- Email: [email protected]
Nafion nanocomposites fabricated using hydrogel microstencils
Microelectronic Engineering 141 (2015) 193–197
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Microelectronic Engineering journal homepage: www.elsevier.com/locate/mee
Enzyme immobilization on microelectrode arrays of CNT/Nafion nanocomposites fabricated using hydrogel microstencils Sung Deuk Choi a, Jin Ho Choi a, Young Ho Kim b, Sung Yeol Kim a, Prabhat K. Dwivedi c, Ashutosh Sharma c, Sanket Goel d, Gyu Man Kim a,⇑ a
School of Mechanical Engineering, Kyungpook National University, Daegu 702-701, South Korea Medical Device Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu 701-310, South Korea Department of Chemical Engineering, Indian Institute of Technology, Kanpur 208016, India d Department of Electrical, Electronics and Instrumentation Engineering, University of Petroleum and Energy Studies (UPES), Dehradun 248007, India b c
a r t i c l e
i n f o
Article history: Received 10 October 2014 Received in revised form 4 February 2015 Accepted 24 March 2015 Available online 28 March 2015 Keywords: Microfluidic biofuel cells 3D microelectrode Carbon nanotube Nanocomposites Hydrogel microstencil
a b s t r a c t Enzyme-modified, three-dimensional (3D) microelectrode arrays for microfluidic biofuel cell applications were fabricated using carbon nanotube (CNT) reinforced NafionÒ nanocomposites and hydrogel microstenciling. CNT/Nafion nanocomposites were prepared by dispersing oxidized, multi-walled CNTs in Nafion solution. A hydrogel microstencil was fabricated using photolithography, soft lithography, and capillary-force lithography. After structuring the nanocomposites with the microstencil, a plasmaetching process was applied to the surface of the nanocomposite in order to obtain a micro-porous structure. Enzymes could be successfully immobilized on the micro-porous structures of nanocomposite surfaces using direct covalent binding. Ó 2015 Published by Elsevier B.V.
1. Introduction Over the past few decades, interest in biofuel cells has increased because of their many advantages including use of low-cost catalysts compared to conventional fuel cells and mild operating conditions [1]. In biofuel cell systems, a biofuel such as glucose is oxidized at the anode side and an oxidant such as O2 is reduced at the cathode side; generally the sides are separated by an ionconducting membrane in order to prevent fuel and oxidant from mixing [2]. In recent years, biofuel cells have employed microfluidic technology in response to the demand for miniaturized fuel cells for portable electronics [3,4]. In a microchannel, the fuel stream and the oxidant stream form a laminar flow without mixing, and therefore microfluidic fuel cells are able to operate efficiently without an ion-conducting membrane which causes ohmic loss [5]. However, most of the microfluidic biofuel cells reported to date have two-dimensional (2D) electrodes, and are thus limited in fuel utilization because of low surface area. Kjeang et al. reported a microfluidic fuel cell incorporating a three-dimensional (3D), ⇑ Corresponding author at: School of Mechanical Engineering, Kyungpook National University, 1370 Sankyuk-dong, Buk-gu, Daegu 702-701, South Korea. Tel.: +82 53 950 7570; fax: +82 53 950 6550. E-mail address: [email protected] (G.M. Kim). http://dx.doi.org/10.1016/j.mee.2015.03.045 0167-9317/Ó 2015 Published by Elsevier B.V.
flow-through, porous electrode made of carbon paper [6]. Although it was not a biofuel cell, fuel utilization could be increased to 94% by using vanadium as the electrolyte material. Similarly, the efficiency of a microfluidic biofuel cell is expected to increase when 3D microelectrodes are employed due to their relatively larger surface area. The commonly used fabrication method of electrodes for microfluidic devices consists of metal deposition and photolithography followed by selective etching. However, this conventional method provides very thin electrodes of less than several hundred nanometers and therefore is not suitable for the fabrication of a thick electrode. An alternative method is to use a microstencil as a template. A microstencil is a membrane that has patterned perforations [7,8]. The use of a microstencil allows patterning with any material, even neural stem cells [7]. Microstencils can be made of solid states of silicon [9], metal, and also flexible polymer. When using hydrogel, it is possible to fabricate a very thick micro-structure greater than several hundred micrometers [10,11]. Furthermore, the patterning process is simple and does not require expensive photolithography equipment. Therefore, a thick electrode can be fabricated readily by adopting a hydrogel-microstencil method. An enzymatic biofuel cell uses a specific enzyme as a biological catalyst. Enzymes such as glucose oxidase or laccase in microfluidic biofuel cells can exist in solution or on an electrode. The former is
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not an effective design choice because it not only requires more enzymes than the latter, but also the enzymes in solution have a short active lifetime [5]. Therefore, enzymes need to be immobilized on electrodes for better enzyme stability and long-term efficiency; an electrode that has high surface permits the immobilization of more enzymes. Immobilization technique on a miniaturized electrode is one of important issues for a microfluidic based fuel cell devices. In this study, we propose methods of fabrication of microporous 3D microelectrode arrays and enzyme immobilization on the microelectrodes for microfluidic biofuel cells. To enhance enzyme immobilization efficiency, the 3D microelectrodes were prepared porous structure and then two enzymes such as glucose oxidase and laccase were immobilized on the surface of porous microelectrodes. 2. Experimental 2.1. Materials Multi-walled carbon nanotubes (MWCNTs, >93% purity, 10–40 nm average diameter, 1–25 lm long) were purchased from CNT Co., Ltd (South Korea). Reagent grade 60% nitric acid was obtained from Duksan Chemical (South Korea). 5% Nafion solution (D521) was acquired from DuPont (USA). Polyethylene glycol diacrylate (PEGDA), 2-hydroxy-2-methylpropiophenone 98% (photo initiator), N-(3-dimethylaminopropyl)-N0 -ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), glucose oxidase from Aspergillus Niger and laccase from Trametes Versicolor were purchased from Sigma–Aldrich (USA). Sodium phosphate monobasic (NaH2PO4) and sodium phosphate dibasic (Na2HPO4) were obtained from Sigma–Aldrich (USA) and used to prepare a phosphate buffer solution. SU-8 2100 and SU-8 developer were obtained from Microchem (USA). Polydimethylsiloxane (PDMS) prepolymer and curing agent (Sylgard 184) were acquired from Dow Corning (USA).
added into Nafion solution, and were then dispersed via sonication for 1 h; a well-dispersed solution of CNT/Nafion nanocomposites was used for fabrication of the 3D microelectrode arrays. 2.4. Fabrication of the 3D microelectrode arrays The 3D microelectrode arrays were fabricated by using a hydrogel microstencil and CNT/Nafion nanocomposites. Fig. 1 shows the fabrication procedure of the 3D microelectrode arrays. Firstly, a PDMS stamp, which was prepared using general photolithography and soft lithography, was placed on a glass as substrate [10]. The space between the PDMS stamp and the substrate was filled using capillary force with PEGDA hydrogel solution containing 2 wt% photo initiator. After curing the hydrogel solution by UV exposure, the PDMS stamp was separated from the substrate. Subsequently, the substrate was rinsed with DI water in order to wash away uncured hydrogel solution, and then dried with nitrogen gas [10]. The CNT/Nafion nanocomposite solution was pipetted onto the hydrogel microstencil, filling the inside of the microstencil pattern. The excess solution was removed using a doctor blade. Subsequently, the substrate was heated on a hotplate (65 °C, 2 min) to remove the solvent of the CNT/Nafion nanocomposites. After separating the hydrogel microstencil from the substrate, the surface of the 3D microelectrode was etched using an oxygen-plasma treatment (V15-G, Plasma Finish, Germany) at high power (120 W) for 10 min [13]. 2.5. Enzyme immobilization GOD and laccase were immobilized on the surface of CNT/ Nafion nanocomposites using direct covalent binding [14]. First, 30 mM EDC and 90 mM NHS were dissolved in a phosphate buffer solution (pH 7.0, 50 mM); the solution was pipetted onto the nanocomposite surface. After 1 h, the nanocomposite surface was dried using nitrogen gas. Then, a phosphate buffer solution (pH 7.0, 50 mM) containing 4 mg/mL of GOD or laccase was dropped onto the surface and allowed to react for 10 h.
2.2. Characterization The structure and size of the 3D microelectrode arrays were examined using a field-emission scanning electron microscope (FE-SEM, S-4800, Hitachi, Japan). The sheet resistance of the CNT/ Nafion nanocomposites was measured using a four-point probe (CMT-SR1060N, Changmin Tech Co., Ltd, South Korea). The electrical conductivity of the CNT/Nafion nanocomposites was calculated by multiplying the sheet resistance and the thickness of nanocomposite film. The atomic concentration of CNT/Nafion nanocomposites was analyzed using X-ray photoelectron spectroscopy (XPS, Quantera SXM, ULVAC-PHI, Japan). 2.3. Preparation of CNT/Nafion nanocomposites The MWCNTs were treated using an oxidation method previously reported [12]. Briefly, 0.5 g of pristine MWCNTs were dispersed in 200 mL of 60% nitric acid using an ultrasonic bath (JAC Ultrasonic 4020P, KODO, South Korea) for 1 h. Then, the solution was refluxed at 120 °C for 12 h under nitrogen gas and was cooled down to room temperature. Subsequently, the MWCNTs in the solution were washed and filtered repeatedly with deionized (DI) water and membrane-filtered (0.22 lm, Nitrocelluose, Millipore, USA) until the pH of the solution was near neutral. After drying in an oven at 60 °C for 48 h, the MWCNTs were stored in a vial before use. To prepare the CNT/Nafion nanocomposites, 10 wt% oxidized MWCNTs (ox-MWCNTs) based on Nafion polymer weight were
3. Results and discussion 3.1. 3D microelectrode arrays with micro-porous structure Electrodes for fuel cells are required to have both electrical conductivity and large surface area in order to react promptly with fuel or oxidant [15]. To enhance the surface area of microelectrode arrays, microelectrodes consist of CNT and Nafion nanocomposites were treated by applying plasma. Fig. 2 shows the plasma-etching effect on CNT/Nafion nanocomposites. When the nanocomposite electrodes are structured by stenciling, most of the CNT surfaces are covered with Nafion as shown in Fig. 2(a). Thus, only a limited area of CNT surface is provided on the electrode for immobilization of enzymes. In order to increase the active CNT surface for the immobilization of enzymes, the Nafion layer on the electrode surface must be etched away. Oxygen-plasma etching was adopted for Nafion removal; Nafion on the electrode surface was removed and a micro-porous structure of CNT/Nafion nanocomposites was obtained, as shown in Fig. 2(b). The surface area of CNT/Nafion nanocomposites is significantly increased because of the microporous structure. The size of the 3D microelectrode arrays was determined by the hydrogel microstencil because the electrode was fabricated using a solution-casting method. Using this method, we prepared 3D microelectrode arrays with different diameters (100 lm and 500 lm) and different thicknesses ranging from several micrometers to hundred micrometers, as shown in Fig. 3. These
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Fig. 1. Schematic images of preparation procedure for hydrogel microstencil (on left) and fabrication procedure of 3D microelectrode arrays from ox-MWCNT/Nafion nanocomposites (on right).
Fig. 2. FE-SEM images of plasma-etching effect on ox-MWCNT/Nafion nanocomposites (10 wt% ox-MWCNT/Nafion nanocomposites, oxygen plasma power: 120 W): (a) before plasma etching, (b) after 10 min of plasma etching.
micro-porous, 3D microelectrode arrays have high surface area and can be applied to microfluidic biofuel cell.
[14]. The GOD- and laccase-modified 3D microelectrodes can be used as the anode and cathode of microfluidic biofuel cells.
3.2. Enzyme immobilization on CNT/Nafion nanocomposites
3.3. Electrical properties of CNT/Nafion nanocomposites
Fig. 4 shows the result of GOD and laccase immobilization on the CNT/Nafion nanocomposites. The ox-MWCNTs have a carboxyl group [16] and are able to be activated by EDC/NHS; enzymes could be immobilized on the exposed surface of the ox-MWCNTs
The electrical conductivity of CNT/Nafion nanocomposites was measured in order to estimate the performance of the 3D microelectrodes. Four samples – ox-MWCNT, Nafion, and ox-MWCNT/ Nafion nanocomposites before and after plasma etching – were
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Fig. 3. FE-SEM images of different-sized 3D microelectrode arrays (10 wt% ox-MWCNT/Nafion nanocomposites, oxygen plasma-etching time:10 min): (a) 100 lm in diameter, 5 lm-thick microelectrode array, (b) 500 lm in diameter, 60 lm-thick microelectrode array.
Fig. 4. FE-SEM images of (a) GOD-modified CNT/Nafion nanocomposites, (b) laccase-modified CNT/Nafion nanocomposites.
prepared and measured for sheet resistance and film thickness. Table 1 shows the results for electrical conductivity. Ox-MWCNT had the highest electrical conductivity of the four samples and Nafion the lowest. In the cases of CNT/Nafion nanocomposites, plasma-etched samples had lower electrical conductivity than that of the non-etched. The decrease in the electrical conductivity of CNT/Nafion nanocomposites is a result of the oxygen-plasma etching process. Kim et al. reported an increase in the electrical resistance of carbon nanotubes caused by oxygen plasma [17]. According to the XPS results for the CNT/Nafion nanocomposites (Fig. 5), the relative atomic-concentration ratio of oxygen and carbon varied with plasma etching. The C1s:O1s atomic concentration Table 1 Electrical conductivity of samples. Sample
Electrical conductivity (S/m)
ox-MWCNT Nafion ox-MWCNT/Nafion (before etching) ox-MWCNT/Nafion (after etching)
1741 0.04305 51.04 ± 1.70 34.49 ± 0.32
ratio is 93.3:6.7% for the non-etched sample and 84.3:15.7% for the plasma-etched sample. The plasma-etching process led to an increase of oxygen percentage in the CNT/Nafion nanocomposites. Thus, the abundance of oxygen functional groups such as carboxyl and carbonyl were increased in CNT/Nafion nanocomposites; therefore, the electrical conduction paths in the carbon nanotubes were broken. Even though the electrical conductivity of the CNT/ Nafion nanocomposites was decreased by the plasma-etching process, the process provided the high surface area needed to immobilize an increased amount of enzymes because of the increase in exposed CNT surface. Therefore, a microfluidic device having microelectrode arrays and possible examples of electrode array integrated into microfluidic channel was designed and prepared as shown in Fig. 6. A schematic image of microfluidic device is shown in Fig. 6(a). Then, 3D microelectrode arrays having micro-porous structure in a microfluidic chip were fabricated by the present method as shown in Fig. 6(b). In the next study, their potential performance as electrodes in a microfluidic chip will be investigated for microfluidic biofuel cells.
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Fig. 5. XPS results for ox-MWCNT/Nafion nanocomposites (10 wt% ox-MWCNT/Nafion nanocomposites, oxygen plasma power: 120 W): (a) before plasma etching, (b) after 10 min of plasma etching.
Fig. 6. (a) Schematic image of microfluidic device having microelectrode arrays and (b) possible examples of electrode array integrated into microfluidic channel.
4. Conclusions
References
We demonstrate the fabrication method of enzyme-modified, 3D microelectrode arrays for microfluidic biofuel cells. The 3D microelectrode arrays were fabricated using CNT/Nafion nanocomposites and hydrogel microstencils. The micro-porous structure of the 3D microelectrode arrays was exposed by removing the Nafion layer on the CNT surface using a plasma-etching process. The plasma-etching process caused some decrease in electrical conductivity, but it provided an increased surface area conducive to the immobilization of enzymes. This enzyme-modified, micro-porous, 3D microelectrode can be applied in microfluidic biofuel cells, possibly improving performance.
[1] J.W. Lee, E. Kjeang, Biomicrofluidics 4 (2010) 041301. [2] A.S. Bedekar, J.J. Feng, S. Krishnamoorthy, K.G. Lim, G.T.R. Palmore, S. Sundaram, Chem. Eng. Comm. 195 (2008) 256–266. [3] E.R. Choban, L.J. Markoski, A. Wieckowski, P.J.A. Kenis, J. Power Sources 128 (2004) 54–60. [4] E. Kjeang, N. Djilali, D. Sinton, J. Power Sources 186 (2009) 353–369. [5] M.J. González-Guerrero, J.P. Esquivel, D. Sánchez-Molas, P. Godignon, F.X. Muñoz, F.J. del Campo, F. Giroud, S.D. Minteer, N. Sabaté, Lab Chip 13 (2013) 2972–2979. [6] E. Kjeang, R. Michel, D.A. Harrington, N. Djilali, D. Sinton, J. Am. Chem. Soc. 130 (2008) 4000–4006. [7] J.H. Choi, H. Lee, H.K. Jin, J.-S. Bae, G.M. Kim, Lab Chip 12 (2012) 5045–5050. [8] J.H. Choi, G.M. Kim, Int. J. Precis. Eng. Manuf. 12 (2011) 165–168. [9] G.M. Kim, M.A.F. van den Boogaart, J. Brugger, Microelectron. Eng. 67 (2003) 609–614. [10] T.D. Dang, Y.H. Kim, J.H. Choi, G.M. Kim, J. Micromech. Microeng. 22 (2012) 015017. [11] J.H. Choi, H.K. Jin, J.-S. Bae, C.W. Park, I.W. Cheong, G.M. Kim, Int. J. Precis. Eng. Manuf. 15 (2014) 945–948. [12] I.D. Rosca, F. Watari, M. Uo, T. Akasaka, Carbon 43 (2005) 3124–3131. [13] S.J. Kim, I.T. Lee, Y.H. Kim, Smart Mater. Struct. 16 (2007) N6–N11. [14] T. Beneyton, I.P.M. Wijaya, C.B. Salem, A.D. Griffiths, V. Taly, Chem. Commun. 49 (2013) 1094–1096. [15] T. Tamaki, Top. Catal. 55 (2012) 1162–1180. [16] L. Stobinski, B. Lesiak, L. Kövér, J. Tóth, S. Biniak, G. Trykowski, J. Judek, J. Alloys Comp. 501 (2010) 77–84. [17] S. Kim, H.J. Kim, H.R. Lee, J.H. Song, S.N. Yi, D.H. Ha, J. Phys. D Appl. Phys. 43 (2010) 305402.
Acknowledgments This work was supported by the National Research Foundation of Korea Grant (NRF-2012K1A3A1A19038448, NRF-2013R1A1A 2060944) funded by the Korean Government (Ministry of Education, Science and Technology). The authors from India thank the Department of Science and Technology, Government of India for financial support under Indo-Korea Project (INT/Korea/P-18/ 2013).
Contents lists available at ScienceDirect
Microelectronic Engineering journal homepage: www.elsevier.com/locate/mee
Enzyme immobilization on microelectrode arrays of CNT/Nafion nanocomposites fabricated using hydrogel microstencils Sung Deuk Choi a, Jin Ho Choi a, Young Ho Kim b, Sung Yeol Kim a, Prabhat K. Dwivedi c, Ashutosh Sharma c, Sanket Goel d, Gyu Man Kim a,⇑ a
School of Mechanical Engineering, Kyungpook National University, Daegu 702-701, South Korea Medical Device Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu 701-310, South Korea Department of Chemical Engineering, Indian Institute of Technology, Kanpur 208016, India d Department of Electrical, Electronics and Instrumentation Engineering, University of Petroleum and Energy Studies (UPES), Dehradun 248007, India b c
a r t i c l e
i n f o
Article history: Received 10 October 2014 Received in revised form 4 February 2015 Accepted 24 March 2015 Available online 28 March 2015 Keywords: Microfluidic biofuel cells 3D microelectrode Carbon nanotube Nanocomposites Hydrogel microstencil
a b s t r a c t Enzyme-modified, three-dimensional (3D) microelectrode arrays for microfluidic biofuel cell applications were fabricated using carbon nanotube (CNT) reinforced NafionÒ nanocomposites and hydrogel microstenciling. CNT/Nafion nanocomposites were prepared by dispersing oxidized, multi-walled CNTs in Nafion solution. A hydrogel microstencil was fabricated using photolithography, soft lithography, and capillary-force lithography. After structuring the nanocomposites with the microstencil, a plasmaetching process was applied to the surface of the nanocomposite in order to obtain a micro-porous structure. Enzymes could be successfully immobilized on the micro-porous structures of nanocomposite surfaces using direct covalent binding. Ó 2015 Published by Elsevier B.V.
1. Introduction Over the past few decades, interest in biofuel cells has increased because of their many advantages including use of low-cost catalysts compared to conventional fuel cells and mild operating conditions [1]. In biofuel cell systems, a biofuel such as glucose is oxidized at the anode side and an oxidant such as O2 is reduced at the cathode side; generally the sides are separated by an ionconducting membrane in order to prevent fuel and oxidant from mixing [2]. In recent years, biofuel cells have employed microfluidic technology in response to the demand for miniaturized fuel cells for portable electronics [3,4]. In a microchannel, the fuel stream and the oxidant stream form a laminar flow without mixing, and therefore microfluidic fuel cells are able to operate efficiently without an ion-conducting membrane which causes ohmic loss [5]. However, most of the microfluidic biofuel cells reported to date have two-dimensional (2D) electrodes, and are thus limited in fuel utilization because of low surface area. Kjeang et al. reported a microfluidic fuel cell incorporating a three-dimensional (3D), ⇑ Corresponding author at: School of Mechanical Engineering, Kyungpook National University, 1370 Sankyuk-dong, Buk-gu, Daegu 702-701, South Korea. Tel.: +82 53 950 7570; fax: +82 53 950 6550. E-mail address: [email protected] (G.M. Kim). http://dx.doi.org/10.1016/j.mee.2015.03.045 0167-9317/Ó 2015 Published by Elsevier B.V.
flow-through, porous electrode made of carbon paper [6]. Although it was not a biofuel cell, fuel utilization could be increased to 94% by using vanadium as the electrolyte material. Similarly, the efficiency of a microfluidic biofuel cell is expected to increase when 3D microelectrodes are employed due to their relatively larger surface area. The commonly used fabrication method of electrodes for microfluidic devices consists of metal deposition and photolithography followed by selective etching. However, this conventional method provides very thin electrodes of less than several hundred nanometers and therefore is not suitable for the fabrication of a thick electrode. An alternative method is to use a microstencil as a template. A microstencil is a membrane that has patterned perforations [7,8]. The use of a microstencil allows patterning with any material, even neural stem cells [7]. Microstencils can be made of solid states of silicon [9], metal, and also flexible polymer. When using hydrogel, it is possible to fabricate a very thick micro-structure greater than several hundred micrometers [10,11]. Furthermore, the patterning process is simple and does not require expensive photolithography equipment. Therefore, a thick electrode can be fabricated readily by adopting a hydrogel-microstencil method. An enzymatic biofuel cell uses a specific enzyme as a biological catalyst. Enzymes such as glucose oxidase or laccase in microfluidic biofuel cells can exist in solution or on an electrode. The former is
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not an effective design choice because it not only requires more enzymes than the latter, but also the enzymes in solution have a short active lifetime [5]. Therefore, enzymes need to be immobilized on electrodes for better enzyme stability and long-term efficiency; an electrode that has high surface permits the immobilization of more enzymes. Immobilization technique on a miniaturized electrode is one of important issues for a microfluidic based fuel cell devices. In this study, we propose methods of fabrication of microporous 3D microelectrode arrays and enzyme immobilization on the microelectrodes for microfluidic biofuel cells. To enhance enzyme immobilization efficiency, the 3D microelectrodes were prepared porous structure and then two enzymes such as glucose oxidase and laccase were immobilized on the surface of porous microelectrodes. 2. Experimental 2.1. Materials Multi-walled carbon nanotubes (MWCNTs, >93% purity, 10–40 nm average diameter, 1–25 lm long) were purchased from CNT Co., Ltd (South Korea). Reagent grade 60% nitric acid was obtained from Duksan Chemical (South Korea). 5% Nafion solution (D521) was acquired from DuPont (USA). Polyethylene glycol diacrylate (PEGDA), 2-hydroxy-2-methylpropiophenone 98% (photo initiator), N-(3-dimethylaminopropyl)-N0 -ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), glucose oxidase from Aspergillus Niger and laccase from Trametes Versicolor were purchased from Sigma–Aldrich (USA). Sodium phosphate monobasic (NaH2PO4) and sodium phosphate dibasic (Na2HPO4) were obtained from Sigma–Aldrich (USA) and used to prepare a phosphate buffer solution. SU-8 2100 and SU-8 developer were obtained from Microchem (USA). Polydimethylsiloxane (PDMS) prepolymer and curing agent (Sylgard 184) were acquired from Dow Corning (USA).
added into Nafion solution, and were then dispersed via sonication for 1 h; a well-dispersed solution of CNT/Nafion nanocomposites was used for fabrication of the 3D microelectrode arrays. 2.4. Fabrication of the 3D microelectrode arrays The 3D microelectrode arrays were fabricated by using a hydrogel microstencil and CNT/Nafion nanocomposites. Fig. 1 shows the fabrication procedure of the 3D microelectrode arrays. Firstly, a PDMS stamp, which was prepared using general photolithography and soft lithography, was placed on a glass as substrate [10]. The space between the PDMS stamp and the substrate was filled using capillary force with PEGDA hydrogel solution containing 2 wt% photo initiator. After curing the hydrogel solution by UV exposure, the PDMS stamp was separated from the substrate. Subsequently, the substrate was rinsed with DI water in order to wash away uncured hydrogel solution, and then dried with nitrogen gas [10]. The CNT/Nafion nanocomposite solution was pipetted onto the hydrogel microstencil, filling the inside of the microstencil pattern. The excess solution was removed using a doctor blade. Subsequently, the substrate was heated on a hotplate (65 °C, 2 min) to remove the solvent of the CNT/Nafion nanocomposites. After separating the hydrogel microstencil from the substrate, the surface of the 3D microelectrode was etched using an oxygen-plasma treatment (V15-G, Plasma Finish, Germany) at high power (120 W) for 10 min [13]. 2.5. Enzyme immobilization GOD and laccase were immobilized on the surface of CNT/ Nafion nanocomposites using direct covalent binding [14]. First, 30 mM EDC and 90 mM NHS were dissolved in a phosphate buffer solution (pH 7.0, 50 mM); the solution was pipetted onto the nanocomposite surface. After 1 h, the nanocomposite surface was dried using nitrogen gas. Then, a phosphate buffer solution (pH 7.0, 50 mM) containing 4 mg/mL of GOD or laccase was dropped onto the surface and allowed to react for 10 h.
2.2. Characterization The structure and size of the 3D microelectrode arrays were examined using a field-emission scanning electron microscope (FE-SEM, S-4800, Hitachi, Japan). The sheet resistance of the CNT/ Nafion nanocomposites was measured using a four-point probe (CMT-SR1060N, Changmin Tech Co., Ltd, South Korea). The electrical conductivity of the CNT/Nafion nanocomposites was calculated by multiplying the sheet resistance and the thickness of nanocomposite film. The atomic concentration of CNT/Nafion nanocomposites was analyzed using X-ray photoelectron spectroscopy (XPS, Quantera SXM, ULVAC-PHI, Japan). 2.3. Preparation of CNT/Nafion nanocomposites The MWCNTs were treated using an oxidation method previously reported [12]. Briefly, 0.5 g of pristine MWCNTs were dispersed in 200 mL of 60% nitric acid using an ultrasonic bath (JAC Ultrasonic 4020P, KODO, South Korea) for 1 h. Then, the solution was refluxed at 120 °C for 12 h under nitrogen gas and was cooled down to room temperature. Subsequently, the MWCNTs in the solution were washed and filtered repeatedly with deionized (DI) water and membrane-filtered (0.22 lm, Nitrocelluose, Millipore, USA) until the pH of the solution was near neutral. After drying in an oven at 60 °C for 48 h, the MWCNTs were stored in a vial before use. To prepare the CNT/Nafion nanocomposites, 10 wt% oxidized MWCNTs (ox-MWCNTs) based on Nafion polymer weight were
3. Results and discussion 3.1. 3D microelectrode arrays with micro-porous structure Electrodes for fuel cells are required to have both electrical conductivity and large surface area in order to react promptly with fuel or oxidant [15]. To enhance the surface area of microelectrode arrays, microelectrodes consist of CNT and Nafion nanocomposites were treated by applying plasma. Fig. 2 shows the plasma-etching effect on CNT/Nafion nanocomposites. When the nanocomposite electrodes are structured by stenciling, most of the CNT surfaces are covered with Nafion as shown in Fig. 2(a). Thus, only a limited area of CNT surface is provided on the electrode for immobilization of enzymes. In order to increase the active CNT surface for the immobilization of enzymes, the Nafion layer on the electrode surface must be etched away. Oxygen-plasma etching was adopted for Nafion removal; Nafion on the electrode surface was removed and a micro-porous structure of CNT/Nafion nanocomposites was obtained, as shown in Fig. 2(b). The surface area of CNT/Nafion nanocomposites is significantly increased because of the microporous structure. The size of the 3D microelectrode arrays was determined by the hydrogel microstencil because the electrode was fabricated using a solution-casting method. Using this method, we prepared 3D microelectrode arrays with different diameters (100 lm and 500 lm) and different thicknesses ranging from several micrometers to hundred micrometers, as shown in Fig. 3. These
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Fig. 1. Schematic images of preparation procedure for hydrogel microstencil (on left) and fabrication procedure of 3D microelectrode arrays from ox-MWCNT/Nafion nanocomposites (on right).
Fig. 2. FE-SEM images of plasma-etching effect on ox-MWCNT/Nafion nanocomposites (10 wt% ox-MWCNT/Nafion nanocomposites, oxygen plasma power: 120 W): (a) before plasma etching, (b) after 10 min of plasma etching.
micro-porous, 3D microelectrode arrays have high surface area and can be applied to microfluidic biofuel cell.
[14]. The GOD- and laccase-modified 3D microelectrodes can be used as the anode and cathode of microfluidic biofuel cells.
3.2. Enzyme immobilization on CNT/Nafion nanocomposites
3.3. Electrical properties of CNT/Nafion nanocomposites
Fig. 4 shows the result of GOD and laccase immobilization on the CNT/Nafion nanocomposites. The ox-MWCNTs have a carboxyl group [16] and are able to be activated by EDC/NHS; enzymes could be immobilized on the exposed surface of the ox-MWCNTs
The electrical conductivity of CNT/Nafion nanocomposites was measured in order to estimate the performance of the 3D microelectrodes. Four samples – ox-MWCNT, Nafion, and ox-MWCNT/ Nafion nanocomposites before and after plasma etching – were
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Fig. 3. FE-SEM images of different-sized 3D microelectrode arrays (10 wt% ox-MWCNT/Nafion nanocomposites, oxygen plasma-etching time:10 min): (a) 100 lm in diameter, 5 lm-thick microelectrode array, (b) 500 lm in diameter, 60 lm-thick microelectrode array.
Fig. 4. FE-SEM images of (a) GOD-modified CNT/Nafion nanocomposites, (b) laccase-modified CNT/Nafion nanocomposites.
prepared and measured for sheet resistance and film thickness. Table 1 shows the results for electrical conductivity. Ox-MWCNT had the highest electrical conductivity of the four samples and Nafion the lowest. In the cases of CNT/Nafion nanocomposites, plasma-etched samples had lower electrical conductivity than that of the non-etched. The decrease in the electrical conductivity of CNT/Nafion nanocomposites is a result of the oxygen-plasma etching process. Kim et al. reported an increase in the electrical resistance of carbon nanotubes caused by oxygen plasma [17]. According to the XPS results for the CNT/Nafion nanocomposites (Fig. 5), the relative atomic-concentration ratio of oxygen and carbon varied with plasma etching. The C1s:O1s atomic concentration Table 1 Electrical conductivity of samples. Sample
Electrical conductivity (S/m)
ox-MWCNT Nafion ox-MWCNT/Nafion (before etching) ox-MWCNT/Nafion (after etching)
1741 0.04305 51.04 ± 1.70 34.49 ± 0.32
ratio is 93.3:6.7% for the non-etched sample and 84.3:15.7% for the plasma-etched sample. The plasma-etching process led to an increase of oxygen percentage in the CNT/Nafion nanocomposites. Thus, the abundance of oxygen functional groups such as carboxyl and carbonyl were increased in CNT/Nafion nanocomposites; therefore, the electrical conduction paths in the carbon nanotubes were broken. Even though the electrical conductivity of the CNT/ Nafion nanocomposites was decreased by the plasma-etching process, the process provided the high surface area needed to immobilize an increased amount of enzymes because of the increase in exposed CNT surface. Therefore, a microfluidic device having microelectrode arrays and possible examples of electrode array integrated into microfluidic channel was designed and prepared as shown in Fig. 6. A schematic image of microfluidic device is shown in Fig. 6(a). Then, 3D microelectrode arrays having micro-porous structure in a microfluidic chip were fabricated by the present method as shown in Fig. 6(b). In the next study, their potential performance as electrodes in a microfluidic chip will be investigated for microfluidic biofuel cells.
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Fig. 5. XPS results for ox-MWCNT/Nafion nanocomposites (10 wt% ox-MWCNT/Nafion nanocomposites, oxygen plasma power: 120 W): (a) before plasma etching, (b) after 10 min of plasma etching.
Fig. 6. (a) Schematic image of microfluidic device having microelectrode arrays and (b) possible examples of electrode array integrated into microfluidic channel.
4. Conclusions
References
We demonstrate the fabrication method of enzyme-modified, 3D microelectrode arrays for microfluidic biofuel cells. The 3D microelectrode arrays were fabricated using CNT/Nafion nanocomposites and hydrogel microstencils. The micro-porous structure of the 3D microelectrode arrays was exposed by removing the Nafion layer on the CNT surface using a plasma-etching process. The plasma-etching process caused some decrease in electrical conductivity, but it provided an increased surface area conducive to the immobilization of enzymes. This enzyme-modified, micro-porous, 3D microelectrode can be applied in microfluidic biofuel cells, possibly improving performance.
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Acknowledgments This work was supported by the National Research Foundation of Korea Grant (NRF-2012K1A3A1A19038448, NRF-2013R1A1A 2060944) funded by the Korean Government (Ministry of Education, Science and Technology). The authors from India thank the Department of Science and Technology, Government of India for financial support under Indo-Korea Project (INT/Korea/P-18/ 2013).