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A Miniature Graphene-based Biosensor for Intracellular Glucose Measurements
Electrochimica Acta 174 (2015) 574–580
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Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta
A Miniature Graphene-based Biosensor for Intracellular Glucose Measurements Kamran ul Hasana,b,* , Muhammad H. Asifa,c, Muhammad Umair Hassand, Mats O. Sandberge, O. Nura , M. Willandera , Siri Fagerholmf , Peter Strålforsf a
Department of Science and Technology, Campus Norrköping, Linköping University, Bredgatan 34, SE-601 74 Norrköping, Sweden Centres of Excellence in Science & Applied Technologies (CESAT), Islamabad, Pakistan Materials Research Lab, Department of Physics, COMSATS Institute of Information Technology, Lahore, 54000, Pakistan d Center for Micro and Nano Devices (CMND), Department of Physics, COMSATS Institute of Information Technology, Islamabad, 44000, Pakistan e Acreo AB Bredgatan 34, SE-602 21 Norrköping, Sweden f Department of Clinical and Experimental Medicine, Linköping University, SE-581 85 Linköping, Sweden b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 12 March 2015 Received in revised form 20 May 2015 Accepted 8 June 2015 Available online 10 June 2015
We report on a small and simple graphene-based potentiometric sensor for the measurement of intracellular glucose concentration. A fine borosilicate glass capillary coated with graphene and subsequently immobilized with glucose oxidase (GOD) enzyme is inserted into the intracellular environment of a single human cell. The functional groups on the edge plane of graphene assist the attachment with the free amine terminals of GOD enzyme, resulting in a better immobilization. The sensor exhibits a glucose-dependent electrochemical potential against an Ag/AgCl reference microelectrode which is linear across the whole concentration range of interest (10 – 1000 mM). Glucose concentration in human fat cell measured by our graphene-based sensor is in good agreement with nuclear magnetic resonance (NMR) spectroscopy. ã2015 Elsevier Ltd. All rights reserved.
Keywords: Graphene Bio-sensors Glucose-oxidase Intracellular sensors Graphene oxide Glucose Sensor
1. Introduction Graphene has been enticing much attention due to its fascinating properties, such as extremely large surface area (2630 m2 g1), high optical transmittance ( 97.7 %), large mechanical fracture strength, ( 125 GPa), excellent thermal conductivity ( 5000 W m1 K1) and superb charge-carrier mobility ( 200,000 cm2 V1s1) [1–8]. Its remarkable properties have opened up a whole realm of applications [3,8–11], amongst which biosensing has been capturing a huge interest [12–14]. This is mainly because of the great promise and performance owning to its tremendously large surface area to volume ratio as a dominating and favorable parameter for sensing applications [8,15,16]. The sensing is usually achieved via immobilization of enzymes onto the sensing surfaces of a suitably modified electrode, which can provide highly selective, sensitive, and rapid analysis of various biological species like DNA [17], Human IgG [18] and glucose [19]. Carbon nanotube (CNT) modified electrodes have already been very successful for biosensing of glucose, which is realized by a
* Corresponding author. E-mail address: [email protected] (K.u. Hasan). http://dx.doi.org/10.1016/j.electacta.2015.06.035 0013-4686/ ã 2015 Elsevier Ltd. All rights reserved.
dramatic decrease in the hydrogen peroxide overpotential, plus the direct electron transfer of glucose oxidase (GOD) observed at asmade electrodes [20,21]. However, using graphene for such applications can offer several advantages over CNTs for its superior properties: Its 2-dimentional nature (single atomic layer of graphite) can maximize the interaction between the surface dopants and adsorbates. It offers much lower Johnson noise and higher sensitivity [22–24], as compared to CNT, therefore, a minute variation of carrier concentration can cause a notable variation of electrical conductivity [25]. Moreover, graphene can be obtained via chemical conversion of graphite, which is a simple and inexpensive synthesis method [26]. We employ the GOD enzyme, which belongs to a family of oxidases that are widely used in many glucose sensors. The binding between GOD and the electrode surface is a critical parameter for the stability and sensitivity of the biosensor. Therefore, it is important to select a suitable electrode material that can favorably bind an enzyme. Various methods, such as covalent binding [27], embedding [28], and cross-linking [29–31], have already been exploited to immobilize the GOD onto different supporting materials. Functionalized graphene has reactive functional groups that can covalently attach with the free NH2-terminals of the enzyme/protein to give an amide linkage [32]. Nevertheless,
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covalent bonding is not obligatory for irreversible protein adsorption to surfaces. There are instances where proteins are partially denatured as they rearrange upon approaching a hydrophobic surface, yet, retaining their enzymatic activity. In general, the enzymatic activity is realized through immobilization of GOD either onto a hydrophilic microenvironment by the covalent attachment method or onto a hydrophobic microenvironment by physisorption. It has been found that the latter microenvironment offers a higher specific activity as compared to the former [33]. Since, graphene is extremely hydrophobic [34], we anticipate good immobilization through physisorption onto its surface that should result in better sensing properties. Recently, a graphene based biosensor has been reported by utilizing polypyrrole (Ppy) to capture the graphene-oxide (GO) and GOD onto the glassy carbon electrode surface [19]. We report a biosensor merely based on graphene, using a graphene coated pipette as a substrate that was inserted into the intracellular environment of a single human fat cell. The diameter of working and the reference electrode was 0.7 mm. To our knowledge this is the first time graphene has been used for this purpose. In this paper, we describe a method in which the GOD is immobilized onto the graphene coated pipette surface (working electrode), shown in Fig. 1. The working electrode was prepared by coating it with commercial graphene-based conductive ink, which can suitably capture GOD on its surface. The peptide bonding between functionalized graphene and GOD molecules provides a biocompatible microenvironment to retain the enzyme in its native structure [35]. Moreover, functionalized graphene nanosheets within the conjugate facilitate the electron transfer between the matrix and electroactive center of the GOD, and
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the percolating network of graphene nanosheets provides multiplexed paths for the rapid charge conduction [36,37]. Furthermore, the amount of GOD that binds to the graphene offers a substantial loading, well enough for these enzymatic biosensing applications [38]. Subsequently, the entire electrode assembly is employed for the detection of intracellular glucose, which is in good agreement with other techniques such as nuclear magnetic resonance spectroscopy (NMR). 2. Experimental 2.1. Electrochemical Setup All experiments were carried out with a conventional twoelectrode system. The working electrode was a graphene coated glass pipette with diameter of 0.7 mm, modified with grapheneGOD composite. The potential of the working electrode was measured against the reference electrode of Ag/AgCl saturated with 3.0 M KCl. All electrochemical experiments were performed at room temperature. 2.2. Synthesis of Graphene Graphite-oxide was made by the modified Hummer’s method [10,39], and mixed with water to yield a yellow-brown suspension. This suspension was then ultrasonicated and treated with hydrazine hydrate. The mixture was heated in an oil bath at about 100 C in a water-cooled condenser for about 24 h. This resulted in the formation of GO which was precipitated as a black solid. This black solid was subsequently filtered and washed with deionized
Fig. 1. a) Schematic and SEM image of the pipet tip used as platform for enzyme immobilization and potentiometric detection of intracellular glucose, b) Schematic illustration of the GOD immobilization on graphene with peptide bond formation between carboxylic groups of GO and GOD and prospective physisorption of GOD on hydrophobic surface of graphene, and c) Representation of the GOD entrapment within a graphene matrix, along with possible electrochemical reaction near the electrode.
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water and methanol. Following this, the precipitate was dried using a continuous N2 flow for about 10 h.
with the previously reported results related to mono-layer graphene nanosheets [42].
2.3. Preparation of Graphene-Glucose Oxidase Conjugate
2.7. X-ray Diffraction
GOD enzyme solution, 10 mg/ml, was prepared in Phosphate Buffered Saline (PBS), using GOD (E.C. 1.1.3.4) from Aspergillus niger type GO3A, 360 U/mg (BBI Enzymes Ltd). PBS 10 mM solution was prepared from Na2HPO4 and KH2PO4 with 0.138 M NaCl, and the pH was adjusted to 7.40. GOD enzyme was electrostatically immobilized by dipping the graphene-coated electrode into 10 mL of the enzyme solution for 15 minutes at room temperature, and then dried in air for approximately 20 minutes.
The X-ray Diffraction (XRD) patterns were recorded in the 2theta (2u) range from 10 to 35 . The XRD patterns of the pristine graphite and graphene oxide are shown in Fig. 3. The pattern of pure graphite shows a peak at 26.5 corresponding to a basal spacing d002 = 3.4 Å. In GO, the intensity of (002) diffraction line (dspace 3.4 Å at 26.23 ) disappears, while the intensity of the diffraction peak at 12 (corresponding to a d-spacing of 7.34 nm) increases with oxidation [43].
2.4. Electrode Modification
2.8. Atomic Force Microscopy
Prior to electrode modification, the glass pipette with a tip diameter of 0.7 mm was coated with gold. The tip of the pipette was immersed in gold etchant in order to remove the metal from the tip before graphene coating. Vorbeck’s commercial graphene-based conductive ink, Vor-inkTM (N62-H15-XP7), was coated to the tip and connected to the gold on the back of pipette (Fig. 1a). Following this, the pipette was coated by graphene-GOD composite.
Height profiles of the GO were measured by AFM after drop casting on a relatively flat SiO2/Si substrate. The average thickness of one GO sheet equal to 1 nm (Fig. 4) confirm that the graphite oxide was completely exfoliated into GO. We observed heights from slightly less than 1 nm to a few nm thick. We assigned the sheets with height 1 nm, 1.5 nm, 2 nm and up to 5 nm to be 1, 2, 3 and few layered graphene sheets, respectively, consistent with the previously reported AFM results [1,44,45].
2.5. Preparation of Adipocytes Human adipocytes were isolated by collagenase digestion of pieces of subcutaneous adipose tissue obtained during elective surgery at the University Hospital in Linköping, Sweden (all patients gave their informed consent and procedures were approved by the local ethics committee). The adipocytes were incubated overnight before use as described by Strålfors and Honor [40], and used in a Krebs-ringer solution buffered with 20 mM HEPES, pH 7.40 and with additives [41]. 2.6. Transmission Electron Microscopy The sample for the transmission electron microscopy (TEM) prepared through simple drop casting of the graphene dispersion onto the holey carbon grid. A fan heater has been utilized with an objective to dry the holey carbon grid containing graphene nanosheets. Low and High resolution TEM results for graphene nanosheets with regular arrangement of hexagonal atomic packing of carbon is shown in Fig. 2, with selected area electron diffraction (SAED) pattern (inset) displaying good crystalline quality. The method of diffraction intensity ratios in the SAED pattern agrees Fig. 3. XRD data of precursor graphite and graphene-oxide, consistent with ref. 43].
Fig. 2. Low (a) and High resolution (b) TEM image of the graphene nanosheets. The inset is corresponding to the SAED pattern confirming the presence of graphene.
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Fig. 4. Tapping mode AFM image of GO with step height profile: The average thickness of a GO sheet, 1 nm, confirm the complete exfoliation of graphite-oxide into GO.
2.9. Raman spectroscopy The Raman spectrum for our GO shows a D-band at 1340 cm1 and the G-band at 1580 cm1, (Fig. 5). The noticeable D-peak evolved from the structural imperfections created due to the attachment of oxygen functional groups on the carbon basal plane. The G-band corresponds to the recovery of the hexagonal network of carbon atoms with defects. The intensity ratio (ID/IG) for the GO increased from 0.84 to 0.95 after functionalization with GOD, indicating that the reduction process altered the structure of GO with many structural defects [11]. These two peaks are visible at 1346 cm1 and 1585 cm1 after introduction of GOD into GO and keeping it over 10 days, confirming the presence of GO in the resulting enzyme electrode and good stability of GO/GOD on the electrode. The relative intensity of D-band increases after incorporation of GOD, pointing towards the interaction between GO and GOD [46]. 3. Results and Discussion 3.1. Electrochemical measurements The generic construction of a two-electrode electrochemical potential cell has the following scheme, reference electrode | reference electrolyte k test electrolyte | indicator electrode which has been adopted in our case, namely AgCl/Ag | Cl k Buffer | Graphene
Fig. 5. Raman spectra of the GO thin film compared with the GO soaked with GOD enzyme.
The variation in the electrochemical cell voltage (emf) is the key to measure the changes in electrolyte composition. The observed changes are then related to the concentration of ions in the test electrolyte via a calibration procedure. 3.2. The Calibration Curve We started our measurements after immobilizing grapheneGOD conjugate onto the graphene coated electrode surface and preparing the Ag/AgCl reference microelectrode in an electrolyte drop. The response of the electrochemical potential difference across as-made electrode assembly (graphene based biosensor) to the changes in buffer electrolyte glucose was measured for the range of 10 mM - 1 mM. We found a linear dependence of the electrochemical potential against the increasing glucose concentration, with a sensitivity of 64 mV/decade, at 23 C (Fig. 6). The biosensor depicted good linearity with a correlation coefficient of 0.993. The linearity suggests a large dynamic for such sensor configuration. This curve was also used as the reference for intracellular glucose measurements (discussed later). The graphene nanosheets within the conjugate, loaded with GOD molecules provide a biocompatible microenvironment for the enzyme to retain its native activity. The bonding between the GOD and graphene brings the graphene surface closer to the reactive center of GOD and assists the direct electron transfer, resulting in the enhanced detection of glucose under physiological conditions
Fig. 6. Calibration curve showing the electrochemical potential difference between the glucose-sensing microelectrode and the Ag/AgCl reference microelectrode. The linearity of the curve suggests a large dynamic range for our sensors.
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[19]. It has been experimentally confirmed that for different number of graphene layers employed for the enzyme conjugation, the extent of p–p interaction exerted by the individual hexagonal cells of the graphene planes onto the backbone of the dimeric protein GOD remains unaffected, suggesting that the amount of enzyme loaded (enzymes conjugation) to the graphene samples is independent of the number of graphene layers [38]. The sensing is based on the enzymatic reaction catalysed by GOD with b d -glucose, given by GOD
H2 O þ O2 þ d Glucose ! d gluconolactone þ H2 O2 The above reaction, termed as half-cell reaction, takes place at the cathode (graphene based electrode) surface in the electrochemical cell [47]. The signal is transferred via multiplexed paths provided by the graphene nanosheets permeating 3D network in the conjugate [35–37], and sent out to the outside world via Au coating underneath. The recorded potential at the electrode-electrolyte interface is then used to quantify the activity (upon concentration change) of glucose in the reaction, according to the fundamentals of potentiometric sensors [47,48]. For the direct potentiometric measurements of the analyte ion concentration, Nernst relationship can be a crosscheck which relates the observed potential with the change in concentration [47–50]. 3.3. Repeatability and Selectivity Our method relies on the pH attenuation monitoring, due to gluconic acid generation, as a result of the enzymetic reaction. Fig. 7 displays the results of three experiments for the same glucose-sensing microelectrode, showing good repeatability and linearity in various glucose concentrations in PBS solution (pH 7.4). Note that for each measurement, working microelectrode was carefully washed with deionized water in order to remove the residual ions (from the preceding measurement) from the surface of the electrode. The selectivity of glucose sensing microelectrode is also very important and relies predominantly upon two major factors: the enzyme analyte reaction, and the selective measurements. The enzyme analyte reaction with glucose is highly selective without any major interfering reaction. It can be useful to control the possible interferences that may be introduced by reducing agents such as ascorbic acid and uric acid, which are well-known interferents with amperometric GOD biosensors [49,51]. The
Fig. 7. Calibration curve from three different experiments showing the electrochemical potential difference at different glucose concentrations, in phosphate buffer saline solution (pH 7.4). Same sensor electrode is used here, exhibiting a good repeatability.
addition of these potential interferents introduces only a negligible change in the signal. Addition of 100 - 500 mM of ascorbic acid or uric acid to 1000 mM glucose only generates a small transient signals and recovers to the original value shortly. We have also analyzed the effect of the varying temperature: the optimum temperature range for the response has been found within 2050 C. Hydrogen peroxide (H2O2) that comes as an enzymatic product of the GOD assisted biological process is usually detected via amperometric type biosensors. It is notable that H2O2 sensing with graphene has already been experimentally well confirmed [35,52– 55]. These sensors offer a good lower limit of detection for H2O2, 0.51 mM, and a wide linear range, 6.5 – 230 mM, which is comparable to its conventional detection approaches [35]. 3.4. Intracellular Measurements The potentiometric sensors offer the advantage of measuring low concentrations in tiny sample volumes (e.g. intracellular as in our case), as they ideally offer the benefits of not chemically influencing the sample and being independent of the analyte solution volume [48]. This advantageous characteristic was exploited to measure the concentration of glucose in single human adipocytes. The glucose selective working microelectrode was mounted on a micromanipulator, and moved into a position at the same level as the cell. The working and the reference microelectrodes were gently pushed through the cell membrane and into the cell, Fig. 8. Once the working and reference microelectrodes were inside the cell which had already been isolated from the surrounding buffer solution, an electrochemical potential difference signal was detected. This signal was recorded and compared with the calibration curve to determine the glucose concentration. Six measurement were carried out simultaneously on different cells with minimum measured value of 57 mM and maximum 68 mM. Based on these measurements, the intracellular
Fig. 8. The experimental setup used for selective measurements of the intracellular glucose concentration (top) with a schematic illustration (bottom). Microscopic images of a single human adipocyte are taken during measurements. As-measured values of intracellular glucose are comparable to other sophisticated techniques such as NMR.
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glucose concentration in a human adipocyte (60 10 mM) corresponded well with intracellular concentration of 70 mM determined by nuclear magnetic resonance spectroscopy in human muscle tissue [56]. 4. Conclusion We purpose and demonstrate a novel functionalized graphenebased selective electrochemical sensor for measuring intracellular glucose. Potentiometric approach has a big advantage in intracellular measurements as the device can be tuned for very low analyte consumption, which is of particular importance in intracellular applications. The as-prepared graphene biosensor exhibited a linear glucose-dependent electrochemical potential difference over the concentration range of interest (10–1000 mM). The measured glucose concentration in human adipocytes by using our graphene based sensor was consistent with values of glucose concentration reported in the literature. These results demonstrate the capability to perform biologically relevant measurements of glucose within living cells. Our proposed sensing scheme is a potential candidate for variety of medical applications. Moreover, the fabrication method is simple and can be extended to immobilize other enzymes and other bioactive molecules for a variety of biosensor designs.
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A Miniature Graphene-based Biosensor for Intracellular Glucose Measurements Kamran ul Hasana,b,* , Muhammad H. Asifa,c, Muhammad Umair Hassand, Mats O. Sandberge, O. Nura , M. Willandera , Siri Fagerholmf , Peter Strålforsf a
Department of Science and Technology, Campus Norrköping, Linköping University, Bredgatan 34, SE-601 74 Norrköping, Sweden Centres of Excellence in Science & Applied Technologies (CESAT), Islamabad, Pakistan Materials Research Lab, Department of Physics, COMSATS Institute of Information Technology, Lahore, 54000, Pakistan d Center for Micro and Nano Devices (CMND), Department of Physics, COMSATS Institute of Information Technology, Islamabad, 44000, Pakistan e Acreo AB Bredgatan 34, SE-602 21 Norrköping, Sweden f Department of Clinical and Experimental Medicine, Linköping University, SE-581 85 Linköping, Sweden b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 12 March 2015 Received in revised form 20 May 2015 Accepted 8 June 2015 Available online 10 June 2015
We report on a small and simple graphene-based potentiometric sensor for the measurement of intracellular glucose concentration. A fine borosilicate glass capillary coated with graphene and subsequently immobilized with glucose oxidase (GOD) enzyme is inserted into the intracellular environment of a single human cell. The functional groups on the edge plane of graphene assist the attachment with the free amine terminals of GOD enzyme, resulting in a better immobilization. The sensor exhibits a glucose-dependent electrochemical potential against an Ag/AgCl reference microelectrode which is linear across the whole concentration range of interest (10 – 1000 mM). Glucose concentration in human fat cell measured by our graphene-based sensor is in good agreement with nuclear magnetic resonance (NMR) spectroscopy. ã2015 Elsevier Ltd. All rights reserved.
Keywords: Graphene Bio-sensors Glucose-oxidase Intracellular sensors Graphene oxide Glucose Sensor
1. Introduction Graphene has been enticing much attention due to its fascinating properties, such as extremely large surface area (2630 m2 g1), high optical transmittance ( 97.7 %), large mechanical fracture strength, ( 125 GPa), excellent thermal conductivity ( 5000 W m1 K1) and superb charge-carrier mobility ( 200,000 cm2 V1s1) [1–8]. Its remarkable properties have opened up a whole realm of applications [3,8–11], amongst which biosensing has been capturing a huge interest [12–14]. This is mainly because of the great promise and performance owning to its tremendously large surface area to volume ratio as a dominating and favorable parameter for sensing applications [8,15,16]. The sensing is usually achieved via immobilization of enzymes onto the sensing surfaces of a suitably modified electrode, which can provide highly selective, sensitive, and rapid analysis of various biological species like DNA [17], Human IgG [18] and glucose [19]. Carbon nanotube (CNT) modified electrodes have already been very successful for biosensing of glucose, which is realized by a
* Corresponding author. E-mail address: [email protected] (K.u. Hasan). http://dx.doi.org/10.1016/j.electacta.2015.06.035 0013-4686/ ã 2015 Elsevier Ltd. All rights reserved.
dramatic decrease in the hydrogen peroxide overpotential, plus the direct electron transfer of glucose oxidase (GOD) observed at asmade electrodes [20,21]. However, using graphene for such applications can offer several advantages over CNTs for its superior properties: Its 2-dimentional nature (single atomic layer of graphite) can maximize the interaction between the surface dopants and adsorbates. It offers much lower Johnson noise and higher sensitivity [22–24], as compared to CNT, therefore, a minute variation of carrier concentration can cause a notable variation of electrical conductivity [25]. Moreover, graphene can be obtained via chemical conversion of graphite, which is a simple and inexpensive synthesis method [26]. We employ the GOD enzyme, which belongs to a family of oxidases that are widely used in many glucose sensors. The binding between GOD and the electrode surface is a critical parameter for the stability and sensitivity of the biosensor. Therefore, it is important to select a suitable electrode material that can favorably bind an enzyme. Various methods, such as covalent binding [27], embedding [28], and cross-linking [29–31], have already been exploited to immobilize the GOD onto different supporting materials. Functionalized graphene has reactive functional groups that can covalently attach with the free NH2-terminals of the enzyme/protein to give an amide linkage [32]. Nevertheless,
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covalent bonding is not obligatory for irreversible protein adsorption to surfaces. There are instances where proteins are partially denatured as they rearrange upon approaching a hydrophobic surface, yet, retaining their enzymatic activity. In general, the enzymatic activity is realized through immobilization of GOD either onto a hydrophilic microenvironment by the covalent attachment method or onto a hydrophobic microenvironment by physisorption. It has been found that the latter microenvironment offers a higher specific activity as compared to the former [33]. Since, graphene is extremely hydrophobic [34], we anticipate good immobilization through physisorption onto its surface that should result in better sensing properties. Recently, a graphene based biosensor has been reported by utilizing polypyrrole (Ppy) to capture the graphene-oxide (GO) and GOD onto the glassy carbon electrode surface [19]. We report a biosensor merely based on graphene, using a graphene coated pipette as a substrate that was inserted into the intracellular environment of a single human fat cell. The diameter of working and the reference electrode was 0.7 mm. To our knowledge this is the first time graphene has been used for this purpose. In this paper, we describe a method in which the GOD is immobilized onto the graphene coated pipette surface (working electrode), shown in Fig. 1. The working electrode was prepared by coating it with commercial graphene-based conductive ink, which can suitably capture GOD on its surface. The peptide bonding between functionalized graphene and GOD molecules provides a biocompatible microenvironment to retain the enzyme in its native structure [35]. Moreover, functionalized graphene nanosheets within the conjugate facilitate the electron transfer between the matrix and electroactive center of the GOD, and
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the percolating network of graphene nanosheets provides multiplexed paths for the rapid charge conduction [36,37]. Furthermore, the amount of GOD that binds to the graphene offers a substantial loading, well enough for these enzymatic biosensing applications [38]. Subsequently, the entire electrode assembly is employed for the detection of intracellular glucose, which is in good agreement with other techniques such as nuclear magnetic resonance spectroscopy (NMR). 2. Experimental 2.1. Electrochemical Setup All experiments were carried out with a conventional twoelectrode system. The working electrode was a graphene coated glass pipette with diameter of 0.7 mm, modified with grapheneGOD composite. The potential of the working electrode was measured against the reference electrode of Ag/AgCl saturated with 3.0 M KCl. All electrochemical experiments were performed at room temperature. 2.2. Synthesis of Graphene Graphite-oxide was made by the modified Hummer’s method [10,39], and mixed with water to yield a yellow-brown suspension. This suspension was then ultrasonicated and treated with hydrazine hydrate. The mixture was heated in an oil bath at about 100 C in a water-cooled condenser for about 24 h. This resulted in the formation of GO which was precipitated as a black solid. This black solid was subsequently filtered and washed with deionized
Fig. 1. a) Schematic and SEM image of the pipet tip used as platform for enzyme immobilization and potentiometric detection of intracellular glucose, b) Schematic illustration of the GOD immobilization on graphene with peptide bond formation between carboxylic groups of GO and GOD and prospective physisorption of GOD on hydrophobic surface of graphene, and c) Representation of the GOD entrapment within a graphene matrix, along with possible electrochemical reaction near the electrode.
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water and methanol. Following this, the precipitate was dried using a continuous N2 flow for about 10 h.
with the previously reported results related to mono-layer graphene nanosheets [42].
2.3. Preparation of Graphene-Glucose Oxidase Conjugate
2.7. X-ray Diffraction
GOD enzyme solution, 10 mg/ml, was prepared in Phosphate Buffered Saline (PBS), using GOD (E.C. 1.1.3.4) from Aspergillus niger type GO3A, 360 U/mg (BBI Enzymes Ltd). PBS 10 mM solution was prepared from Na2HPO4 and KH2PO4 with 0.138 M NaCl, and the pH was adjusted to 7.40. GOD enzyme was electrostatically immobilized by dipping the graphene-coated electrode into 10 mL of the enzyme solution for 15 minutes at room temperature, and then dried in air for approximately 20 minutes.
The X-ray Diffraction (XRD) patterns were recorded in the 2theta (2u) range from 10 to 35 . The XRD patterns of the pristine graphite and graphene oxide are shown in Fig. 3. The pattern of pure graphite shows a peak at 26.5 corresponding to a basal spacing d002 = 3.4 Å. In GO, the intensity of (002) diffraction line (dspace 3.4 Å at 26.23 ) disappears, while the intensity of the diffraction peak at 12 (corresponding to a d-spacing of 7.34 nm) increases with oxidation [43].
2.4. Electrode Modification
2.8. Atomic Force Microscopy
Prior to electrode modification, the glass pipette with a tip diameter of 0.7 mm was coated with gold. The tip of the pipette was immersed in gold etchant in order to remove the metal from the tip before graphene coating. Vorbeck’s commercial graphene-based conductive ink, Vor-inkTM (N62-H15-XP7), was coated to the tip and connected to the gold on the back of pipette (Fig. 1a). Following this, the pipette was coated by graphene-GOD composite.
Height profiles of the GO were measured by AFM after drop casting on a relatively flat SiO2/Si substrate. The average thickness of one GO sheet equal to 1 nm (Fig. 4) confirm that the graphite oxide was completely exfoliated into GO. We observed heights from slightly less than 1 nm to a few nm thick. We assigned the sheets with height 1 nm, 1.5 nm, 2 nm and up to 5 nm to be 1, 2, 3 and few layered graphene sheets, respectively, consistent with the previously reported AFM results [1,44,45].
2.5. Preparation of Adipocytes Human adipocytes were isolated by collagenase digestion of pieces of subcutaneous adipose tissue obtained during elective surgery at the University Hospital in Linköping, Sweden (all patients gave their informed consent and procedures were approved by the local ethics committee). The adipocytes were incubated overnight before use as described by Strålfors and Honor [40], and used in a Krebs-ringer solution buffered with 20 mM HEPES, pH 7.40 and with additives [41]. 2.6. Transmission Electron Microscopy The sample for the transmission electron microscopy (TEM) prepared through simple drop casting of the graphene dispersion onto the holey carbon grid. A fan heater has been utilized with an objective to dry the holey carbon grid containing graphene nanosheets. Low and High resolution TEM results for graphene nanosheets with regular arrangement of hexagonal atomic packing of carbon is shown in Fig. 2, with selected area electron diffraction (SAED) pattern (inset) displaying good crystalline quality. The method of diffraction intensity ratios in the SAED pattern agrees Fig. 3. XRD data of precursor graphite and graphene-oxide, consistent with ref. 43].
Fig. 2. Low (a) and High resolution (b) TEM image of the graphene nanosheets. The inset is corresponding to the SAED pattern confirming the presence of graphene.
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Fig. 4. Tapping mode AFM image of GO with step height profile: The average thickness of a GO sheet, 1 nm, confirm the complete exfoliation of graphite-oxide into GO.
2.9. Raman spectroscopy The Raman spectrum for our GO shows a D-band at 1340 cm1 and the G-band at 1580 cm1, (Fig. 5). The noticeable D-peak evolved from the structural imperfections created due to the attachment of oxygen functional groups on the carbon basal plane. The G-band corresponds to the recovery of the hexagonal network of carbon atoms with defects. The intensity ratio (ID/IG) for the GO increased from 0.84 to 0.95 after functionalization with GOD, indicating that the reduction process altered the structure of GO with many structural defects [11]. These two peaks are visible at 1346 cm1 and 1585 cm1 after introduction of GOD into GO and keeping it over 10 days, confirming the presence of GO in the resulting enzyme electrode and good stability of GO/GOD on the electrode. The relative intensity of D-band increases after incorporation of GOD, pointing towards the interaction between GO and GOD [46]. 3. Results and Discussion 3.1. Electrochemical measurements The generic construction of a two-electrode electrochemical potential cell has the following scheme, reference electrode | reference electrolyte k test electrolyte | indicator electrode which has been adopted in our case, namely AgCl/Ag | Cl k Buffer | Graphene
Fig. 5. Raman spectra of the GO thin film compared with the GO soaked with GOD enzyme.
The variation in the electrochemical cell voltage (emf) is the key to measure the changes in electrolyte composition. The observed changes are then related to the concentration of ions in the test electrolyte via a calibration procedure. 3.2. The Calibration Curve We started our measurements after immobilizing grapheneGOD conjugate onto the graphene coated electrode surface and preparing the Ag/AgCl reference microelectrode in an electrolyte drop. The response of the electrochemical potential difference across as-made electrode assembly (graphene based biosensor) to the changes in buffer electrolyte glucose was measured for the range of 10 mM - 1 mM. We found a linear dependence of the electrochemical potential against the increasing glucose concentration, with a sensitivity of 64 mV/decade, at 23 C (Fig. 6). The biosensor depicted good linearity with a correlation coefficient of 0.993. The linearity suggests a large dynamic for such sensor configuration. This curve was also used as the reference for intracellular glucose measurements (discussed later). The graphene nanosheets within the conjugate, loaded with GOD molecules provide a biocompatible microenvironment for the enzyme to retain its native activity. The bonding between the GOD and graphene brings the graphene surface closer to the reactive center of GOD and assists the direct electron transfer, resulting in the enhanced detection of glucose under physiological conditions
Fig. 6. Calibration curve showing the electrochemical potential difference between the glucose-sensing microelectrode and the Ag/AgCl reference microelectrode. The linearity of the curve suggests a large dynamic range for our sensors.
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[19]. It has been experimentally confirmed that for different number of graphene layers employed for the enzyme conjugation, the extent of p–p interaction exerted by the individual hexagonal cells of the graphene planes onto the backbone of the dimeric protein GOD remains unaffected, suggesting that the amount of enzyme loaded (enzymes conjugation) to the graphene samples is independent of the number of graphene layers [38]. The sensing is based on the enzymatic reaction catalysed by GOD with b d -glucose, given by GOD
H2 O þ O2 þ d Glucose ! d gluconolactone þ H2 O2 The above reaction, termed as half-cell reaction, takes place at the cathode (graphene based electrode) surface in the electrochemical cell [47]. The signal is transferred via multiplexed paths provided by the graphene nanosheets permeating 3D network in the conjugate [35–37], and sent out to the outside world via Au coating underneath. The recorded potential at the electrode-electrolyte interface is then used to quantify the activity (upon concentration change) of glucose in the reaction, according to the fundamentals of potentiometric sensors [47,48]. For the direct potentiometric measurements of the analyte ion concentration, Nernst relationship can be a crosscheck which relates the observed potential with the change in concentration [47–50]. 3.3. Repeatability and Selectivity Our method relies on the pH attenuation monitoring, due to gluconic acid generation, as a result of the enzymetic reaction. Fig. 7 displays the results of three experiments for the same glucose-sensing microelectrode, showing good repeatability and linearity in various glucose concentrations in PBS solution (pH 7.4). Note that for each measurement, working microelectrode was carefully washed with deionized water in order to remove the residual ions (from the preceding measurement) from the surface of the electrode. The selectivity of glucose sensing microelectrode is also very important and relies predominantly upon two major factors: the enzyme analyte reaction, and the selective measurements. The enzyme analyte reaction with glucose is highly selective without any major interfering reaction. It can be useful to control the possible interferences that may be introduced by reducing agents such as ascorbic acid and uric acid, which are well-known interferents with amperometric GOD biosensors [49,51]. The
Fig. 7. Calibration curve from three different experiments showing the electrochemical potential difference at different glucose concentrations, in phosphate buffer saline solution (pH 7.4). Same sensor electrode is used here, exhibiting a good repeatability.
addition of these potential interferents introduces only a negligible change in the signal. Addition of 100 - 500 mM of ascorbic acid or uric acid to 1000 mM glucose only generates a small transient signals and recovers to the original value shortly. We have also analyzed the effect of the varying temperature: the optimum temperature range for the response has been found within 2050 C. Hydrogen peroxide (H2O2) that comes as an enzymatic product of the GOD assisted biological process is usually detected via amperometric type biosensors. It is notable that H2O2 sensing with graphene has already been experimentally well confirmed [35,52– 55]. These sensors offer a good lower limit of detection for H2O2, 0.51 mM, and a wide linear range, 6.5 – 230 mM, which is comparable to its conventional detection approaches [35]. 3.4. Intracellular Measurements The potentiometric sensors offer the advantage of measuring low concentrations in tiny sample volumes (e.g. intracellular as in our case), as they ideally offer the benefits of not chemically influencing the sample and being independent of the analyte solution volume [48]. This advantageous characteristic was exploited to measure the concentration of glucose in single human adipocytes. The glucose selective working microelectrode was mounted on a micromanipulator, and moved into a position at the same level as the cell. The working and the reference microelectrodes were gently pushed through the cell membrane and into the cell, Fig. 8. Once the working and reference microelectrodes were inside the cell which had already been isolated from the surrounding buffer solution, an electrochemical potential difference signal was detected. This signal was recorded and compared with the calibration curve to determine the glucose concentration. Six measurement were carried out simultaneously on different cells with minimum measured value of 57 mM and maximum 68 mM. Based on these measurements, the intracellular
Fig. 8. The experimental setup used for selective measurements of the intracellular glucose concentration (top) with a schematic illustration (bottom). Microscopic images of a single human adipocyte are taken during measurements. As-measured values of intracellular glucose are comparable to other sophisticated techniques such as NMR.
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glucose concentration in a human adipocyte (60 10 mM) corresponded well with intracellular concentration of 70 mM determined by nuclear magnetic resonance spectroscopy in human muscle tissue [56]. 4. Conclusion We purpose and demonstrate a novel functionalized graphenebased selective electrochemical sensor for measuring intracellular glucose. Potentiometric approach has a big advantage in intracellular measurements as the device can be tuned for very low analyte consumption, which is of particular importance in intracellular applications. The as-prepared graphene biosensor exhibited a linear glucose-dependent electrochemical potential difference over the concentration range of interest (10–1000 mM). The measured glucose concentration in human adipocytes by using our graphene based sensor was consistent with values of glucose concentration reported in the literature. These results demonstrate the capability to perform biologically relevant measurements of glucose within living cells. Our proposed sensing scheme is a potential candidate for variety of medical applications. Moreover, the fabrication method is simple and can be extended to immobilize other enzymes and other bioactive molecules for a variety of biosensor designs.
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