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pH sensing characteristics and biosensing application of solution-gated reduced graphene oxide field-effect transistors
Biosensors and Bioelectronics 45 (2013) 70–76
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pH sensing characteristics and biosensing application of solution-gated reduced graphene oxide field-effect transistors Il-Yung Sohn a, Duck-Jin Kim a, Jin-Heak Jung b, Ok Ja Yoon a, Tien Nguyen Thanh a, Trung Tran Quang a, Nae-Eung Lee a,b,c,n a
School of Advanced Materials Science & Engineering, Sungkyunkwan University, 300 Chunchun-dong, Suwon, Gyeonggi-do 440-746, South Korea SKKU Advanced Institute of Nanotechnology (SAINT), Sungkyunkwan University, 300 Chunchun-dong, Suwon, Gyeonggi-do 440-746, South Korea c Samsung Advanced Institute for Health Sciences and Technology (SAIHST), Sungkyunkwan University, 300 Chunchun-dong, Suwon, Gyeonggi-do 440-746, South Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 July 2012 Received in revised form 28 September 2012 Accepted 23 January 2013 Available online 4 February 2013
Solution-gated reduced graphene oxide field-effect transistors (R-GO FETs) were investigated for pH sensing and biochemical sensing applications. A channel of a networked R-GO film formed by selfassembly was incorporated as a sensing layer into a solution-gated FET structure for pH sensing and the detection of acetylcholine (Ach), which is a neurotransmitter in the nerve system, through enzymatic reactions. The fabricated R-GO FET was sensitive to protons (H þ ) with a pH sensitivity of 29 mV/pH in terms of the shift of the charge neutrality point (CNP), which is attributed to changes in the surface potential caused by the interaction of protons with OH surface functional groups present on the R-GO surface. The R-GO FET immobilized with acetylcholinesterase (AchE) was used to detect Ach in the concentration range of 0.1–10 mM by sensing protons generated during the enzymatic reactions. The results indicate that R-GO FETs provide the capability to detect protons, demonstrating their applicability as a biosensing device for enzymatic reactions. & 2013 Elsevier B.V. All rights reserved.
Keywords: Reduced graphene oxide Field effect transistor pH sensor Acetylcholine
1. Introduction Recently, graphene (Gr) and reduced graphene oxide (R-GO) have garnered a great deal of attention due to possible applications in optoelectronic devices (Bonaccorso et al., 2010), energy conversion and storage devices (Sun et al., 2011), nanoelectromechanical resonators (Chen et al., 2009a, 2009b), and sensors (Hill et al., 2011) due to their outstanding electronic, mechanical, and electrochemical properties (Rao et al., 2009; Zhu et al., 2010). Among the applications, biochemical sensors based on Gr or R-GO have been investigated because chemical and biological species can interact with pristine or functionalized Gr and R-GO surfaces (Shao et al., 2010; Pumera, 2011; Kuila et al., 2011). Interactions between biomolecules or adsorption of biomolecules on Gr or R-GO surfaces modulate electrical charge transport via changes of the carrier concentration and carrier mobilities caused by electrostatic gating or the charge doping effect in the channel (Heller et al., 2010; Dong et al., 2010; Kim et al., 2013). For this reason, several theoretical and experimental works involving solutiongated Gr or R-GO FETs, in which the channel conductance is n Corresponding author at: Samsung Advanced Institute for Health Sciences and Technology (SAIHST), Sungkyunkwan University, 300 Chunchun-dong, Suwon, Gyeonggi-do 440-746, South Korea. Tel.: þ 82 31 290 7398; fax: þ 82 31 290 7410. E-mail address: [email protected] (N.-E. Lee).
0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.01.051
modulated by applying a gate potential from a reference electrode through the electrolyte, have been reported for pH sensing of ions (Ang et al., 2008; Ohno et al., 2009; Ristein et al., 2010; Fu et al., 2011; Sudibya et al., 2011), DNA (Dong et al., 2010; Stine et al., 2010), proteins (Huang et al., 2010; He et al., 2011), and cellular activities (He et al., 2010; Huang et al., 2011). Even though it has outstanding electrical properties, mechanically exfoliated or thermally grown Gr has some drawbacks such as its difficulty of fabrication and risk of contamination due to polymer residue during the transfer and patterning process, which affects the surface cleanliness (Lin et al., 2011). Surface contamination of transferred and patterned Gr presumably limits the adsorption site density of probe molecules which significantly affects the detection limit, sensitivity, and dynamic range due to disturbed binding of chemical and biological species (Sheehan and Whitman, 2005; Squires et al., 2008; Arlett et al., 2011). To solve these problems, several studies have been conducted involving dry transfer techniques (Bae et al., 2010) as well as the perfect removal of polymeric residues (Pirkle et al., 2011; Moser et al., 2012; Goossens et al., 2012). Furthermore, the sensitivity of Gr to protons (H þ ) is drastically affected by its different surface conditions, which depend on the preparation method (Fu et al., 2011). On the other hand, a R-GO networked film has been utilized as a transducing material of a FET sensor because of its low-cost fabrication, facile patterning, and thickness control
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(He et al., 2010; Wang et al., 2010, ). Even though FETs with a R-GO channel have low field-effect channel mobilities, ambipolar electrical behaviors similar to that of a FET can be obtained. Also, FETs with a R-GO channel for chemical sensing of metal ions (Sudibya et al., 2011) and detection of DNA (Stine et al., 2010) and proteins (He et al., 2010, 2011; Kim et al., 2013) have been reported. Therefore, solution-gated R-GO FETs for biochemical sensing applications are of great scientific interest and promising for practical sensing applications. However, there have been no reports on the detection of H þ ions using R-GO FETs and their application for biosensing based on H þ detection. The sensing mechanism of conventional pH sensors using gate dielectrics as an ion sensitive layer in silicon ion-sensitive FETs (ISFET) is often explained by electrostatic gating effects based on the site-binding theory (Siu and Cobbold, 1979; Fung et al., 1986). In Gr FETs, where the Gr channel directly contacts the electrolyte, shifts of the charge neutrality point (CNP) as a sensing parameter have been explained to be due to electrostatic gating effects (Heller et al., 2010). As previously mentioned, however, there are still some discrepancies in regards to the sensitivity and sensing mechanism in Gr FET pH sensors. Fu et al. (2011) reported no pH sensitivity of a pristine Gr FET while a large pH sensitivity in a FET containing a epitaxial Gr channel (Ang et al., 2008) and mechanically exfoliated Gr (Ohno et al., 2009) was reported. Discrepancies such as these imply the importance of the surface conditions of Gr and R-GO for chemical sensing in Gr and R-GO FET devices. Herein, we describe solution-gated FETs employing a channel of a R-GO networked film with oxygen-containing functional groups on the surface and edges of the nanosheets, which may enhance the pH responsiveness as a channel material for the detection of H þ ions. Our pH measurement results using the solution-gated R-GO FET demonstrated reliable pH sensing capability with good linearity and repeatability. In addition, we confirmed that H þ detection in R-GO FETs can be used for the biosensing of target acetylcholine (Ach) biomolecules from their reactions with probe acetylcholinesterase (AchE) enzyme molecules immobilized on the R-GO channel surface.
2. Experimental The networked R-GO channel in R-GO FETs was formed by self-assembly of graphene oxide (GO) nanosheets and subsequent reduction of the networked GO nanosheets. GO nanosheets were manufactured from graphite powder (99.9999%, 325 mesh, Alfa Aesar). Graphite oxide was synthesized by a modified Hummer’s method (Kovtyukhova et al., 1999) using graphite powder dispersed in deionized (DI) water (18.2 MO, ELGA). Then, the graphite oxide was exfoliated to single-layer GO nanosheets by sonication for 2 h. The resulting GO aqueous solution (2 mg/ml) was subjected to three centrifuging steps: once at 2000 rpm for 90 min and twice at 4000 rpm for 60 min. At each step, the upper part of the solution was collected in order to remove the multilayer GO nanosheets and remaining graphite oxide. A SiO2 (100 nm)/Si substrate (Waferkorea) was cleaned using a NH4OH:H2O2:DI water solution with a ratio of 1:1:6 for 30 min at 85 1C followed by rinsing with DI water and drying with N2 gas. Then, the substrate was soaked in a 3% 3-aminopropyltriethoxysilane (APTES) (Sigma–Aldrich) aqueous solution for 30 min in order to form amino functional groups which produce a positively charged SiO2 surface. Then, washing with DI water and drying with N2 gas were performed. A GO solution was dropped on the substrate surface where the channel area is formed. An hour later, the remaining GO nanosheets were thoroughly washed out by gun-type DI water so that a very thin, networked film of GO nanosheets was assembled on the APTES-modified SiO2 surface. The obtained networked film of GO
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was reduced in hydrazine monohydrate (99.9%, Alfa Aesar) vapor at 60 1C for 12 h to produce the conductive R-GO channel. The R-GO film was thermally annealed for 2 h at 200 1C in an Ar atmosphere. The Au/Cr (65 nm/15 nm) drain–source electrodes were deposited on the R-GO film by a thermal evaporator using a shadow mask. The channel length (L) between the drain–source electrodes was kept constant at 400 mm with a width/length (W/L) ratio of 20. Silicone rubber (Sylgard 184, Dow Corning) was introduced for the encapsulation of the metal drain–source electrodes in order to minimize the leakage current due to exposure of the source and drain electrodes to the electrolyte. The silicone rubber layer covered the metal electrodes completely and defined the final channel area. The sensing channel length exposed to the solution was about 200 mm. A solution loading chamber of silicone rubber was also formed and attached. All curing processes of the silicone rubber were carried out at 80 1C. Topographic and chemical characterization of the formed GO and R-GO films were performed. Surface chemical analysis was carried out in an X-ray photoelectron spectroscopy (XPS) system (ESCA2000, VG Microtech). The surface topography of the R-GO nanosheets and networked films were examined by an atomic force microscope (AFM) (diInnova, Veeco) in the tapping mode with a silicon tip (NCHR 30, Nano World) in air. Electrical characterization of the solution-gated R-GO FETs was performed using a semiconductor analyzer (4145B, Agilent). An Ag/AgCl reference electrode was used for gate biasing to the R-GO FET through the electrolytes. In order to make sure that the leakage current was maintained far below the sensing signal, the gate and drain currents were monitored at fixed drain–source potentials of 0 and 0.1 V, respectively, with a sweeping gate potential in the range of 0.2 to 0.2 V. The devices showing a small leakage current ( o0.1% of the signal current) were used in the sensing measurements. Various buffers with different pH values were prepared using mixtures of a 0.1 M phosphate buffer (PB, NaH2PO4, and Na2HPO4) and a 0.1 M NaCl solution. The potential shift in the R-GO FET was measured in the different pH buffers with a volume of 200 ml. Then, to adjust the pH value, the initial pH buffer was removed and replaced by a new pH buffer. The real-time pH response was measured by monitoring the drain–source current while half of the initial pH buffer was removed and replaced by a new pH buffer in sequence. The pH in the PB solutions was monitored by a pH meter (Orion 3 Star, Thermo Scientific). For functionalization of the R-GO surface by linker molecules, the R-GO channel was soaked in a dimethylformamide (DMF, 99.9% Sigma–Aldrich) solution containing 5 mM 1-pyrenebuthanoic acid succinimidyl ester (PBSE, Invitrogen) for 2 h at RT (Chen et al., 2001; Huang et al., 2010, 2011; He et al., 2011; Sudibya et al., 2011). The PBSE linker molecules bound to the R-GO surface through p–p interactions chemically react with enzyme probe molecules. The resulting R-GO surface was immersed in a 100 U/ml AchE solution (dissolved from 1000 U lyophilized powder, Sigma–Aldrich) in a phosphate buffered saline (PBS, pH 7.4, Cellgro) solution for 12 h at 4 1C followed by PBS washing. For the sensing experiments, the analyte solutions were prepared by dissolving Ach chloride powder (99.9%, Sigma–Aldrich) in a 0.1 M PB solution at a pH of 7. For the electrical measurements, the drain–source current was monitored during continuous injection of each concentrated analyte solution (2 ml) into the initial 200 ml PB solution in the detection chamber.
3. Results and discussion To obtain surface topographical information of the GO nanosheets synthesized using the modified Hummer’s method, the GO aqueous solution was spin-coated on a SiO2/Si substrate.
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The AFM image (Fig. 1a) shows that the GO nanosheets had thicknesses of approximately 1 nm and diameters of 0.5–1.5 mm. In addition, no big particles were observed, indicating that GO
Fig. 1. (a) Morphological characteristics of the R-GO nanosheets. The height profile scanned across the line indicates a R-GO thickness of 1 nm. (b) 3D mapping image of the area marked by the dotted boxes in (a). XPS C1s spectra of (c) GO and (d) R-GO nanosheets. Before reduction of the GO nanosheets, C–C ( 284.5 eV), C–O ( 286.5 eV), and C ¼O ( 287.8 eV) peaks are present. After hydrazine-vapor reduction of GO, the C–O and C ¼ O peaks still remained but were reduced significantly. These peaks in the C1s XPS spectra indicate the presence of –OH and –COOH functional groups on the R-GO surface.
nanosheets with a one-atomic layer were synthesized from graphite. In order to form a well-networked R-GO film as the channel in the device, the formation of positively charged surfaces by APTES treatment of the SiO2/Si substrate is very effective (Li et al., 2009). The treatment prevents agglomeration of GO nanosheets in the solution and induces good coverage forming a few-layer, networked channel on the substrate. After the GO nanosheets were self-assembled, a reduction process using hydrazine vapor was carried out. Surface chemical analysis of GO (Fig. 1c) and RGO (Fig. 1d) was carried out using XPS measurements. The results indicate that the relative intensities of the C–O ( 286.5 eV) and C¼O peaks ( 287.8 eV) quantitatively decreased after the hydrazine vapor reduction. The XPS spectra also illustrated that the O/C atomic ratio decreased by a factor 7 after the reduction of GO. After deposition of the drain–source electrodes on the R-GO film, the fabricated FET showed good Ohmic behavior between the contact metals and the R-GO film along with a consistent channel resistance of 10–20 kO, despite great diversity of erratic junctions in between many R-GO nanosheets in the channel. A schematic illustration of the electrical measurements used to evaluate the sensing is shown in Fig. 2a. The solution-gate biasing effectively modulated the drain–source current (IDS) in the R-GO FET through the electrolyte and the CNP was positioned near the zero gate voltage in the pH 7 PB solution (Fig. 2b). An average channel mobility of 0.5 cm2/V-s was achieved, indicating lower performance in terms of the field-effect channel mobility in R-GO
Fig. 2. (a) Experimental set-up employed for the measurement of H þ and biosensing using the solution-gated R-GO FET. (b) Typical transfer characteristics of the fabricated solution-gated R-GO FET. A Ag/AgCl reference electrode was used as a gate electrode to control the gate potential through the electrolyte. The transfer curve shows a CNP near zero in the pH 7 PB solution.
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FETs due to the high density of defects and the presence of oxygenated functional groups on the channel surface compared to Gr FETs. However, the lower field-effect channel mobility is not a critical factor in the sensing performance of solution-gated R-GO FETs. The fabricated pH sensor showed a good electrical stability in pH sensing (Supplementary Fig. S1).
Fig. 3. Electrical characterization of the pH dependence of the R-GO FET. (a) The transfer characteristics obtained at a source–drain voltage (VDS) of 0.1 V show the changes in the channel conductance (DG/Gmin) by varying the gate potential at the reference electrode (VAg/AgCl). (b) CNP shifts in different buffers with pH values ranging from 6 to 9 where a sensitivity of 29.2 mV/pH was obtained. The inset shows a plot of the shifts in the CNP when the pH value of the solution was cycled from 9 to 6 and then from 6 to 9. (c) The drain–source current (IDS) was monitored in real-time by adding different pH buffers sequentially into the detection chamber. The inset shows the linear response of the IDS to the pH of the PB solution. All pH values of the PB solutions were measured by a pH meter in the detection chamber.
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Fig. 3a shows the pH-dependent transfer characteristics of the R-GO FET under different pH values ranging from 6 to 9. Electrical measurements at a source–drain voltage (VDS) of 0.1 V were used to monitor changes of the channel conductance (DG/Gmin) as the gate potential was varied at the reference electrode (VAg/AgCl) where Gmin is the minimum channel conductance in the transfer characteristics. A negative shift of the CNP, determined as the VAg/AgCl value where Gmin occurs with no significant changes of the slopes, was certainly observed with decreasing pH value in the electrolyte. This observation indicates that the field-effect mobility did not change but carrier doping occurred as the H þ concentration in the solution changed. A calibration curve of the CNP shifts was obtained from the transfer curves measured from many devices (n¼6) and the results shown in Fig. 3b represent typical Nernstian behavior. As shown in Fig. 3b, linearity in the pH range of 6–9 and a sensitivity of 29.2 mV/pH were obtained. Also, when the pH value of the PB solution was decreased from 9 to 6 and then increased from 6 to 9, reversible pH responses were achieved indicating good reproducibility of the sensor in pH sensing (see the inset of Fig. 3b). Current monitoring while varying the pH environment was introduced to achieve real-time sensing. The IDS was continuously monitored under a fixed gate bias (VAg/AgCl ¼ 0.2 V, the p-branch of the transfer curves) and a drain–source bias (VDS ¼ þ0.1 V) with periodic injection of the different pH buffers (Fig. 3c). The IDS value was decreased at the point of injection of the lower pH buffer. From the results displayed in Fig. 3c, calibration curves for the current modulation (inset of Fig. 3c) were plotted and the results demonstrated Nernstian behavior with a sensitivity of 1.47 in terms of DG/Gmin per pH. The results in Fig. 3 indicate that the increasing H þ ion concentration, i.e. decreasing pH, in the buffer solution leads to an increase of electrons in the R-GO channel. The sensing mechanism for H þ ions in a conventional Si ISFET can be explained by electrostatic gating effects based on the Gouy-Chapman-SternGraham model (Fung et al., 1986). H þ ions in the solution interact with hydroxyl species (–OH) and metal anions (M ) on the gate dielectric surface in a Si ISFET and therefore different H þ concentrations can induce changes of the surface charge density on the gate dielectric, which can be explained by the site-binding theory (Yates et al., 1973; Siu and Cobbold, 1979; Fung et al., 1986). As a result in the change of the surface potential, various carrier concentrations in the channel can induce shifts in the threshold voltage and modulate the source–drain current in a Si ISFET. Similar to the case of a Si ISFET, the pH sensing mechanism of the R-GO FET may be explained by the electrostatic gating effect. Plentiful functional groups such as –OH and –COOH on the R-GO surface, as implied from the XPS spectra (Fig. 1d), can interact with H þ ions in the electrolyte. In the buffer with a reduced pH, more H þ ions bind with O and COO , resulting in an increase of positive charges on the R-GO surface. This causes an increase in the amount of electrons in the channel, which causes the shift in the CNP in the left direction. By contrast, a sensitivity of 99 mV/pH was obtained (Ang et al., 2008) but much reduced sensitivities of 0–20 mV/pH were recently reported for pH sensing based on a Gr FET (Cheng et al., 2010; Heller et al., 2010; Ristein et al., 2010; Fu et al., 2011). FETs with a pristine pure Gr channel with no surface functional groups do not show pH sensitivity because pristine Gr has no available bonding sites for charged H þ ions (Fu et al., 2011). Our R-GO FET results imply the inherent sensitivity of R-GO channels to H þ ions. The ion detection capability of R-GO FETs can also be used as a biosensing platform in which a change of the H þ ion concentration is required. In our work, as a biosensing application of pH sensitive R-GO FETs, detection of Ach, was carried out. Various strategies for enzyme-based electrochemical detection of Ach are well
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presented in other literatures (Silvana and Jean, 2006; Perry et al., 2009). Specifically, potentiometric biosensors based on ISFETs, having operation principle similar to R-GO FET, have been extensively investigated by using pH sensitivity of oxide gate insulators (Van der Schoot and Bergveld, 1987; Schoning and Poghossian, 2002; Dzyadevych et al., 2006; Hai et al., 2006; Xue and Cui, 2008; Goykhman et al., 2009). Ach is a neurotransmitter secreted by a neural cell which functions both in the peripheral nervous system and in the central nervous system (Perry et al., 1999). A lack of Ach in the nervous system causes diseases such as myasthenia and Alzheimer’s disease
(Perry et al., 1999). In an attempt to test the detection capability of Ach, AchE molecules, which are specific enzymes which dissociate Ach, were immobilized by using a 1-pyrenebuthanoic acid succinimidyl ester (PBSE) linker on the R-GO surface. As shown schematically in Fig. 4a, the pyrene terminal can bind with the R-GO surface through p–p interactions by sharing p-electrons in the sp3 orbitals on the R-GO surface. In addition, succinimidyl groups are able to bind with the amine groups in the AchE molecule leading to the formation of amide bonding by nucleophilic substitution (Chen et al., 2001; Huang et al., 2010, 2011; He et al., 2011; Sudibya et al., 2011). Through the enzymatic reaction between Ach and AchE, H þ ions are produced during the hydrolysis of Ach (Fig. 4a). Sensing of H þ ions can therefore be used for the detection of Ach molecules. As observed in the AFM images in Fig. 4b, after AchE immobilization, obvious changes in the surface topography were observed. The height difference between the AchE-immobilized surface and bare SiO2 increased from 1.0 nm before immobilization to an average of 3.2 nm (Fig. 4b). For AFM measurement purposes, R-GO nanosheets were intentionally not fully networked in order to confirm the height change upon adsorption of AchE molecules on the R-GO surface. The 3D mapping image of the AchE-immobilized R-GO surface measured over an area of 1 1 mm2 shown in Fig. 4c indicates uniform attachment of the AchE molecules on the R-GO surface. These results confirm that AchE enzyme molecules specifically bind to the R-GO surface through the PBSE linker. Electrochemical impedance spectroscopy measurements were also performed to confirm immobilization of AchE molecules on the R-GO film (Supplementary Fig. S2). Significant increase in the charge transfer resistance was observed originating from the association of the AchE molecules onto the PBSE functionalized R-GO film. Immobilization of AchE molecules on the R-GO channel in the FET structure causes changes including a decrease in the sensitivity to pH presumably because the pyrene terminals of the PBSE linker cover a fraction of the R-GO surface through p–p stacking (Huang et al., 2011). As shown in Fig. 5, the sensitivity to PH in terms of the CNP shift decreased slightly from 29.2 to 15.4 mV/pH. The estimated sensitivity in terms of DG/Gmin per pH decreased from 1.47 to 0.8. In other words, the interactions of H þ ions with the R-GO surface were affected presumably because some fraction of the functional groups containing oxygen are covered by pyrene terminals in the linker molecules resulting in a
Fig. 4. (a) Schematic illustration of the enzyme immobilization and enzymatic reaction, (b) Morphological characteristics of AchE-immobilized R-GO nanosheets, and (c) 3D mapping image of the area marked by the dotted boxes in (a).
Fig. 5. Comparison of the pH sensitivity of the R-GO FET before and after AchE immobilization. A decrease in the pH sensitivity after AchE immobilization on the R-GO surface was observed.
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reduction of the number of sites for protonation and deprotonation on the R-GO surface. It was reported that blocking of surface functional groups on Gr surface by aromatic molecules also reduces the sensitivity of Gr to pH (Fu et al., 2011). For sensing Ach with an AchE-immobilized R-GO FET, the conductance change of the R-GO channel was monitored at fixed gate and drain–source biases (VAg/AgCl ¼ 0.2 V and VDS ¼ þ 0.1 V) with periodic injection of the analyte solution with various Ach concentrations into the initial pH buffer (Fig. 6a). The conductance of the R-GO channel was significantly reduced with increasing Ach concentration, which indicates generation of H þ ions leading to a lower pH. These results agree well with the sensing mechanism of the R-GO FET, in which released H þ ions from the enzymatic reaction interact with functional groups on the R-GO channel and create a positive surface potential, inducing a decrease of holes in the channel. During monitoring, some peaks in the current response were observed upon injection of the
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analyte solution with a high Ach concentration, indicating a sudden decrease in the conductance while the conductance gradually decreased with the injection of the analyte solution with a low Ach concentration. It is presumed that immobilized AchE dissociates Ach molecules quickly near the surface and therefore the H þ ion concentration suddenly increases within a narrow distance between the AchE and R-GO surface. Then, H þ ions diffuse out into the solution away from the R-GO surface and the signal is stabilized after reaching steady-state. The plot of the sensor signal vs. the Ach concentration shown in the inset of Fig. 6a demonstrates Nernstian behavior with good linearity in response to the Ach concentration in the range of 0.1–10 mM. The sensitivity estimated from the data in the inset of Fig. 6a was 1.06 in terms of DG/Gmin per decade. Reproducibility of Ach sensing by R-GO FETs was also investigated by measuring 10 mM Ach solution (Supplementary Fig. S3). Even though variation in response signals between different batches was observed, the R-GO FETs in each batch showed no significant variation in the response. In order to verify the time-dependent ambipolar response to a specific Ach concentration, a 10 mM solution of Ach was injected and the time responses of the p- and n-branch regions were monitored, as shown in the results of Fig. 6b. After injection of the Ach solution into the detection chamber, the conductance increased at þ 0.2 VAg/AgCl and decreased at 0.2 VAg/AgCl, where the current is transported by major carriers of electrons and holes corresponding to the n-branch and p-branch of the ambipolar transfer characteristics, respectively. With injection of the PB solution, no conductance changes were detected at þ0.2 VAg/AgCl. The measured change of DG/Gmin in the 10 mM solution, which was about 2.3 70.2 (Fig. 6b), indicates a pH change of about 2.9 (i.e. reduction from a pH of 7.0 to 4.1) based on the pre-calibration curve obtained for the AchE-immobilized R-GO FET. The DG/Gmin change in Fig. 6b is very consistent with the data obtained in Fig. 6a. These results confirm the response of the sensor in the analyte solution containing Ach molecules.
4. Conclusions
Fig. 6. Ach sensing characteristics of the AchE-immobilized R-GO FET. (a) An increase of the Ach concentration induces a conductance decrease due to increased H þ ion generation from the increased enzymatic reaction. The data in the inset of Fig. 6a shows a plot of the change of the channel conductance as the Ach concentration was varied. (b) Conductance changes at þ0.2 and 0.2 VAg/AgCl with injection of the 10 mM Ach solution and at þ 0.2 VAg/AgCl with injection of the PB solution.
In this work, a fast responding, highly sensitive, and reliable R-GO FET-based sensing device with a very simple and easy to make device structure was demonstrated. The sensing mechanism of H þ ions can be presumably explained by the site-binding theory due to the presence of plentiful functional groups on the R-GO channel compared to pristine graphene. In addition, we applied our pH sensitive R-GO FET to enzyme-based biosensing in which a neurotransmitter, Ach, was sensed through the detection of H þ ions produced during the enzymatic reaction. For practical and commercial implementation of pH sensitive R-GO FET as a transducer for biosensing, obtaining selectivity of H þ ions to other ions, lower limit of detection, and stability in the real samples would be challenging issues. For selective sensing of H þ ions to other ions in the physiological fluids, a device structure with capability of selective delivery of H þ ions onto the R-GO surface, for example, by employing H þ selective membrane impregnated with enzymes, can be possibly used. Obtaining lower limit of detection for Ach sensing would be required in future work by adjusting the density of functional groups on R-GO surface or by constructing hybrid channel of R-GO and H þ sensitive oxide nanostructures. In case of instability caused by p–p interaction of proteins in real samples with R-GO surface, the use of H þ selective membrane would also help to block proteins in real samples from their attachment on the R-GO surface. With further optimization for real sample measurements, the capability
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of sensing the enzymatic reaction may enable R-GO FETs to be applied as a secondary transducer in cell-based biosensors.
Acknowledgments This work was supported by the Basic Science Research Program (Grant No. 2009-0083540) and the WCU Program (Grant No. R32-2008-000-10124-0) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (MEST). This work was also supported by Yeonam Foundation, Korea.
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.2013.01.051.
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pH sensing characteristics and biosensing application of solution-gated reduced graphene oxide field-effect transistors Il-Yung Sohn a, Duck-Jin Kim a, Jin-Heak Jung b, Ok Ja Yoon a, Tien Nguyen Thanh a, Trung Tran Quang a, Nae-Eung Lee a,b,c,n a
School of Advanced Materials Science & Engineering, Sungkyunkwan University, 300 Chunchun-dong, Suwon, Gyeonggi-do 440-746, South Korea SKKU Advanced Institute of Nanotechnology (SAINT), Sungkyunkwan University, 300 Chunchun-dong, Suwon, Gyeonggi-do 440-746, South Korea c Samsung Advanced Institute for Health Sciences and Technology (SAIHST), Sungkyunkwan University, 300 Chunchun-dong, Suwon, Gyeonggi-do 440-746, South Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 July 2012 Received in revised form 28 September 2012 Accepted 23 January 2013 Available online 4 February 2013
Solution-gated reduced graphene oxide field-effect transistors (R-GO FETs) were investigated for pH sensing and biochemical sensing applications. A channel of a networked R-GO film formed by selfassembly was incorporated as a sensing layer into a solution-gated FET structure for pH sensing and the detection of acetylcholine (Ach), which is a neurotransmitter in the nerve system, through enzymatic reactions. The fabricated R-GO FET was sensitive to protons (H þ ) with a pH sensitivity of 29 mV/pH in terms of the shift of the charge neutrality point (CNP), which is attributed to changes in the surface potential caused by the interaction of protons with OH surface functional groups present on the R-GO surface. The R-GO FET immobilized with acetylcholinesterase (AchE) was used to detect Ach in the concentration range of 0.1–10 mM by sensing protons generated during the enzymatic reactions. The results indicate that R-GO FETs provide the capability to detect protons, demonstrating their applicability as a biosensing device for enzymatic reactions. & 2013 Elsevier B.V. All rights reserved.
Keywords: Reduced graphene oxide Field effect transistor pH sensor Acetylcholine
1. Introduction Recently, graphene (Gr) and reduced graphene oxide (R-GO) have garnered a great deal of attention due to possible applications in optoelectronic devices (Bonaccorso et al., 2010), energy conversion and storage devices (Sun et al., 2011), nanoelectromechanical resonators (Chen et al., 2009a, 2009b), and sensors (Hill et al., 2011) due to their outstanding electronic, mechanical, and electrochemical properties (Rao et al., 2009; Zhu et al., 2010). Among the applications, biochemical sensors based on Gr or R-GO have been investigated because chemical and biological species can interact with pristine or functionalized Gr and R-GO surfaces (Shao et al., 2010; Pumera, 2011; Kuila et al., 2011). Interactions between biomolecules or adsorption of biomolecules on Gr or R-GO surfaces modulate electrical charge transport via changes of the carrier concentration and carrier mobilities caused by electrostatic gating or the charge doping effect in the channel (Heller et al., 2010; Dong et al., 2010; Kim et al., 2013). For this reason, several theoretical and experimental works involving solutiongated Gr or R-GO FETs, in which the channel conductance is n Corresponding author at: Samsung Advanced Institute for Health Sciences and Technology (SAIHST), Sungkyunkwan University, 300 Chunchun-dong, Suwon, Gyeonggi-do 440-746, South Korea. Tel.: þ 82 31 290 7398; fax: þ 82 31 290 7410. E-mail address: [email protected] (N.-E. Lee).
0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.01.051
modulated by applying a gate potential from a reference electrode through the electrolyte, have been reported for pH sensing of ions (Ang et al., 2008; Ohno et al., 2009; Ristein et al., 2010; Fu et al., 2011; Sudibya et al., 2011), DNA (Dong et al., 2010; Stine et al., 2010), proteins (Huang et al., 2010; He et al., 2011), and cellular activities (He et al., 2010; Huang et al., 2011). Even though it has outstanding electrical properties, mechanically exfoliated or thermally grown Gr has some drawbacks such as its difficulty of fabrication and risk of contamination due to polymer residue during the transfer and patterning process, which affects the surface cleanliness (Lin et al., 2011). Surface contamination of transferred and patterned Gr presumably limits the adsorption site density of probe molecules which significantly affects the detection limit, sensitivity, and dynamic range due to disturbed binding of chemical and biological species (Sheehan and Whitman, 2005; Squires et al., 2008; Arlett et al., 2011). To solve these problems, several studies have been conducted involving dry transfer techniques (Bae et al., 2010) as well as the perfect removal of polymeric residues (Pirkle et al., 2011; Moser et al., 2012; Goossens et al., 2012). Furthermore, the sensitivity of Gr to protons (H þ ) is drastically affected by its different surface conditions, which depend on the preparation method (Fu et al., 2011). On the other hand, a R-GO networked film has been utilized as a transducing material of a FET sensor because of its low-cost fabrication, facile patterning, and thickness control
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(He et al., 2010; Wang et al., 2010, ). Even though FETs with a R-GO channel have low field-effect channel mobilities, ambipolar electrical behaviors similar to that of a FET can be obtained. Also, FETs with a R-GO channel for chemical sensing of metal ions (Sudibya et al., 2011) and detection of DNA (Stine et al., 2010) and proteins (He et al., 2010, 2011; Kim et al., 2013) have been reported. Therefore, solution-gated R-GO FETs for biochemical sensing applications are of great scientific interest and promising for practical sensing applications. However, there have been no reports on the detection of H þ ions using R-GO FETs and their application for biosensing based on H þ detection. The sensing mechanism of conventional pH sensors using gate dielectrics as an ion sensitive layer in silicon ion-sensitive FETs (ISFET) is often explained by electrostatic gating effects based on the site-binding theory (Siu and Cobbold, 1979; Fung et al., 1986). In Gr FETs, where the Gr channel directly contacts the electrolyte, shifts of the charge neutrality point (CNP) as a sensing parameter have been explained to be due to electrostatic gating effects (Heller et al., 2010). As previously mentioned, however, there are still some discrepancies in regards to the sensitivity and sensing mechanism in Gr FET pH sensors. Fu et al. (2011) reported no pH sensitivity of a pristine Gr FET while a large pH sensitivity in a FET containing a epitaxial Gr channel (Ang et al., 2008) and mechanically exfoliated Gr (Ohno et al., 2009) was reported. Discrepancies such as these imply the importance of the surface conditions of Gr and R-GO for chemical sensing in Gr and R-GO FET devices. Herein, we describe solution-gated FETs employing a channel of a R-GO networked film with oxygen-containing functional groups on the surface and edges of the nanosheets, which may enhance the pH responsiveness as a channel material for the detection of H þ ions. Our pH measurement results using the solution-gated R-GO FET demonstrated reliable pH sensing capability with good linearity and repeatability. In addition, we confirmed that H þ detection in R-GO FETs can be used for the biosensing of target acetylcholine (Ach) biomolecules from their reactions with probe acetylcholinesterase (AchE) enzyme molecules immobilized on the R-GO channel surface.
2. Experimental The networked R-GO channel in R-GO FETs was formed by self-assembly of graphene oxide (GO) nanosheets and subsequent reduction of the networked GO nanosheets. GO nanosheets were manufactured from graphite powder (99.9999%, 325 mesh, Alfa Aesar). Graphite oxide was synthesized by a modified Hummer’s method (Kovtyukhova et al., 1999) using graphite powder dispersed in deionized (DI) water (18.2 MO, ELGA). Then, the graphite oxide was exfoliated to single-layer GO nanosheets by sonication for 2 h. The resulting GO aqueous solution (2 mg/ml) was subjected to three centrifuging steps: once at 2000 rpm for 90 min and twice at 4000 rpm for 60 min. At each step, the upper part of the solution was collected in order to remove the multilayer GO nanosheets and remaining graphite oxide. A SiO2 (100 nm)/Si substrate (Waferkorea) was cleaned using a NH4OH:H2O2:DI water solution with a ratio of 1:1:6 for 30 min at 85 1C followed by rinsing with DI water and drying with N2 gas. Then, the substrate was soaked in a 3% 3-aminopropyltriethoxysilane (APTES) (Sigma–Aldrich) aqueous solution for 30 min in order to form amino functional groups which produce a positively charged SiO2 surface. Then, washing with DI water and drying with N2 gas were performed. A GO solution was dropped on the substrate surface where the channel area is formed. An hour later, the remaining GO nanosheets were thoroughly washed out by gun-type DI water so that a very thin, networked film of GO nanosheets was assembled on the APTES-modified SiO2 surface. The obtained networked film of GO
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was reduced in hydrazine monohydrate (99.9%, Alfa Aesar) vapor at 60 1C for 12 h to produce the conductive R-GO channel. The R-GO film was thermally annealed for 2 h at 200 1C in an Ar atmosphere. The Au/Cr (65 nm/15 nm) drain–source electrodes were deposited on the R-GO film by a thermal evaporator using a shadow mask. The channel length (L) between the drain–source electrodes was kept constant at 400 mm with a width/length (W/L) ratio of 20. Silicone rubber (Sylgard 184, Dow Corning) was introduced for the encapsulation of the metal drain–source electrodes in order to minimize the leakage current due to exposure of the source and drain electrodes to the electrolyte. The silicone rubber layer covered the metal electrodes completely and defined the final channel area. The sensing channel length exposed to the solution was about 200 mm. A solution loading chamber of silicone rubber was also formed and attached. All curing processes of the silicone rubber were carried out at 80 1C. Topographic and chemical characterization of the formed GO and R-GO films were performed. Surface chemical analysis was carried out in an X-ray photoelectron spectroscopy (XPS) system (ESCA2000, VG Microtech). The surface topography of the R-GO nanosheets and networked films were examined by an atomic force microscope (AFM) (diInnova, Veeco) in the tapping mode with a silicon tip (NCHR 30, Nano World) in air. Electrical characterization of the solution-gated R-GO FETs was performed using a semiconductor analyzer (4145B, Agilent). An Ag/AgCl reference electrode was used for gate biasing to the R-GO FET through the electrolytes. In order to make sure that the leakage current was maintained far below the sensing signal, the gate and drain currents were monitored at fixed drain–source potentials of 0 and 0.1 V, respectively, with a sweeping gate potential in the range of 0.2 to 0.2 V. The devices showing a small leakage current ( o0.1% of the signal current) were used in the sensing measurements. Various buffers with different pH values were prepared using mixtures of a 0.1 M phosphate buffer (PB, NaH2PO4, and Na2HPO4) and a 0.1 M NaCl solution. The potential shift in the R-GO FET was measured in the different pH buffers with a volume of 200 ml. Then, to adjust the pH value, the initial pH buffer was removed and replaced by a new pH buffer. The real-time pH response was measured by monitoring the drain–source current while half of the initial pH buffer was removed and replaced by a new pH buffer in sequence. The pH in the PB solutions was monitored by a pH meter (Orion 3 Star, Thermo Scientific). For functionalization of the R-GO surface by linker molecules, the R-GO channel was soaked in a dimethylformamide (DMF, 99.9% Sigma–Aldrich) solution containing 5 mM 1-pyrenebuthanoic acid succinimidyl ester (PBSE, Invitrogen) for 2 h at RT (Chen et al., 2001; Huang et al., 2010, 2011; He et al., 2011; Sudibya et al., 2011). The PBSE linker molecules bound to the R-GO surface through p–p interactions chemically react with enzyme probe molecules. The resulting R-GO surface was immersed in a 100 U/ml AchE solution (dissolved from 1000 U lyophilized powder, Sigma–Aldrich) in a phosphate buffered saline (PBS, pH 7.4, Cellgro) solution for 12 h at 4 1C followed by PBS washing. For the sensing experiments, the analyte solutions were prepared by dissolving Ach chloride powder (99.9%, Sigma–Aldrich) in a 0.1 M PB solution at a pH of 7. For the electrical measurements, the drain–source current was monitored during continuous injection of each concentrated analyte solution (2 ml) into the initial 200 ml PB solution in the detection chamber.
3. Results and discussion To obtain surface topographical information of the GO nanosheets synthesized using the modified Hummer’s method, the GO aqueous solution was spin-coated on a SiO2/Si substrate.
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The AFM image (Fig. 1a) shows that the GO nanosheets had thicknesses of approximately 1 nm and diameters of 0.5–1.5 mm. In addition, no big particles were observed, indicating that GO
Fig. 1. (a) Morphological characteristics of the R-GO nanosheets. The height profile scanned across the line indicates a R-GO thickness of 1 nm. (b) 3D mapping image of the area marked by the dotted boxes in (a). XPS C1s spectra of (c) GO and (d) R-GO nanosheets. Before reduction of the GO nanosheets, C–C ( 284.5 eV), C–O ( 286.5 eV), and C ¼O ( 287.8 eV) peaks are present. After hydrazine-vapor reduction of GO, the C–O and C ¼ O peaks still remained but were reduced significantly. These peaks in the C1s XPS spectra indicate the presence of –OH and –COOH functional groups on the R-GO surface.
nanosheets with a one-atomic layer were synthesized from graphite. In order to form a well-networked R-GO film as the channel in the device, the formation of positively charged surfaces by APTES treatment of the SiO2/Si substrate is very effective (Li et al., 2009). The treatment prevents agglomeration of GO nanosheets in the solution and induces good coverage forming a few-layer, networked channel on the substrate. After the GO nanosheets were self-assembled, a reduction process using hydrazine vapor was carried out. Surface chemical analysis of GO (Fig. 1c) and RGO (Fig. 1d) was carried out using XPS measurements. The results indicate that the relative intensities of the C–O ( 286.5 eV) and C¼O peaks ( 287.8 eV) quantitatively decreased after the hydrazine vapor reduction. The XPS spectra also illustrated that the O/C atomic ratio decreased by a factor 7 after the reduction of GO. After deposition of the drain–source electrodes on the R-GO film, the fabricated FET showed good Ohmic behavior between the contact metals and the R-GO film along with a consistent channel resistance of 10–20 kO, despite great diversity of erratic junctions in between many R-GO nanosheets in the channel. A schematic illustration of the electrical measurements used to evaluate the sensing is shown in Fig. 2a. The solution-gate biasing effectively modulated the drain–source current (IDS) in the R-GO FET through the electrolyte and the CNP was positioned near the zero gate voltage in the pH 7 PB solution (Fig. 2b). An average channel mobility of 0.5 cm2/V-s was achieved, indicating lower performance in terms of the field-effect channel mobility in R-GO
Fig. 2. (a) Experimental set-up employed for the measurement of H þ and biosensing using the solution-gated R-GO FET. (b) Typical transfer characteristics of the fabricated solution-gated R-GO FET. A Ag/AgCl reference electrode was used as a gate electrode to control the gate potential through the electrolyte. The transfer curve shows a CNP near zero in the pH 7 PB solution.
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FETs due to the high density of defects and the presence of oxygenated functional groups on the channel surface compared to Gr FETs. However, the lower field-effect channel mobility is not a critical factor in the sensing performance of solution-gated R-GO FETs. The fabricated pH sensor showed a good electrical stability in pH sensing (Supplementary Fig. S1).
Fig. 3. Electrical characterization of the pH dependence of the R-GO FET. (a) The transfer characteristics obtained at a source–drain voltage (VDS) of 0.1 V show the changes in the channel conductance (DG/Gmin) by varying the gate potential at the reference electrode (VAg/AgCl). (b) CNP shifts in different buffers with pH values ranging from 6 to 9 where a sensitivity of 29.2 mV/pH was obtained. The inset shows a plot of the shifts in the CNP when the pH value of the solution was cycled from 9 to 6 and then from 6 to 9. (c) The drain–source current (IDS) was monitored in real-time by adding different pH buffers sequentially into the detection chamber. The inset shows the linear response of the IDS to the pH of the PB solution. All pH values of the PB solutions were measured by a pH meter in the detection chamber.
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Fig. 3a shows the pH-dependent transfer characteristics of the R-GO FET under different pH values ranging from 6 to 9. Electrical measurements at a source–drain voltage (VDS) of 0.1 V were used to monitor changes of the channel conductance (DG/Gmin) as the gate potential was varied at the reference electrode (VAg/AgCl) where Gmin is the minimum channel conductance in the transfer characteristics. A negative shift of the CNP, determined as the VAg/AgCl value where Gmin occurs with no significant changes of the slopes, was certainly observed with decreasing pH value in the electrolyte. This observation indicates that the field-effect mobility did not change but carrier doping occurred as the H þ concentration in the solution changed. A calibration curve of the CNP shifts was obtained from the transfer curves measured from many devices (n¼6) and the results shown in Fig. 3b represent typical Nernstian behavior. As shown in Fig. 3b, linearity in the pH range of 6–9 and a sensitivity of 29.2 mV/pH were obtained. Also, when the pH value of the PB solution was decreased from 9 to 6 and then increased from 6 to 9, reversible pH responses were achieved indicating good reproducibility of the sensor in pH sensing (see the inset of Fig. 3b). Current monitoring while varying the pH environment was introduced to achieve real-time sensing. The IDS was continuously monitored under a fixed gate bias (VAg/AgCl ¼ 0.2 V, the p-branch of the transfer curves) and a drain–source bias (VDS ¼ þ0.1 V) with periodic injection of the different pH buffers (Fig. 3c). The IDS value was decreased at the point of injection of the lower pH buffer. From the results displayed in Fig. 3c, calibration curves for the current modulation (inset of Fig. 3c) were plotted and the results demonstrated Nernstian behavior with a sensitivity of 1.47 in terms of DG/Gmin per pH. The results in Fig. 3 indicate that the increasing H þ ion concentration, i.e. decreasing pH, in the buffer solution leads to an increase of electrons in the R-GO channel. The sensing mechanism for H þ ions in a conventional Si ISFET can be explained by electrostatic gating effects based on the Gouy-Chapman-SternGraham model (Fung et al., 1986). H þ ions in the solution interact with hydroxyl species (–OH) and metal anions (M ) on the gate dielectric surface in a Si ISFET and therefore different H þ concentrations can induce changes of the surface charge density on the gate dielectric, which can be explained by the site-binding theory (Yates et al., 1973; Siu and Cobbold, 1979; Fung et al., 1986). As a result in the change of the surface potential, various carrier concentrations in the channel can induce shifts in the threshold voltage and modulate the source–drain current in a Si ISFET. Similar to the case of a Si ISFET, the pH sensing mechanism of the R-GO FET may be explained by the electrostatic gating effect. Plentiful functional groups such as –OH and –COOH on the R-GO surface, as implied from the XPS spectra (Fig. 1d), can interact with H þ ions in the electrolyte. In the buffer with a reduced pH, more H þ ions bind with O and COO , resulting in an increase of positive charges on the R-GO surface. This causes an increase in the amount of electrons in the channel, which causes the shift in the CNP in the left direction. By contrast, a sensitivity of 99 mV/pH was obtained (Ang et al., 2008) but much reduced sensitivities of 0–20 mV/pH were recently reported for pH sensing based on a Gr FET (Cheng et al., 2010; Heller et al., 2010; Ristein et al., 2010; Fu et al., 2011). FETs with a pristine pure Gr channel with no surface functional groups do not show pH sensitivity because pristine Gr has no available bonding sites for charged H þ ions (Fu et al., 2011). Our R-GO FET results imply the inherent sensitivity of R-GO channels to H þ ions. The ion detection capability of R-GO FETs can also be used as a biosensing platform in which a change of the H þ ion concentration is required. In our work, as a biosensing application of pH sensitive R-GO FETs, detection of Ach, was carried out. Various strategies for enzyme-based electrochemical detection of Ach are well
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presented in other literatures (Silvana and Jean, 2006; Perry et al., 2009). Specifically, potentiometric biosensors based on ISFETs, having operation principle similar to R-GO FET, have been extensively investigated by using pH sensitivity of oxide gate insulators (Van der Schoot and Bergveld, 1987; Schoning and Poghossian, 2002; Dzyadevych et al., 2006; Hai et al., 2006; Xue and Cui, 2008; Goykhman et al., 2009). Ach is a neurotransmitter secreted by a neural cell which functions both in the peripheral nervous system and in the central nervous system (Perry et al., 1999). A lack of Ach in the nervous system causes diseases such as myasthenia and Alzheimer’s disease
(Perry et al., 1999). In an attempt to test the detection capability of Ach, AchE molecules, which are specific enzymes which dissociate Ach, were immobilized by using a 1-pyrenebuthanoic acid succinimidyl ester (PBSE) linker on the R-GO surface. As shown schematically in Fig. 4a, the pyrene terminal can bind with the R-GO surface through p–p interactions by sharing p-electrons in the sp3 orbitals on the R-GO surface. In addition, succinimidyl groups are able to bind with the amine groups in the AchE molecule leading to the formation of amide bonding by nucleophilic substitution (Chen et al., 2001; Huang et al., 2010, 2011; He et al., 2011; Sudibya et al., 2011). Through the enzymatic reaction between Ach and AchE, H þ ions are produced during the hydrolysis of Ach (Fig. 4a). Sensing of H þ ions can therefore be used for the detection of Ach molecules. As observed in the AFM images in Fig. 4b, after AchE immobilization, obvious changes in the surface topography were observed. The height difference between the AchE-immobilized surface and bare SiO2 increased from 1.0 nm before immobilization to an average of 3.2 nm (Fig. 4b). For AFM measurement purposes, R-GO nanosheets were intentionally not fully networked in order to confirm the height change upon adsorption of AchE molecules on the R-GO surface. The 3D mapping image of the AchE-immobilized R-GO surface measured over an area of 1 1 mm2 shown in Fig. 4c indicates uniform attachment of the AchE molecules on the R-GO surface. These results confirm that AchE enzyme molecules specifically bind to the R-GO surface through the PBSE linker. Electrochemical impedance spectroscopy measurements were also performed to confirm immobilization of AchE molecules on the R-GO film (Supplementary Fig. S2). Significant increase in the charge transfer resistance was observed originating from the association of the AchE molecules onto the PBSE functionalized R-GO film. Immobilization of AchE molecules on the R-GO channel in the FET structure causes changes including a decrease in the sensitivity to pH presumably because the pyrene terminals of the PBSE linker cover a fraction of the R-GO surface through p–p stacking (Huang et al., 2011). As shown in Fig. 5, the sensitivity to PH in terms of the CNP shift decreased slightly from 29.2 to 15.4 mV/pH. The estimated sensitivity in terms of DG/Gmin per pH decreased from 1.47 to 0.8. In other words, the interactions of H þ ions with the R-GO surface were affected presumably because some fraction of the functional groups containing oxygen are covered by pyrene terminals in the linker molecules resulting in a
Fig. 4. (a) Schematic illustration of the enzyme immobilization and enzymatic reaction, (b) Morphological characteristics of AchE-immobilized R-GO nanosheets, and (c) 3D mapping image of the area marked by the dotted boxes in (a).
Fig. 5. Comparison of the pH sensitivity of the R-GO FET before and after AchE immobilization. A decrease in the pH sensitivity after AchE immobilization on the R-GO surface was observed.
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reduction of the number of sites for protonation and deprotonation on the R-GO surface. It was reported that blocking of surface functional groups on Gr surface by aromatic molecules also reduces the sensitivity of Gr to pH (Fu et al., 2011). For sensing Ach with an AchE-immobilized R-GO FET, the conductance change of the R-GO channel was monitored at fixed gate and drain–source biases (VAg/AgCl ¼ 0.2 V and VDS ¼ þ 0.1 V) with periodic injection of the analyte solution with various Ach concentrations into the initial pH buffer (Fig. 6a). The conductance of the R-GO channel was significantly reduced with increasing Ach concentration, which indicates generation of H þ ions leading to a lower pH. These results agree well with the sensing mechanism of the R-GO FET, in which released H þ ions from the enzymatic reaction interact with functional groups on the R-GO channel and create a positive surface potential, inducing a decrease of holes in the channel. During monitoring, some peaks in the current response were observed upon injection of the
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analyte solution with a high Ach concentration, indicating a sudden decrease in the conductance while the conductance gradually decreased with the injection of the analyte solution with a low Ach concentration. It is presumed that immobilized AchE dissociates Ach molecules quickly near the surface and therefore the H þ ion concentration suddenly increases within a narrow distance between the AchE and R-GO surface. Then, H þ ions diffuse out into the solution away from the R-GO surface and the signal is stabilized after reaching steady-state. The plot of the sensor signal vs. the Ach concentration shown in the inset of Fig. 6a demonstrates Nernstian behavior with good linearity in response to the Ach concentration in the range of 0.1–10 mM. The sensitivity estimated from the data in the inset of Fig. 6a was 1.06 in terms of DG/Gmin per decade. Reproducibility of Ach sensing by R-GO FETs was also investigated by measuring 10 mM Ach solution (Supplementary Fig. S3). Even though variation in response signals between different batches was observed, the R-GO FETs in each batch showed no significant variation in the response. In order to verify the time-dependent ambipolar response to a specific Ach concentration, a 10 mM solution of Ach was injected and the time responses of the p- and n-branch regions were monitored, as shown in the results of Fig. 6b. After injection of the Ach solution into the detection chamber, the conductance increased at þ 0.2 VAg/AgCl and decreased at 0.2 VAg/AgCl, where the current is transported by major carriers of electrons and holes corresponding to the n-branch and p-branch of the ambipolar transfer characteristics, respectively. With injection of the PB solution, no conductance changes were detected at þ0.2 VAg/AgCl. The measured change of DG/Gmin in the 10 mM solution, which was about 2.3 70.2 (Fig. 6b), indicates a pH change of about 2.9 (i.e. reduction from a pH of 7.0 to 4.1) based on the pre-calibration curve obtained for the AchE-immobilized R-GO FET. The DG/Gmin change in Fig. 6b is very consistent with the data obtained in Fig. 6a. These results confirm the response of the sensor in the analyte solution containing Ach molecules.
4. Conclusions
Fig. 6. Ach sensing characteristics of the AchE-immobilized R-GO FET. (a) An increase of the Ach concentration induces a conductance decrease due to increased H þ ion generation from the increased enzymatic reaction. The data in the inset of Fig. 6a shows a plot of the change of the channel conductance as the Ach concentration was varied. (b) Conductance changes at þ0.2 and 0.2 VAg/AgCl with injection of the 10 mM Ach solution and at þ 0.2 VAg/AgCl with injection of the PB solution.
In this work, a fast responding, highly sensitive, and reliable R-GO FET-based sensing device with a very simple and easy to make device structure was demonstrated. The sensing mechanism of H þ ions can be presumably explained by the site-binding theory due to the presence of plentiful functional groups on the R-GO channel compared to pristine graphene. In addition, we applied our pH sensitive R-GO FET to enzyme-based biosensing in which a neurotransmitter, Ach, was sensed through the detection of H þ ions produced during the enzymatic reaction. For practical and commercial implementation of pH sensitive R-GO FET as a transducer for biosensing, obtaining selectivity of H þ ions to other ions, lower limit of detection, and stability in the real samples would be challenging issues. For selective sensing of H þ ions to other ions in the physiological fluids, a device structure with capability of selective delivery of H þ ions onto the R-GO surface, for example, by employing H þ selective membrane impregnated with enzymes, can be possibly used. Obtaining lower limit of detection for Ach sensing would be required in future work by adjusting the density of functional groups on R-GO surface or by constructing hybrid channel of R-GO and H þ sensitive oxide nanostructures. In case of instability caused by p–p interaction of proteins in real samples with R-GO surface, the use of H þ selective membrane would also help to block proteins in real samples from their attachment on the R-GO surface. With further optimization for real sample measurements, the capability
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of sensing the enzymatic reaction may enable R-GO FETs to be applied as a secondary transducer in cell-based biosensors.
Acknowledgments This work was supported by the Basic Science Research Program (Grant No. 2009-0083540) and the WCU Program (Grant No. R32-2008-000-10124-0) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (MEST). This work was also supported by Yeonam Foundation, Korea.
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.2013.01.051.
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