carbon nanotubes has been successfully used in glucose sensors as electrode by Ye et al.37 It has also been reported that nano carbon materials such as SWNT, OMC have potential applications in constructing highly sensitive, stable, and fast response glucose sensors without enzyme loading.38 The reduction peak potential of GOx immobilized-OMC/PEDOT–PSS (curve 4) was much lower than curve 1, 2, and 3. It indicated that the immobilization on OMC can improve the reduction ability of glucose oxidase as the anode catalyst.39 ΔEp is the potential difference between the oxidation peak potential and reduction peak potential, which is inversely proportional to the electron transfer rate.40 The quasi-reversible CV with ΔEp of 210 mV was obtained at GOx immobilized-OMC/PEDOT–PSS (curve 4). It indicated that the immobilization of GOx in the OMC can improve the electron transfer between the electrolyte and the electrode surface. Moreover, the peak current density of GOx immobilized-OMC/PEDOT–PSS was 85.12 μA cm−2, which is much higher than GOx-OMC modified electrode such as GOx/TiO2/FePc/CNT (about 65.3 μA cm−2).41 Recently OMC is mainly used to modify electrode for making the direct electron transfer (DET). For example, Pt/OMC and GOx were used to modify GC electrode,21 Pt/OMC and GOx were used to modify gold electrode,42 Nafion/OMC-FDU-15 powder and GOx were used to modify GC electrode.21 However, in our research, the electrode was only made of GOx immobilized OMC without any other electrode material such as Pt, GC or gold. As a result, the electron transfer can be more efficient, and the enzyme loading in the same volume can be more, compared with those electrodes modified with GOx immobilized OMC. Considering the CV studies, significant enhancement of GOx performance as the catalyst on electrode has been observed when it was immobilized on OMC, and also, direct electron transfer of GOx can be realized by immobilizing GOx on OMC to fabricate electrode.
image file: c5ra27111h-f4.tif
Fig. 4 Cyclic voltammetric test of (1) OMC/PEDOT–PSS, (2) GOx/PEDOT–PSS, (3) GOx/OMC/PEDOT–PSS, and (4) GOx immobilized-OMC/PEDOT–PSS in O2-saturated 0.1 M PBS pH 7.0 in 5 mM glucose at 100 mV s−1.

4. Conclusion

To overcome the problem of low activity and stability of glucose oxidase served as the anode catalyst in biofuel cell, the glucose oxidase was immobilized on ordered mesoporous carbon with the maximum enzyme load of 37.38 mg g−1 OMC (activity 8531.59 U g−1). Improved enzyme activity stabilities in higher temperature and pH value were observed after immobilization, which indicated the great potential for the utilization in the physiological environment such as human body. The overall performance of immobilized GOx-OMC with respect to catalyst stability and efficiency is promising compared with its free form.

Acknowledgements

This work was supported by the Dean’s Enrichment Program of the College of Agriculture and Life Sciences at North Carolina State University, ​the National Natural Science Foundation of China (31470431, 41176106), Guangdong Natural Science Foundation for Major cultivation project (2014A030308017), Shenzhen Grant Plan for Science & Technology (JCYJ20120613112512654, JSGG20130411160539208), Shenzhen special funds for Bio-industry development (NYSW20140327010012).

References

  1. L. Xia, B. Liang, L. Li, X. Tang, I. Palchetti, M. Mascini and A. Liu, Biosens. Bioelectron., 2013, 44, 160–163 CrossRef CAS PubMed.
  2. X. Wei and J. Liu, Front Energ Power Eng China, 2008, 2, 1–13 CrossRef.
  3. K. Rabaey, G. Lissens, S. D. Siciliano and W. Verstraete, Biotechnol. Lett., 2003, 25, 1531–1535 CrossRef CAS PubMed.
  4. K. Shoji, M. Suzuki, Y. Akiyama, T. Hoshino, N. Nakamura, H. Ohno and K. Morishima, PowerMEMS, 2011, pp. 27–30 Search PubMed.
  5. M. R. Mousavi, S. Chamanian, I. Ahadzadeh, M. Bahrami and M. Hosseini, 21st International Conference on Systems Engineering (ICSEng), 2011, p. 315 Search PubMed.
  6. M. Falk, S. Shleev, C. W. Narváez Villarrubia, S. Babanova and P. Atanassov, Enzym. Fuel Cells, 2014, 422 CAS.
  7. C. H. Kwon, S.-H. Lee, Y.-B. Choi, J. A. Lee, S. H. Kim, H.-H. Kim, G. M. Spinks, G. G. Wallace, M. D. Lima and M. E. Kozlov, Nat. Commun., 2014, 5, 3928 CAS.
  8. F. Tasca, Development of Second and Third Generation Bioelectronics, Lund University, 2010 Search PubMed.
  9. F. Gao, L. Viry, M. Maugey, P. Poulin and N. Mano, Nat. Commun., 2010, 1, 2 Search PubMed.
  10. P. Cinquin, C. Gondran, F. Giroud, S. Mazabrard, A. Pellissier, F. Boucher, J.-P. Alcaraz, K. Gorgy, F. Lenouvel and S. Mathé, PLoS One, 2010, 5, e10476 Search PubMed.
  11. S. D. Minteer, B. Y. Liaw and M. J. Cooney, Curr. Opin. Biotechnol., 2007, 18, 228–234 CrossRef CAS PubMed.
  12. P. Atanassov, C. Apblett, S. Banta, S. Brozik, S. C. Barton, M. Cooney, B. Y. Liaw, S. Mukerjee and S. D. Minteer, Interface, 2007, 16, 28–31 CAS.
  13. H. Liu and B. E. Logan, Environ. Sci. Technol., 2004, 38, 4040–4046 CrossRef CAS PubMed.
  14. S. Fan, C. Hou, B. Liang, R. Feng and A. Liu, Bioresour. Technol., 2015, 192, 821–825 CrossRef CAS PubMed.
  15. C. Hou, D. Yang, B. Liang and A. Liu, Anal. Chem., 2014, 86, 6057–6063 CrossRef CAS PubMed.
  16. J. Kim, H. Jia and P. Wang, Biotechnol. Adv., 2006, 24, 296–308 CrossRef CAS PubMed.
  17. L. Deng, L. Shang, Y. Wang, T. Wang, H. Chen and S. Dong, Electrochem. Commun., 2008, 10, 1012–1015 CrossRef CAS.
  18. Y. Yan, W. Zheng, L. Su and L. Mao, Adv. Mater., 2006, 18, 2639–2643 CrossRef CAS.
  19. C. Liu, S. Alwarappan, Z. Chen, X. Kong and C.-Z. Li, Biosens. Bioelectron., 2010, 25, 1829–1833 CrossRef CAS PubMed.
  20. F. Gao, X. Guo, J. Yin, D. Zhao, M. Li and L. Wang, RSC Adv., 2011, 1, 1301–1309 RSC.
  21. J. Yu, D. Yu, T. Zhao and B. Zeng, Talanta, 2008, 74, 1586–1591 CrossRef CAS PubMed.
  22. K. Wang, H. Yang, L. Zhu, Z. Ma, S. Xing, Q. Lv, J. Liao, C. Liu and W. Xing, Electrochim. Acta, 2009, 54, 4626–4630 CrossRef CAS.
  23. S. Jun, S. H. Joo, R. Ryoo, M. Kruk, M. Jaroniec, Z. Liu, T. Ohsuna and O. Terasaki, J. Am. Chem. Soc., 2000, 122, 10712–10713 CrossRef CAS.
  24. A. Vinu, C. Streb, V. Murugesan and M. Hartmann, J. Phys. Chem. B, 2003, 107, 8297–8299 CrossRef CAS.
  25. H.-Y. Kim, J.-W. Lee, T. W. Jeffries and I.-G. Choi, Bioresour. Technol., 2011, 102, 1440–1446 CrossRef CAS PubMed.
  26. K. Besteman, J.-O. Lee, F. G. Wiertz, H. A. Heering and C. Dekker, Nano Lett., 2003, 3, 727–730 CrossRef CAS.
  27. R. Parthasarathy and C. R. Martin, Synthesis of polymeric microcapsule arrays and their use for enzyme immobilization, DTIC Document, 1994 Search PubMed.
  28. S. Wang, Q. Zhang, R. Wang, S. Yoon, J. Ahn, D. Yang, J. Tian, J. Li and Q. Zhou, Electrochem. Commun., 2003, 5, 800–803 CrossRef CAS.
  29. M. K. Gouda and M. A. Abdel-Naby, Microbiol. Res., 2002, 157, 275–281 CrossRef CAS PubMed.
  30. N. Ramanathan, J. Appl. Physiol., 1964, 19, 531–533 CAS.
  31. A. Zebda, S. Cosnier, J.-P. Alcaraz, M. Holzinger, A. Le Goff, C. Gondran, F. Boucher, F. Giroud, K. Gorgy and H. Lamraoui, Sci. Rep., 2013, 3, 1–5 Search PubMed.
  32. N. Ortega, M. Perez-Mateos, M. C. Pilar and M. D. Busto, J. Agric. Food Chem., 2008, 57, 109–115 CrossRef PubMed.
  33. M. Sardar, I. Roy and M. N. Gupta, Enzyme Microb. Technol., 2000, 27, 672–679 CrossRef CAS PubMed.
  34. F. Ramaekers, D. Haag, A. Kant, O. Moesker, P. Jap and G. Vooijs, Proc. Natl. Acad. Sci. U. S. A., 1983, 80, 2618–2622 CrossRef CAS.
  35. H. Razmi and R. Mohammad-Rezaei, Biosens. Bioelectron., 2013, 41, 498–504 CrossRef CAS PubMed.
  36. K. E. Toghill and R. G. Compton, Int. J. Electrochem. Sci., 2010, 5, 1246–1301 CAS.
  37. B.-B. Jiang, X.-W. Wei, F.-H. Wu, K.-L. Wu, L. Chen, G.-Z. Yuan, C. Dong and Y. Ye, Microchim. Acta, 2014, 181, 1463–1470 CrossRef CAS.
  38. G. Wang, X. He, L. Wang, A. Gu, Y. Huang, B. Fang, B. Geng and X. Zhang, Microchim. Acta, 2013, 180, 161–186 CrossRef CAS.
  39. P. Jenkins, S. Tuurala, A. Vaari, M. Valkiainen, M. Smolander and D. Leech, Bioelectrochemistry, 2012, 87, 172–177 CrossRef CAS PubMed.
  40. J. J. Harris and M. L. Bruening, Langmuir, 2000, 16, 2006–2013 CrossRef CAS.
  41. H.-F. Cui, K. Zhang, Y.-F. Zhang, Y.-L. Sun, J. Wang, W.-D. Zhang and J. H. Luong, Biosens. Bioelectron., 2013, 46, 113–118 CrossRef CAS PubMed.
  42. X. Jiang, Y. Wu, X. Mao, X. Cui and L. Zhu, Sens. Actuators, B, 2011, 153, 158–163 CrossRef CAS.

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