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Three-dimensional graphene micropillar based electrochemical sensor for phenol detection
Biosensors and Bioelectronics 50 (2013) 387–392
Contents lists available at SciVerse ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Short communication
Three-dimensional graphene micropillar based electrochemical sensor for phenol detection Fei Liu, Yunxian Piao, Jong Seob Choi, Tae Seok Seo n Department of Chemical and Biomolecular Engineering (BK21 Program), Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea
art ic l e i nf o
a b s t r a c t
Article history: Received 9 April 2013 Received in revised form 7 June 2013 Accepted 26 June 2013 Available online 4 July 2013
A three-dimensional (3D) graphene incorporated electrochemical sensor was constructed for sensitive enzyme based phenol detection. To form the 3D graphene structure, polydimethylsiloxane (PDMS) micropillars were fabricated in the microchannel by using a conventional photolithography and the surface was modified with 3-aminopropyltriethoxysilane. Then, the negatively charged graphene oxide sheets were electrostatically adsorbed on the PDMS micropillar surface, and reduced in the hydrazine vapor. The resultant 3D graphene film provides a conductive working electrode as well as an enzymemediated sensor with a large surface area. After bonded with an electrode patterned glass wafer, the 3D graphene based electrochemical sensor was produced. Using the 3D graphene as a working electrode, an excellent electron transfer property was demonstrated by cyclic voltammetry measurement in an electrolyte solution containing 1 mM K3Fe(CN)6 and 0.1 M KCl. To utilize the 3D graphene as an enzyme sensor, tyrosinase enzymes were immobilized on the surface of the graphene micropillar, and the target phenol was injected in the microchannel. The enzyme catalytic reaction process was monitored by amperometric responses and the limit of detection for phenol was obtained as 50 nM, thereby suggesting that the 3D graphene micropillar structure enhances the enzyme biosensing capability not only by increasing the surface area for enzyme immobilization, but also by the superlative graphene conductivity property. & 2013 Published by Elsevier B.V.
Keywords: Graphene Micropilar Electrochemical sensor Phenol detection Enzyme
1. Introduction Graphene and its derivatives have attracted considerable attention due to its super large surface area, biocompatibility, excellent electrocatalytic activity, fast electron transfer, strong mechanical strength and high chemical stability (Geim., 2009). Due to these extraordinary properties, graphene has been extensively applied for supercapacitors, solar cells, batteries, and flexible electronic devices (Zhu et al., 2010; Eda and Chhowalla, 2010). In terms of the synthetic method for graphene, reduction via graphene oxide (GO) is widely accepted due to its facile chemical process, mass production capability, and no use of complicated instruments, although some defects remain in the reduced graphene structure (Dreyer et al., 2010). The GO nanomaterials have enriched multifarious applications, in particular, in the fields of nanocomposites with polymer, and optical and electrical sensors (Shao et al., 2010; Pumera et al., 2010). Regarding the electrochemical sensors, the majority of graphene and its
n
Corresponding author. Tel.: +82 42 350 3933; fax: +82 42 350 3910. E-mail address: [email protected] (T.S. Seo).
0956-5663/$ - see front matter & 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.bios.2013.06.055
nanocomposites have been applied to modify a glassy carbon electrode (GCE) for immobilizing various biomolecules (Guo et al., 2010; Zhou et al., 2009; Shan et al., 2009a, 2009b; Wang et al., 2010; Song et al., 2011). For instance, Kang et al. reported a glucose electrochemical biosensor by using a graphene–chitosan film modified GCE (Kang et al., 2009). Shan et al. presented a graphene–glucose oxidase (GOD) biosensor by utilizing a GODpolyethylenimine-functionalized GCE (Shan et al., 2009a, 2009b). Liu et al. demonstrated a poly(diallyl-dimethylammonium chloride)/graphene film covered on the GCE as an electrochemical biosensor for detecting nitrate (Liu et al., 2010a, 2010b). At the same time, multi-layered graphene films have also been applied as working electrodes instead of the GCE in the electrochemical system, and have shown good electrochemical performance. Choi et al. prepared a free-standing reduced GO/Nafion hybrid film for detecting organophosphate (Choi et al., 2010a, 2010b). Our group has also reported an electrochemical biosensor based on the freestanding graphene/gold nanoparticle composite film for phenol detection, and found that the addition of the gold nanoparticles contributed to the synergistic effect on the electrochemical response of the graphene film (Liu et al., 2011, 2012a, 2012b). Thus, most of the graphene based electrochemical sensors have
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two designs up to now: the assembly of two-dimensional (2D) GOs on the GCE or multi-stacked graphene films. Since the biosensing reaction occurs on the surface of graphene, the ideal structure of graphene would be three-dimensional to enhance the detection sensitivity. In this study, we presented a 3D graphene micropillar structure to serve as an electrochemical sensor to detect phenol with high sensitivity. The PDMS micropillars were patterned in the microfluidic channel, and the GO was self-assembled and reduced on the surface of the micropillars to form the 3D graphene film. The resultant 3D graphene provides an efficient working electrode with a large surface area for enzyme immobilization. Tyrosinase (TYR) was loaded on the 3D graphene, and the produced 3D graphene micropillar/TYR biosensor was utilized for detecting phenol, which is considered one of the important environmental pollutants due to high toxicity.
2. Material and methods The GO suspension was prepared by the modified Hummers method following our previously published works, and the 3D PDMS micropillar structure was fabricated in Fig. S1. Using the GO and the 3D PDMS micropillars, the 3D graphene micropillar was constructed as shown in Fig. S2. The electrode patterned glass substrate was prepared following a standard photolithography process (Fig. S3). All the detailed information was described in the Supplementary Information.
2.1. Assembly of the patterned PDMS layer with the glass substrate The prepared electrode-patterned glass substrate was cleaned with acetone, isopropanol, and distilled water, and dried in N2 gas. After O2 plasma treatment for 1 min, 200 mL of a FeCl3 aqueous solution (0.1 M) was dropped on the patterned Ag electrode and maintained for 1 min until the Ag electrode changed to black, resulting in an R electrode (Ag/AgCl) (Polk et al., 2006; Ha et al., 2005). After washing with distilled water and drying in N2 gas, the glass substrate was permanently bonded with the PDMS layer at 80 1C overnight. The detailed design and dimension of the microchannel and micropillars in the PDMS layer are shown in Fig. 1a and b. The function of the assembled device was tested by cyclic voltammetry (CV) after injection with an electrolyte solution containing 1 mM K3Fe(CN)6 and 0.1 M KCl. To avoid bubble generation during measurement, we pretreated the device with O2 plasma to make the surface hydrophilic.
3. Results and discussion 3.1. Characterization of the 3D graphene micropillar The 2D GO sheets were prepared by the modified Hummers method using the graphite flakes (Hummers et al., 1958; Liu et al., 2012a, 2012b). The size of the resultant GOs ranged from several hundred nanometers to a few micrometers, and they mostly exist as a monolayer (Fig. S4). Due to oxidation process, the negative charged functional groups such as carboxylic acid, hydroxy, and epoxy are dominant on the GO surface. Since the PDMS micropillars were modified with APTES, positive charged amine groups were available which led to strong electrostatic interaction with the 2D GO. The self-assembled GO forms the multi-stacked film which results in the low sheet resistance than a single layered GO (Hummers et al., 1958). Recovery to the pristine graphene from the GO is essential to properly perform the electrochemical sensor. There are several reports for the reduction process of GO including thermal annealing, microwave based reduction, and chemical and electrochemical reduction (Compton et al., 2010; Pei et al., 2012). The reduced GO showed good conductivity, high chemical stability, and electrochemical property (Mao et al., 2012; Chen et al., 2012; Yang et al., 2009; Tang et al., 2009). Since the GO in our case was coated on the PDMS polymer materials, we employed a hydrazine hydrate vapor method for the reduction instead of using a thermal method. Fig. 1c and d showed C 1s XPS spectra before and after reduction of the 3D GO micropillar to estimate the recovering level of sp2 structure. The C1s XPS spectrum of the 3D GO micropillar displays the presence of three kinds of carbon atoms (Fig. 1c): nonoxygenated ring carbons (C–C) at 284.5 eV (45.49%), carbons in epoxy/ether (C–O) at 286.7 eV (42.72%), and carboxylate carbons (O–CQO) at 288.7 eV (11.78%). After 10 h reduction in hydrazine hydrate vapor, the C1s XPS date of the 3D graphene micropillar represents a dominant C–C carbon peak with high suppression of oxygenated carbon peaks. After reduction in hydrazine hydrate vapor, the morphology of the 3D graphene micropillars was investigated by SEM (Fig. 2a–d). Fig. 2a and b show that PDMS micropillars were well patterned in the middle and at the edge in a large area, and the graphene sheets were self-assembled on the surface of the micropillars without any defects. Fig. 2c displays a magnified SEM image for single graphene micropillar. Owing to the strongly and densely packed graphene sheets, microwave structures were clearly observed. The height and diameter of the 3D graphene micropillar were 38 and 27 mm, respectively, which were well matched with our design. If the PDMS micropillars were not completely covered by the graphene film, the charging effect became serious, revealing uncharacteristically bright in the SEM image as shown in Fig. 2d.
2.2. Enzyme-mediated electrochemical sensor for phenol detection
3.2. Electrochemical behavior of the 3D graphene sensor
The TYR obtained from the mushroom enzyme (Sigma) was immobilized on the surface of the 3D graphene micropillars by physical adsorption. Five mL of the TYR solution dissolved in a phosphate buffer (PB) (10 mg/mL) were dropped on the 3D graphene micropillar surface and incubated at 30 1C in a humid chamber for 4 h. Washing with a 0.1 M PB solution and drying in N2 gas were subsequently followed. The enzyme-loaded PDMS layer was bonded with the glass layer on which the O2 plasma was pretreated. Phenol samples were prepared in a 0.1 M PB solution with various concentration (0, 10, 50, 100, 500, 1000, 1500 and 2000 nM), and injected into the microfluidic channel from the sample inlet hole. After 10 min incubation, the plot of the amperometric current vs. time was obtained under the potential of 100 mV at room temperature.
The image of the experimental setup using the 3D graphene micropillar based electrochemical sensor chip was shown in Fig. 2e. The device consists of the microchannel for the sample injection, the 3D graphene micropillar, and the two Au and one Ag/AgCl electrodes. The dimension of our device is similar to that of a 100 Korean coin (Diameter 2.4 cm) (Inset of Fig. 2e). Cyclic voltammetric response was monitored by filling the microchannel with an electrolyte solution containing 1 mM K3Fe(CN)6 and 0.1 M KCl. The 3D graphene micropillar worked as a W electrode, and the patterned Ag/AgCl functioned as an R electrode. CV was carried out at different scan rates from 3 to 10 mV/s with a potential range from 0.2 to 0.6 V (Fig. 3a). A series of reversible CV curves were obtained with distinct redox peaks, and the CV area became larger with increasing the scan rate. While the anodic peak potential (Epa)
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Fig. 1. (a) Detailed design and dimension of the microchannel in the PDMS layer and the patterned electrodes in the glass substrate (unit: μm). (b) Illustration of a 3D graphene micropillar incorporated electrochemical sensor device. The microfluidic channel was patterned in the PDMS layer for sample loading, and the graphene micropillar was fabricated in the middle of the microchannel. The working (W), counter (C) and reference (R) electrodes were deposited on a glass substrate. C 1s XPS spectra of (c) the 3D GO on the PDMS micropillars and (d) the reduced 3D GO on the PDMS micropillars.
moved in a positive direction as the scan rate increased, the cathodic peak potential (Epc) moved in a negative direction, thereby producing gradual increment of a peak-to-peak potential separation (△Ep). The △Ep as 418 mV at 10 mV/s scan rate, and 201 mV at 3 mV/ s. In proportion to the scan rate, both the absolute value of anodic (Ipa) and cathodic peak current (Ipc) became larger, while maintaining the ratio of the Ipa to Ipc as 1.0, and showed a linear relationship with the potential scan rate. The linear regression equations can be expressed as Ipa ¼0.3636v+0.5808 (Ipa: mA, v: mV/s, R2: 0.9935) and Ipc ¼ 0.1431v–0.5219 (Ipc: mA, v: mV/s, R2: 0.9850) (Fig. 3b). The perfect liner relationship indicates that the electrochemical reaction is controlled by a semi-infinite linear diffusion based redox process from the electrolyte to the 3D graphene film electrode surface. Compared with the 2D graphene film, both Ipa and Ipc of the 3D graphene micropillar showed 3.8-fold enhancement (Fig. S5). The stability of the graphene micropillar electrode was evaluated by performing the continuous scan (100 cycles) at the high scan rate of 100 mV s 1, and the change of the current response was recorded only within 5% variation, demonstrating excellent stability of the 3D graphene micropillar electrode (Fig. S6). 3.3. Electrochemical 3D graphene sensor for phenol detection Taking full advantages of the rapid electron transfer property and a large area for enzyme immobilization of the as-synthesized 3D graphene micropillar, we performed the TYR-mediated phenol detection. The TYR, well known as a polyphenol oxidase, can
catalyze two reactions of phenol in the presence of oxygen. Phenol is hydroxylated to form catechol, and catechol is further oxidized to form o-quinone, which is electrochemically active and can be reduced back to catechol at the electrode (Song et al., 2011). The sensing mechanism of our proposed 3D graphene micropillar/TYR biosensor relies on the reduction of the oquinone, the enzymatic oxidation product, whose amperometric detection occurs at negative potential ( 100 mV). Such a low reduction potential is quite different from that of the interfering substances such as uric acid and ascorbic acid. Moreover, the tyrosinase enzyme-mediated biosensor was specific to the target phenol, thereby preventing the interference issues from undesirable molecules. (Jackowska and Krysinski, 2013; Zhou et al., 2007). Firstly, we confirmed the TYR immobilization and the phenol detection on the TYR immobilized 3D graphene micropillar electrode by monitoring the current change in the CV measurement. As shown in Fig. S7, after the TYR immobilization, the current was reduced compared with that of the pristine 3D graphene micropillar due to the electron transfer resistance by the enzyme, meaning the successful attachment of enzymes on the 3D graphene micropillar. However, upon the addition of phenol on the TYR immobilized 3D graphene micropillar electrode, the CV redox signal was enhanced, indicating that the immobilized TYR enzymes on the surface of the 3D graphene micropillars were electrochemically active and the phenol detection could be performed on the proposed graphene device.
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Fig. 2. (a, b) Low- and (c) high-magnification SEM images of the 3D graphene micropillars. (d) The PDMS micropillars uncovered with graphene are shown bright due to the charging effect. (e) Digital image of the graphene micropillar integrated electrochemical sensor device for phenol detection.
Owing to the micropillar structure, the amount of the enzyme loading could increase by 3-fold over the plane 2D graphene. To evaluate the detection sensitivity of our proposed device, various concentrations of phenol (0, 10, 50, 100, 500, 1000, 1500, 2000 nM), was injected into the microchannel in a 0.1 M PB solution, and the phenol-induced amperometric current–time responses were monitored for 100 s (Fig. 3c). The reference time point in our experiments was taken with the switch-on time of the measuring voltage. As shown in Fig. 3c, the amperometric response of the 3D graphene micropillar/TYR was rapid by showing that the reduction current was saturated within 20 s. The current shift (the current difference before and after phenol addition) was linearly proportional to the concentration of the injected phenol ranging from 50 nM to 2 mM with a linear regression equation of y¼0.0039x+0.5511 (R² ¼0.9427) (Fig. 3d). The detection sensitivity was determined as 3.9 nA mM 1 cm 1,
and the lowest detection limit was 50 nM, which is better than that of the gold nanoparticles-modified glassy carbon electrode method (Carralero Sanz et al., 2005) and that of the electropolymerized para-toluene sulfonate-doped polypyrrole film method (Rajesh et al., 2004). The reproducibility of the 3D graphene micropillar/TYR sensor was evaluated by detecting 2 mM phenol at 100 mV. The average steadystate current was 4.68 nA with a relative standard deviation (R.S.D.) of 5.36% which was calculated from five independent measurements. We found that the 3D graphene micropillar/TYR biosensor was stable at least for two weeks without loss of enzymatic activity when stored at 4 1C. The Michaelis–Menten constant (Km) for the immobilized tyrosinase enzyme was obtained from Fig. 3d. From the Lineweaver– Burk equation of Imax/Is ¼Km/C+1 where C is the concentration of the substrate, the tyrosinase-catalyzed conversion of a substrate has a Michaelis constant of 1.4 μM. These results imply that the 3D graphene
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Fig. 3. (a) CV curves of the graphene micropillar integrated electrochemical sensor at different scan rate from 3 to 10 mV/s in an electrolyte solution containing 1 mM K3Fe (CN)6 and 0.1 M KCl. (b) The plot of the anodic and cathodic peak current vs. the scan rate. (c) Amperometric current–time curve to detect phenol at various concentrations (0, 10, 50, 100, 500, 1000, 1500, and 2000 nM from top to bottom). (d) The calibration curve by plotting the amperometric current shift vs. the concentration of phenol.
micropillars with a large surface area could provide an advanced electrochemical platform by using high conductive property of graphene as well as increasing the amount of the immobilized enzymes.
the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012-0004343) and the Converging Research Center Program funded by the Ministry of Education, Science and Technology (2011K000837).
4. Conclusions In this study, we fabricated a 3D graphene based electrochemical sensor, and demonstrated its high performance for phenol detection. The 2D GO was multi-stacked on the 3D PDMS micropillars, and was reduced to form the conductive 3D graphene structure. After assembled with the electrode-patterned glass substrate, the 3D graphene could serve as a working electrode in the electrochemical sensing. The enzyme loading on the 3D graphene could be enhanced compared with the 2D structure, thereby improving the detection sensitivity. The TYR enzyme was immobilized and the TYR-mediated phenol detection was successfully performed with a limit of detection of 50 nM. This is the first demonstration of using 3D graphene as an excellent electrochemical biosensor in the microfluidic system. We expect that our proposed device could expand its applications in the fields of pathogen detection, healthcare screening, and environmental pollutant monitoring.
Acknowledgments This work was supported by the Converging Technology Project funded by the Korean Ministry of Environment (M112-00061-0002-0),
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2013.06.055.
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Contents lists available at SciVerse ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Short communication
Three-dimensional graphene micropillar based electrochemical sensor for phenol detection Fei Liu, Yunxian Piao, Jong Seob Choi, Tae Seok Seo n Department of Chemical and Biomolecular Engineering (BK21 Program), Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea
art ic l e i nf o
a b s t r a c t
Article history: Received 9 April 2013 Received in revised form 7 June 2013 Accepted 26 June 2013 Available online 4 July 2013
A three-dimensional (3D) graphene incorporated electrochemical sensor was constructed for sensitive enzyme based phenol detection. To form the 3D graphene structure, polydimethylsiloxane (PDMS) micropillars were fabricated in the microchannel by using a conventional photolithography and the surface was modified with 3-aminopropyltriethoxysilane. Then, the negatively charged graphene oxide sheets were electrostatically adsorbed on the PDMS micropillar surface, and reduced in the hydrazine vapor. The resultant 3D graphene film provides a conductive working electrode as well as an enzymemediated sensor with a large surface area. After bonded with an electrode patterned glass wafer, the 3D graphene based electrochemical sensor was produced. Using the 3D graphene as a working electrode, an excellent electron transfer property was demonstrated by cyclic voltammetry measurement in an electrolyte solution containing 1 mM K3Fe(CN)6 and 0.1 M KCl. To utilize the 3D graphene as an enzyme sensor, tyrosinase enzymes were immobilized on the surface of the graphene micropillar, and the target phenol was injected in the microchannel. The enzyme catalytic reaction process was monitored by amperometric responses and the limit of detection for phenol was obtained as 50 nM, thereby suggesting that the 3D graphene micropillar structure enhances the enzyme biosensing capability not only by increasing the surface area for enzyme immobilization, but also by the superlative graphene conductivity property. & 2013 Published by Elsevier B.V.
Keywords: Graphene Micropilar Electrochemical sensor Phenol detection Enzyme
1. Introduction Graphene and its derivatives have attracted considerable attention due to its super large surface area, biocompatibility, excellent electrocatalytic activity, fast electron transfer, strong mechanical strength and high chemical stability (Geim., 2009). Due to these extraordinary properties, graphene has been extensively applied for supercapacitors, solar cells, batteries, and flexible electronic devices (Zhu et al., 2010; Eda and Chhowalla, 2010). In terms of the synthetic method for graphene, reduction via graphene oxide (GO) is widely accepted due to its facile chemical process, mass production capability, and no use of complicated instruments, although some defects remain in the reduced graphene structure (Dreyer et al., 2010). The GO nanomaterials have enriched multifarious applications, in particular, in the fields of nanocomposites with polymer, and optical and electrical sensors (Shao et al., 2010; Pumera et al., 2010). Regarding the electrochemical sensors, the majority of graphene and its
n
Corresponding author. Tel.: +82 42 350 3933; fax: +82 42 350 3910. E-mail address: [email protected] (T.S. Seo).
0956-5663/$ - see front matter & 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.bios.2013.06.055
nanocomposites have been applied to modify a glassy carbon electrode (GCE) for immobilizing various biomolecules (Guo et al., 2010; Zhou et al., 2009; Shan et al., 2009a, 2009b; Wang et al., 2010; Song et al., 2011). For instance, Kang et al. reported a glucose electrochemical biosensor by using a graphene–chitosan film modified GCE (Kang et al., 2009). Shan et al. presented a graphene–glucose oxidase (GOD) biosensor by utilizing a GODpolyethylenimine-functionalized GCE (Shan et al., 2009a, 2009b). Liu et al. demonstrated a poly(diallyl-dimethylammonium chloride)/graphene film covered on the GCE as an electrochemical biosensor for detecting nitrate (Liu et al., 2010a, 2010b). At the same time, multi-layered graphene films have also been applied as working electrodes instead of the GCE in the electrochemical system, and have shown good electrochemical performance. Choi et al. prepared a free-standing reduced GO/Nafion hybrid film for detecting organophosphate (Choi et al., 2010a, 2010b). Our group has also reported an electrochemical biosensor based on the freestanding graphene/gold nanoparticle composite film for phenol detection, and found that the addition of the gold nanoparticles contributed to the synergistic effect on the electrochemical response of the graphene film (Liu et al., 2011, 2012a, 2012b). Thus, most of the graphene based electrochemical sensors have
388
F. Liu et al. / Biosensors and Bioelectronics 50 (2013) 387–392
two designs up to now: the assembly of two-dimensional (2D) GOs on the GCE or multi-stacked graphene films. Since the biosensing reaction occurs on the surface of graphene, the ideal structure of graphene would be three-dimensional to enhance the detection sensitivity. In this study, we presented a 3D graphene micropillar structure to serve as an electrochemical sensor to detect phenol with high sensitivity. The PDMS micropillars were patterned in the microfluidic channel, and the GO was self-assembled and reduced on the surface of the micropillars to form the 3D graphene film. The resultant 3D graphene provides an efficient working electrode with a large surface area for enzyme immobilization. Tyrosinase (TYR) was loaded on the 3D graphene, and the produced 3D graphene micropillar/TYR biosensor was utilized for detecting phenol, which is considered one of the important environmental pollutants due to high toxicity.
2. Material and methods The GO suspension was prepared by the modified Hummers method following our previously published works, and the 3D PDMS micropillar structure was fabricated in Fig. S1. Using the GO and the 3D PDMS micropillars, the 3D graphene micropillar was constructed as shown in Fig. S2. The electrode patterned glass substrate was prepared following a standard photolithography process (Fig. S3). All the detailed information was described in the Supplementary Information.
2.1. Assembly of the patterned PDMS layer with the glass substrate The prepared electrode-patterned glass substrate was cleaned with acetone, isopropanol, and distilled water, and dried in N2 gas. After O2 plasma treatment for 1 min, 200 mL of a FeCl3 aqueous solution (0.1 M) was dropped on the patterned Ag electrode and maintained for 1 min until the Ag electrode changed to black, resulting in an R electrode (Ag/AgCl) (Polk et al., 2006; Ha et al., 2005). After washing with distilled water and drying in N2 gas, the glass substrate was permanently bonded with the PDMS layer at 80 1C overnight. The detailed design and dimension of the microchannel and micropillars in the PDMS layer are shown in Fig. 1a and b. The function of the assembled device was tested by cyclic voltammetry (CV) after injection with an electrolyte solution containing 1 mM K3Fe(CN)6 and 0.1 M KCl. To avoid bubble generation during measurement, we pretreated the device with O2 plasma to make the surface hydrophilic.
3. Results and discussion 3.1. Characterization of the 3D graphene micropillar The 2D GO sheets were prepared by the modified Hummers method using the graphite flakes (Hummers et al., 1958; Liu et al., 2012a, 2012b). The size of the resultant GOs ranged from several hundred nanometers to a few micrometers, and they mostly exist as a monolayer (Fig. S4). Due to oxidation process, the negative charged functional groups such as carboxylic acid, hydroxy, and epoxy are dominant on the GO surface. Since the PDMS micropillars were modified with APTES, positive charged amine groups were available which led to strong electrostatic interaction with the 2D GO. The self-assembled GO forms the multi-stacked film which results in the low sheet resistance than a single layered GO (Hummers et al., 1958). Recovery to the pristine graphene from the GO is essential to properly perform the electrochemical sensor. There are several reports for the reduction process of GO including thermal annealing, microwave based reduction, and chemical and electrochemical reduction (Compton et al., 2010; Pei et al., 2012). The reduced GO showed good conductivity, high chemical stability, and electrochemical property (Mao et al., 2012; Chen et al., 2012; Yang et al., 2009; Tang et al., 2009). Since the GO in our case was coated on the PDMS polymer materials, we employed a hydrazine hydrate vapor method for the reduction instead of using a thermal method. Fig. 1c and d showed C 1s XPS spectra before and after reduction of the 3D GO micropillar to estimate the recovering level of sp2 structure. The C1s XPS spectrum of the 3D GO micropillar displays the presence of three kinds of carbon atoms (Fig. 1c): nonoxygenated ring carbons (C–C) at 284.5 eV (45.49%), carbons in epoxy/ether (C–O) at 286.7 eV (42.72%), and carboxylate carbons (O–CQO) at 288.7 eV (11.78%). After 10 h reduction in hydrazine hydrate vapor, the C1s XPS date of the 3D graphene micropillar represents a dominant C–C carbon peak with high suppression of oxygenated carbon peaks. After reduction in hydrazine hydrate vapor, the morphology of the 3D graphene micropillars was investigated by SEM (Fig. 2a–d). Fig. 2a and b show that PDMS micropillars were well patterned in the middle and at the edge in a large area, and the graphene sheets were self-assembled on the surface of the micropillars without any defects. Fig. 2c displays a magnified SEM image for single graphene micropillar. Owing to the strongly and densely packed graphene sheets, microwave structures were clearly observed. The height and diameter of the 3D graphene micropillar were 38 and 27 mm, respectively, which were well matched with our design. If the PDMS micropillars were not completely covered by the graphene film, the charging effect became serious, revealing uncharacteristically bright in the SEM image as shown in Fig. 2d.
2.2. Enzyme-mediated electrochemical sensor for phenol detection
3.2. Electrochemical behavior of the 3D graphene sensor
The TYR obtained from the mushroom enzyme (Sigma) was immobilized on the surface of the 3D graphene micropillars by physical adsorption. Five mL of the TYR solution dissolved in a phosphate buffer (PB) (10 mg/mL) were dropped on the 3D graphene micropillar surface and incubated at 30 1C in a humid chamber for 4 h. Washing with a 0.1 M PB solution and drying in N2 gas were subsequently followed. The enzyme-loaded PDMS layer was bonded with the glass layer on which the O2 plasma was pretreated. Phenol samples were prepared in a 0.1 M PB solution with various concentration (0, 10, 50, 100, 500, 1000, 1500 and 2000 nM), and injected into the microfluidic channel from the sample inlet hole. After 10 min incubation, the plot of the amperometric current vs. time was obtained under the potential of 100 mV at room temperature.
The image of the experimental setup using the 3D graphene micropillar based electrochemical sensor chip was shown in Fig. 2e. The device consists of the microchannel for the sample injection, the 3D graphene micropillar, and the two Au and one Ag/AgCl electrodes. The dimension of our device is similar to that of a 100 Korean coin (Diameter 2.4 cm) (Inset of Fig. 2e). Cyclic voltammetric response was monitored by filling the microchannel with an electrolyte solution containing 1 mM K3Fe(CN)6 and 0.1 M KCl. The 3D graphene micropillar worked as a W electrode, and the patterned Ag/AgCl functioned as an R electrode. CV was carried out at different scan rates from 3 to 10 mV/s with a potential range from 0.2 to 0.6 V (Fig. 3a). A series of reversible CV curves were obtained with distinct redox peaks, and the CV area became larger with increasing the scan rate. While the anodic peak potential (Epa)
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Fig. 1. (a) Detailed design and dimension of the microchannel in the PDMS layer and the patterned electrodes in the glass substrate (unit: μm). (b) Illustration of a 3D graphene micropillar incorporated electrochemical sensor device. The microfluidic channel was patterned in the PDMS layer for sample loading, and the graphene micropillar was fabricated in the middle of the microchannel. The working (W), counter (C) and reference (R) electrodes were deposited on a glass substrate. C 1s XPS spectra of (c) the 3D GO on the PDMS micropillars and (d) the reduced 3D GO on the PDMS micropillars.
moved in a positive direction as the scan rate increased, the cathodic peak potential (Epc) moved in a negative direction, thereby producing gradual increment of a peak-to-peak potential separation (△Ep). The △Ep as 418 mV at 10 mV/s scan rate, and 201 mV at 3 mV/ s. In proportion to the scan rate, both the absolute value of anodic (Ipa) and cathodic peak current (Ipc) became larger, while maintaining the ratio of the Ipa to Ipc as 1.0, and showed a linear relationship with the potential scan rate. The linear regression equations can be expressed as Ipa ¼0.3636v+0.5808 (Ipa: mA, v: mV/s, R2: 0.9935) and Ipc ¼ 0.1431v–0.5219 (Ipc: mA, v: mV/s, R2: 0.9850) (Fig. 3b). The perfect liner relationship indicates that the electrochemical reaction is controlled by a semi-infinite linear diffusion based redox process from the electrolyte to the 3D graphene film electrode surface. Compared with the 2D graphene film, both Ipa and Ipc of the 3D graphene micropillar showed 3.8-fold enhancement (Fig. S5). The stability of the graphene micropillar electrode was evaluated by performing the continuous scan (100 cycles) at the high scan rate of 100 mV s 1, and the change of the current response was recorded only within 5% variation, demonstrating excellent stability of the 3D graphene micropillar electrode (Fig. S6). 3.3. Electrochemical 3D graphene sensor for phenol detection Taking full advantages of the rapid electron transfer property and a large area for enzyme immobilization of the as-synthesized 3D graphene micropillar, we performed the TYR-mediated phenol detection. The TYR, well known as a polyphenol oxidase, can
catalyze two reactions of phenol in the presence of oxygen. Phenol is hydroxylated to form catechol, and catechol is further oxidized to form o-quinone, which is electrochemically active and can be reduced back to catechol at the electrode (Song et al., 2011). The sensing mechanism of our proposed 3D graphene micropillar/TYR biosensor relies on the reduction of the oquinone, the enzymatic oxidation product, whose amperometric detection occurs at negative potential ( 100 mV). Such a low reduction potential is quite different from that of the interfering substances such as uric acid and ascorbic acid. Moreover, the tyrosinase enzyme-mediated biosensor was specific to the target phenol, thereby preventing the interference issues from undesirable molecules. (Jackowska and Krysinski, 2013; Zhou et al., 2007). Firstly, we confirmed the TYR immobilization and the phenol detection on the TYR immobilized 3D graphene micropillar electrode by monitoring the current change in the CV measurement. As shown in Fig. S7, after the TYR immobilization, the current was reduced compared with that of the pristine 3D graphene micropillar due to the electron transfer resistance by the enzyme, meaning the successful attachment of enzymes on the 3D graphene micropillar. However, upon the addition of phenol on the TYR immobilized 3D graphene micropillar electrode, the CV redox signal was enhanced, indicating that the immobilized TYR enzymes on the surface of the 3D graphene micropillars were electrochemically active and the phenol detection could be performed on the proposed graphene device.
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Fig. 2. (a, b) Low- and (c) high-magnification SEM images of the 3D graphene micropillars. (d) The PDMS micropillars uncovered with graphene are shown bright due to the charging effect. (e) Digital image of the graphene micropillar integrated electrochemical sensor device for phenol detection.
Owing to the micropillar structure, the amount of the enzyme loading could increase by 3-fold over the plane 2D graphene. To evaluate the detection sensitivity of our proposed device, various concentrations of phenol (0, 10, 50, 100, 500, 1000, 1500, 2000 nM), was injected into the microchannel in a 0.1 M PB solution, and the phenol-induced amperometric current–time responses were monitored for 100 s (Fig. 3c). The reference time point in our experiments was taken with the switch-on time of the measuring voltage. As shown in Fig. 3c, the amperometric response of the 3D graphene micropillar/TYR was rapid by showing that the reduction current was saturated within 20 s. The current shift (the current difference before and after phenol addition) was linearly proportional to the concentration of the injected phenol ranging from 50 nM to 2 mM with a linear regression equation of y¼0.0039x+0.5511 (R² ¼0.9427) (Fig. 3d). The detection sensitivity was determined as 3.9 nA mM 1 cm 1,
and the lowest detection limit was 50 nM, which is better than that of the gold nanoparticles-modified glassy carbon electrode method (Carralero Sanz et al., 2005) and that of the electropolymerized para-toluene sulfonate-doped polypyrrole film method (Rajesh et al., 2004). The reproducibility of the 3D graphene micropillar/TYR sensor was evaluated by detecting 2 mM phenol at 100 mV. The average steadystate current was 4.68 nA with a relative standard deviation (R.S.D.) of 5.36% which was calculated from five independent measurements. We found that the 3D graphene micropillar/TYR biosensor was stable at least for two weeks without loss of enzymatic activity when stored at 4 1C. The Michaelis–Menten constant (Km) for the immobilized tyrosinase enzyme was obtained from Fig. 3d. From the Lineweaver– Burk equation of Imax/Is ¼Km/C+1 where C is the concentration of the substrate, the tyrosinase-catalyzed conversion of a substrate has a Michaelis constant of 1.4 μM. These results imply that the 3D graphene
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Fig. 3. (a) CV curves of the graphene micropillar integrated electrochemical sensor at different scan rate from 3 to 10 mV/s in an electrolyte solution containing 1 mM K3Fe (CN)6 and 0.1 M KCl. (b) The plot of the anodic and cathodic peak current vs. the scan rate. (c) Amperometric current–time curve to detect phenol at various concentrations (0, 10, 50, 100, 500, 1000, 1500, and 2000 nM from top to bottom). (d) The calibration curve by plotting the amperometric current shift vs. the concentration of phenol.
micropillars with a large surface area could provide an advanced electrochemical platform by using high conductive property of graphene as well as increasing the amount of the immobilized enzymes.
the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012-0004343) and the Converging Research Center Program funded by the Ministry of Education, Science and Technology (2011K000837).
4. Conclusions In this study, we fabricated a 3D graphene based electrochemical sensor, and demonstrated its high performance for phenol detection. The 2D GO was multi-stacked on the 3D PDMS micropillars, and was reduced to form the conductive 3D graphene structure. After assembled with the electrode-patterned glass substrate, the 3D graphene could serve as a working electrode in the electrochemical sensing. The enzyme loading on the 3D graphene could be enhanced compared with the 2D structure, thereby improving the detection sensitivity. The TYR enzyme was immobilized and the TYR-mediated phenol detection was successfully performed with a limit of detection of 50 nM. This is the first demonstration of using 3D graphene as an excellent electrochemical biosensor in the microfluidic system. We expect that our proposed device could expand its applications in the fields of pathogen detection, healthcare screening, and environmental pollutant monitoring.
Acknowledgments This work was supported by the Converging Technology Project funded by the Korean Ministry of Environment (M112-00061-0002-0),
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2013.06.055.
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