functionalized cvd monolayer graphene for label-free

Nano Res

1

Functionalized CVD Monolayer Graphene for

Label-Free Impedimetric Biosensing

Shimaa Eissa1,2, Gaston Contreras Jimenez2, Farzaneh Mahvash2,3, Abdeladim Guermoune2, Chaker

Tlili1, Thomas Szkopek3, Mohammed Zourob4, and Mohamed Siaj2 ()

Nano Res., Just Accepted Manuscript • DOI: 10.1007/s12274-014-0671-0

http://www.thenanoresearch.com on December 2 2014

© Tsinghua University Press 2014

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Nano Research

DOI 10.1007/s12274-014-0671-0

Nano Res.

f

Functionalized CVD monolayer graphene for label-free

impedimetric biosensing

Shimaa Eissa,a,b Gaston Contreras Jimenez,b Farzaneh

Mahvash,b,c Abdeladim Guermoune,b Chaker Tlili,a

Thomas Szkopek,c Mohammed Zourob,d and Mohamed

Siaj *,b

a Institut national de la recherche scientifique, Centre –

Energie, Matériaux et Télécommunications, 1650, Boul.

Lionel Boulet, Varennes (Québec) J3X 1S2, Canada b Département de Chimie et Biochimie, Université du

Québec à Montréal, Montréal, Québec, H3C 3P8,

Canada cCranfield Health, Vincent Building, Cranfield University,

Cranfield, Bedfordshire, UK d Dept. of Electrical and Computer Engineering, McGill

University, Montréal, Québec, H3A 2A7, Canada

Facile strategy for the covalent functionalization of chemical vapor

deposited monolayer graphene supported on glass substrate as

electrode material for electrochemical biosensing application.

Nano Res.

Functionalized CVD Monolayer Graphene for Label-Free Impedimetric Biosensing

Shimaa Eissa1,2, Gaston Contreras Jimenez2, Farzaneh Mahvash2,3, Abdeladim Guermoune2, Chaker Tlili1, Thomas Szkopek3, Mohammed Zourob4, and Mohamed Siaj2 () 1

Received: day month year Revised: day month year Accepted: day month year (automatically inserted by the publisher) © Tsinghua University Press and Springer-Verlag Berlin Heidelberg 2014 KEYWORDS CVD graphene, electrochemical impedance spectroscopy, Biosensor, Diazonium functionalization

ABSTRACT Recent advances in large area graphene growth have led to tremendous applications in a variety of areas. In the present study, chemical vapor deposited (CVD) monolayer graphene supported on glass substrate is examined as electrode material for electrochemical biosensing applications. We report here a facile strategy for the covalent functionalization of CVD monolayer graphene by electrochemical reduction of carboxyphenyl diazonium salt prepared in situ in acidic aqueous solution. The carboxyphenyl-modified graphene is characterized using Raman spectroscopy, X- ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM) as well as electrochemical impedance spectroscopy (EIS). We also show that the number of grafted carboxyphenyl groups on the graphene surface can be controlled by the number of cyclic voltammetry (CV) scans used for the electrografting step. We further present the fabrication and characterization of an immunosensor based on immobilization of ovalbumin antibody on the graphene surface after the activation of the grafted carboxylic groups via an EDC/NHS chemistry. The binding between the surface-immobilized antibodies and ovalbumin was then monitored by faradaic electrochemical impedance spectroscopy (EIS) in [Fe(CN)6]3-/4- solution. The percentage change of charge-transfer resistance (Rct) after binding shows a linear response with the ovalbumin concentrations in the range of 1.0 pgmL-1 to 100 ngmL−1, with a detection limit of 0.9 pgmL−1. Our results indicate good sensitivity of the developed functionalized CVD graphene platform which opens the way for use of CVD monolayer graphene in a wide range of electrochemical biosensing devices.

Nano Research DOI (automatically inserted by the publisher) Research Article

Nano Res.

1.Introduction

Graphene is a one atom thick two dimensional allotrope of carbon with exceptionally high electrical conductivity, elasticity, electron mobility [1], thermal conductivity [2] and mechanical strength [3]. Since its successful isolation in 2004 [1], graphene has been extensively incorporated into wide range of applications including nanoelectronics, solar cells, fuel cells, photovoltaic devices as well as biosensors [4]. Particularly, the combination of graphene and biomolecules for the fabrication of biosensing platforms has gained great research interest in recent years. Graphene has been explored in developing optical biosensors using fluorescence [5-7], surface plasmon resonance [8,9] and colorimetric [10-12] methods. Several biosensors based on graphene field effect transistors [13,14,15] have been also demonstrated. The superior advantages offered by electrochemical transducers for sensing, such as their lower cost, easier operation, capability of automation and miniaturization, have also led to significant interest in developing graphene-based electrochemical biosensors [16]. Graphene has been employed as electrode material in various electrochemical biosensors using enzymes, antibodies, ssDNA or aptamers as recognition receptors [17]. Moreover, graphene has been utilized as a label nanocarrier in different biosensing assays showing outstanding electrochemical signal enhancement [18]. Superior electrochemical biosensing performance of graphene to carbon nanotubes has been also reported in terms of sensitivity, signal-to-noise ratio, electron transfer kinetics and stability [19,20]. However, the majority of graphene based materials used in the reported electrochemical biosensing platforms are produced by the reduction of graphene oxide (GO). The GO is typically prepared by Hummer’s method [21], reduced using chemical, thermal or electrochemical techniques [22] and drop casted on the surface of carbon electrodes. These synthetic methods result in abundant structural defects and functional groups on the graphene surface which significantly affects its electrochemistry and consequently its sensing behaviour [16]. However, with the great advances in graphene synthesis methodologies, CVD has emerged as effective method for producing large surface area, uniform, easily transferred, low cost,

and high quality graphene sheets [23]. Recently, the electrochemical behaviour of CVD monolayer graphene has been investigated in some reports. Li et al. [24] have reported the electrochemical behaviour of CVD-grown graphene towards the redox reaction of ferrocenemethanol showing 10-fold electron transfer rate compared with the basal plane of bulk graphite. A superior electrocatalytic activity of nitrogen doped CVD-grown graphene towards the electrochemical reduction of oxygen has been presented by Quet al. [25]. On the other hand, the application of CVD graphene in electrochemical biosensors has not been reported intensively. Specifically, we report here the first application of CVD monolayer graphene as a label-free electrochemical biosensing platform. An important factor in the application of a material as electrode in electrochemical biosensors is the functionalization method and the subsequent immobilization of biomolecules. Recently, significant effort has been devoted to graphene functionalization using various covalent and noncovalent approaches [26]. However, covalent functionalization schemes of graphene appear to be more promising due to their more robust nature as well as their more ability to significantly change the electronic properties of the graphene sheet. Among the reported approaches for the covalent modification of graphene, the use of aryl diazonium salt has been well investigated [27]. This functionalization method is easy to achieve and to control, and enables a wide variety of grafting groups that can be used to carry out further chemistries. The covalent functionalization of graphene using aryl diazonium salt has been reported with epitaxial graphene (EG) [28], graphene nanoribbons [29], chemically converted graphene (CCG) [30], mechanically exfoliated graphene (MEG) [31] as well as CVD graphene. The reactivity of CVD-grown graphene towards the diazonium functionalization has been recently reported [32]. Jin et al. [33] have also shown the successful functionalization of CVD graphene with 4-propargyloxybenzenediazonium tetrafluoroborate and the subsequent attachment of a short chain polyethylene glycol with a terminal carboxylic group. In all these reports, the graphene diazonium functionalization occurred via spontaneous electron transfer mechanism, typically requiring several hours

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for completion [28,34] and sometimes heat or surfactants [30, 34]. We have recently reported a robust, fast, and controlled modification of graphene modified screen printed carbon electrodes by electrochemical reduction of aryl diazonium salt [35-37]. Similarly, an electrografting method of nitrophenyl groups using diazonium salt reduction on CVD monolayer graphene transferred to silicon substrate was also reported [38]. The authors have shown the successful grafting of uniform thin films using electrochemistry and demonstrated the modulation of the electronic properties of graphene by opening a band gap upon functionalization [38]. More recently, a mechanistic investigation of electrografted diazonium-based films on few layered graphene (vapour-deposited) on nickel has been reported [39]. However, diazonium functionalized monolayer CVD graphene sheets has yet to be applied to biosensing. In this work, we demonstrate the controlled modification of a CVD monolayer graphene by electrochemical reduction of carboxyphenyl diazonium salt in acidic aqueous solution. We then utilize for the first time the developed carboxyphenyl modified graphene as an impedimetric immunosening platform for label-free detection of protein. Here, we employ Faradaic electrochemical impedance spectroscopy (EIS) as a sensitive technique that probes the change in interfacial properties on the graphene electrode surface that enables both the detailed monitoring of the graphene modification steps and the subsequent biorecognition events. 2. Experimental:

2.1 materials and Reagents

Twenty-five micrometer thick, Cu foils (Alfa Aesar, 25 µm thick, N# 13382) were used as the substrate for graphene growth. H2 (99.9 %) and CH4 (99.9 %) used in graphene growth were directly connected to the CVD system. Poly (methyl methacrylate) (PMMA, 950 kDa, 4% solution in anisole) was obtained from MicroChem (Newton, USA). Kapton® Tape, (6.4mm), Copper Conductive Tape (6.3 mm) and Silver Paint were obtained from Ted Pella, Inc (CA, USA). Anti-Ovalbumin monoclonal antibody

(anti-OVA-MAb) was obtained from Abcam (Cambridge, USA). Ovalbumin from chicken egg white, 4-aminobenzoic acid, sodium nitrite, potassium ferrocyanide (K4Fe(CN)6), potassium ferricyanide (K3Fe(CN)6), dipotassium hydrogen orthophosphate, potassium dihydrogen orthophosphate, sodium chloride, hydrochloric acid, 2-(N-morpholino) ethanesulfonic acid (MES), ammonium persulfate, bovine serum albumin (BSA), chicken egg lysozyme, and β-lactoglobulin (β-LG) were obtained from Sigma (Ontario, Canada). 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were obtained from Fisher Scientific (Ontario, Canada). A phosphate buffered saline PBS solution (10 mM, pH 7.4) was used for the preparation of the ovalbumin antibody and protein standard solutions and dilutions. A 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution (100 mM, pH 5.0) was used in the activation of the carboxylic groups. All aqueous solutions were prepared with deionized distilled water obtained from a Milli-Q water purifying system (18 MΩcm). 2.2 Apparatus

All electrochemical experiments were performed using an Autolab PGSTAT302N (Eco Chemie, The Netherlands) potentiostat/galvanostat controlled by NOVA software version 1.9. X-ray photoelectron spectroscopy (XPS) measurements were performed with VG Escalab220iXL instrument using a Mg polychromatic source (MgKα=1253.6 eV) at a base pressure between 5×10-10 and 1×10-9 mbar. Raman spectroscopy measurements were performed using a micro-Raman spectrometer (RenishawInVia Reflex, Model: RM3000) with excitation from an argon ion laser beam (514 nm) in a backscattering geometry. Atomic force microscopy images were obtained using Veeco/Bruker AFM instrument in ScanAsyst mode.

2.3 Graphene synthesis by chemical vapor deposition

Sheets of copper foil ∼ (2cm ×2 cm) were introduced into a 1 inch diameter quartz tube and heated to 1000 ◦C under vacuum in a 700 mTorr H2 flow for 20 min. Then, CH4 was introduced to the system with a total

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tube pressure of 1 Torr. Graphene growth was carried out for 90 min, then, CH4 flow was cut off and the tube was cooled down to room temperature while maintaining H2 pressure at 700 mTorr. The graphene grown on copper was then removed from the CVD reactor.

2.4 Graphene transfer onto glass substrate

After growth, polymethyl methacrylate (PMMA) was spin coated onto the surface of the graphene coated Cu and baked at 120 oC for 1 min. The sample was then immersed in ammonium persulfate (0.1 M) solution to etch the Cu foil at room temperature. The floating PMMA-supported graphene film was then carefully transferred to a de-ionized water bath to remove the residual etchant. The transfer of the PMMA-graphene film to a clean DI water solution is repeated at least three times to ensure thorough washing. A glass sheet is then used to extract the PMMA-supported graphene from the water bath. The graphene sample was then dried at ambient temperature for 48 h in a clean environment to ensure complete adhesion to the glass. The PMMA was then dissolved in a warm acetone bath to produce a pristine graphene layer on glass sheet.

2.5 Connection of graphene sheet as working electrode

The graphene sheet was connected using conductive tape and silver paste is used to improve the connection between the copper and the graphene sheet. A nonconductive adhesive polyimide tape was then used to insulate the metal contacts and to expose a well defined area of the graphene surface (3

×5 mm) to the solution during the electrochemical measurements. The electrode was then connected to the potentiostat through the copper contact.

2.6 Carboxyphenyl functionalization of the CVD graphene electrode surface and the immunosensor construction

As shown in Scheme 1, the 4-carboxyphenyl diazonium salt was prepared in-situ by mixing 2 mM sodium nitrite solution with 2 mM 4-aminobenzoic acid in 0.5 M HCl. The mixture was then stirred for 5 min at room temperature. The functionalization of CVD graphene electrodes (GE) was then performed by electroreduction of the carboxyphenyl diazonium cations using three cyclic voltammetric scans from +0.3 to − 0.6 V at a scan rate of 100 mV s-1. The electrode was then thoroughly rinsed with Milli-Q water. The 4-carboxyphenyl (CP) modified graphene electrode was activated by incubating the electrodes in MES buffer (pH 5.0) containing 100 mM EDC and 20 mM NHS for 60 min. After washing with MES buffer, the modified electrode surface was covered with anti-OVA-MAb solution (10 µg mL-1 in 10 mM PBS buffer, pH 7.4) and incubated for 2 hours at room temperature in water-saturated atmosphere. The removal of the unreacted antibodies was performed by gently washing the electrodes with PBS buffer (10 mM, pH 7.4). Then, the electrodes were incubated in 0.1 % of BSA in PBS buffer, pH 7.4 for 30 min to deactivate the remaining active groups and to block the free graphene surface in order to minimize nonspecific adsorption. The prepared immunosensor was then rinsed with PBS and used in the assay or kept at 4◦C in PBS buffer pH 7.4 for later use.

Scheme1 The electrografting process of carboxyphenyl groups on the graphene electrode and the subsequent antibody immobilization.


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2.7 Electrochemical measurements

All electrochemical measurements were performed at room temperature using a three-electrode configuration. A platinum electrode served as an auxiliary electrode, an Ag/AgCl electrode served as a reference electrode, while the CVD graphene sheet was used as working electrode. All electrochemical experiments were performed in a 5 mL glass cell. For the ovalbumin measurements and the negative control experiments, 50 µL of ovalbumin or control proteins (lysozyme and β-LG) of the required concentration in PBS buffer pH 7.4 were incubated on the immunosensor surface for 45 min in water-saturated atmosphere. The immunosensor was then washed thoroughly with PBS buffer pH 7.4 and subjected to electrochemical measurements. Impedance measurements were recorded in a 0.1 M PBS buffer solution containing 10 mM [Fe(CN)6]3−/4− redox pair (1:1 molar ratio) over a frequency range from 100 kHz to 0.1 Hz with a 10 mV sinusoidal potential and a DC potential of 0.2 V vs the Ag/AgCl reference electrode. The equivalent circuit shown in Fig. S1† was used to fit the obtained impedance spectra represented as Nyquist plots in the complex planes. The goodness-of-fit (χ2) was calculated to be in the range of 0.001-0.32 which indicates good fit between the theoretical and the experimental data (Fig. S1). 3. Results and discussion

High quality and uniform graphene sheets were grown by a CVD process using Cu foil as catalytic substrate (Fig.1A) as shown in the scanning electron microscopy (SEM) image (Fig. 1B). The graphene sheets were then transferred onto a clean glass substrate (Fig. 1C). We used here glass as an insulating substrate to avoid the possible influence of the underlying metal substrate on the electrochemical behaviour of graphene. The characteristics of the graphene films were analysed before and after the transfer process by optical microscopy, Raman spectroscopy and X-ray photoelectron spectroscopy (Fig. S2). No significant changes were observed, indicating that the chemical and structural composition of the graphene films was maintained through the transfer process. graphene

films were then connected as a working electrode using copper tape and silver paint and subsequently insulated to expose a well defined area of graphene as described in detail in the experimental section (Fig. 1D). This simple design ensures that graphene is the only conductive surface exposed to the solution during electrochemical measurements.

Fig. 1 (A) Optical microscopic image of monolayer graphene film on Cu foil. (B) SEM image of graphene (C) Monolayer graphene film transferred onto a glass sheet, (D) Schematic of the procedure for connecting monolayer graphene sheets into working electrodes for electrochemical measurements.

The functionalization of the CVD graphene surface was then performed by electrochemical reduction of in situ generated carboxyphenyldiazonium salt in acidic aqueous solution (Scheme 1). The electrochemical reduction was done using three consecutive cyclic voltammetry scans from 0.4 to -0.6 V at scan rate of 100 mV/s. Fig. 2 shows a single irreversible cathodic peak in the first cyclic voltammetric cycle at -0.34 V vs. Ag/AgCl. This observed cathodic peak is attributed to the reduction of the diazoniumcations via a single electron transfer process. The diazonium salt reduction led to the elimination of a nitrogen molecule and the formation of an aryl radical which forms a covalent bond with the graphene surface. The cathodic peak current then gradually decreased in the subsequent CV scans indicating the gradual decrease of electron transfer between the diazonium cations in the solution and the carboxyphenyl modified graphene electrode. We interpret these observations to the increase in the number of aryl groups attached to the graphene


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surface with increasing number of CV cycles, resulting in retardation of electron transfer. In the third CV cycle, the cathodic peak has almost disappeared, indicating coverage of the CVD graphene surface with the carboxyphenyl groups. This behaviour is in agreement with that previously reported for the reduction of diazonium cations on graphene-modified screen printed carbon electrodes [36].

Fig. 2 Successive cyclic voltammograms for the in situ generated 4-carboxyphenyl diazonium salt in the diazotation mixture (2 mM NaNO2 + 2 mM 4-aminobenzoic acid in 0.5 M HCl) at scan rate of 100 mVs−1 on graphene.

We examined the graphene electrodes before and after the carboxyphenyl functionalization using Raman spectroscopy, X-ray photoelectron spectroscopy and atomic force microscopy. The Raman spectra of graphene electrodes before and after functionalization are shown in Fig. 3A. The bare graphene (black curve), exhibits two prominent Raman Stokes peaks, a single symmetric 2D peak at ∼2700 cm-1 and a G peak at ∼1580 cm−1. The intensity of the 2D peak is almost twice the intensity of the G peak over most of the sample area, characteristic of monolayer graphene and indicative that the graphene sheets transferred onto the glass substrate were continuous and uniform [40,41]. A small D peak at ∼1350 cm-1 was also observed in the Raman spectra of the bare graphene, corresponding to a low defect density. After subsequent modification of graphene using one (Fig. 3, red curve) to three CV (Fig. 3, blue curve) scans in the diazonium solution, the intensity of the D peak was gradually enhanced

and D′ and D + D* peaks appeared at ∼1620 cm−1 and ∼2950 cm−1, respectively. These changes are attributable to the breaking of translational symmetry of the honeycomb lattice of C–C sp2 bonds on the basal plane of the graphene due to the formation of localized C–C sp3 bonds by carboxyphenyl grafting. Moreover, the defect density, σ, on the graphene lattice after covalent functionalization was quantified using the ID/IG ratio based on the empirical equation [42]:

where LD is the mean defect spacing, rS is the radius of the structurally damage region around each sp3 bond / defect site, and rA is the radius of the activated region around it. We used CA (=4.56), CS (=0.86), rA (=1.0 nm), and rS (=0.07 nm) as previously reported for aryl groups covalently attached to the graphene lattice [32].

Fig. 3 (A) Raman spectra of the graphene electrode before (Black curve) and after subjected to the electrgrafting process using one (red curve) and three (blue curve) CV cycles and (b) XPS C1s core level spectra of graphene electrode before (bottom) and after (top) the carboxyphenyl functionalization. Table 1. Values of ID/IG from the Raman spectra and the calculated values of the defect density, σ, for the graphene before and after the diazonium.

Electrode ID/IG σ (cm-2) σ (mol cm-2)

Bare graphene 0.10 7.1×1011 1.2×10-12

CP-GE, 1CV 0.55 4.1×1012 6.8×10-12

CP-GE, 3CV 1.42 1.1×1013 1.9×10-11

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As shown in Table 1, The initial defect density, σ, is estimated for pristine CVD graphene to be very low (7.1×1011 cm-2), whereas upon carboxyphenyl functionalization with one and three CV cycles, the defect density is increased to 4.1×1012 cm-2 and 1.1×1013 cm-2, respectively. These results confirm the gradual increase of the carboxyphenyl groups covalently attached to the graphene surface with the gradual increase of the number of CV cycles used in the electrografting procedure. After the third cycle, no further change in the Raman spectra was observed which indicates that the coverage of the graphene surface with monolayer of aryl groups, a figure in good agreement with the observed saturation in CV Faradaic current data explained above. It is worth noting that the estimated defect density value obtained from our method at three CV cycles is consistent with that reported for the grafting of carboxyphenyl tetrafluporoborate on graphene supported on SiO2 using spontaneous electron transfer chemistry [32] as well as on Ni using an electrografting method in acetonitrile [39]. For graphene/Ni electrografting, five CV scans were shown to be necessary to realize the maximum modification of the graphene surface. However, here we show that three CV scans were enough to cover the graphene surface with monolayer of aryl groups which indicates that the electrografting reaction in our case is faster. The slow reactivity of the graphene/ Ni towards the electrografting was explained to be due to the stabilizing lattice match between the monolayer graphene and the underlaying Ni which makes the attachment of the aryl radicals on the basal plane more difficult [39]. The modification of the graphene electrodes was also confirmed using X-ray photoelectron spectroscopy. Fig. 3B shows the XPS C1s core level spectra of the graphene monolayers before and after the carboxyphenyl functionalization. For the bare graphene, a sharp peak at a binding energy of 284.2 eV was observed as expected for the sp2 hybridized carbon atoms of graphene. However, after the carboxyphenyl modification, an additional peak centered at 288.8 eV appeared, typical of the carboxylic acid groups grafted on the graphene surface. Examination of the modified CVD graphene electrodes by atomic force microscopy has also

proved the formation of a carboxyphenyl layer. As shown in Fig. S3, the AFM images obtained in ScanAsyst mode reveals significant difference between bare and modified graphene using three CV scans with an estimated average thickness value of 6.9 nm of the grafted layer. This value is consistent with the theoretical length of 0.69 nm for the carboxyphenyl moiety estimated by the ChemDraw Ultra 13.0 software (Fig. S4) which indicates that the grafted thin film composed of monolayers. XPS, Raman spectroscopy and AFM thus confirms the covalent modification of graphene. However, it is well known that one of the common characteristics of functionalization by reduction of diazonium salts is the possibility of the continuous growth of multilayers of aryl groups on the surface [43,44]. Careful optimization of the electrografting conditions is required to avoid multilayer formation, and thus, electrode passivation. Hence, we employed EIS to provide more insight into the blocking effect of the electrografted carboxyphenyl groups formed by different CV cycles. EIS is a non-destructive technique widely used to probe the interfacial properties of electrode surfaces upon modification [45]. The impedimetric behaviour of the graphene electrodes was studied in the presence of the [Fe(CN)6]3−/4− redox couple before and after the carboxyphenyl modification. Fig. 4A shows the Nyquist plots of a bare graphene electrode and various 4-carboxyphenyl-modified graphene electrodes prepared using different CV cycles. A semi-circle in the high frequency domain characterizes the Nyquist plot of the bare graphene electrode, which corresponds to an interfacial charge transfer mechanism. However, a Warburg line was not observed in the low frequency limit, indicating that charge transfer dominates over mass transport effects [46]. The impedance spectra were fitted with an equivalent circuit shown in the inset of Fig. 4A. This circuit consists of the solution resistance, Rs, which represent the bulk properties of the electrolyte solution, the double-layer capacitance, C, and charge-transfer resistance, RCT. The semicircle corresponds to the parallel combination of the charge-transfer resistance, RCT, with the double layer capacitance, C. Among the electrical parameters, we focused on the change of the RCT which corresponds to the diameter of the semicircle as it was more

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Fig. 4 (A) Nyquist plot of bare GE and 4-CP-GE functionalized by diazonium salt reduction using different CV cycles in 10 mM [Fe(CN)6]4−/3− at 100 mV/s. Inset: The equivalent circuit used for fitting the experimental results; (B) Plot of Rct of 4-CP-GE in 10 mM [Fe(CN)6]4−/3− vs. the number of CV scanning cycles for diazonium reduction on GE.

significantly influenced by the modification of the graphene surface. As shown in Fig. 4A, the diameter of the semicircle, corresponding to RCT, is significantly larger in the case of the carboxyphenyl modified graphene electrodes as compared to bare graphene. This increase in the impedance can be attributed to two factors: the thickness of the grafted organic layer and the negative charge of the carboxylic groups that repel the [Fe(CN)6]3−/4− redox anions. A gradual increase in RCT was observed with increasing the number of CV scans used in the electrografting process even after the third scan as can be seen in Fig. 4B.These results may indicate the continuous growth of the organic film forming multilayers, leading in turn to more hindrance for the electron transfer process of the [Fe(CN)6]4−/3−. In this work, three electrografting CV scans were chosen for impedimetric sensing on the basis of the saturation in Raman spectra following three CV scans. We thus ensure maximum monolayer coverage with carboxylphenyl groups while minimizing the formation of multilayers that may lead to electrode passivation that negatively affects biosensor performance [47,48]. EIS was also used to characterize the further fabrication steps for the immunosensor. Fig. 5 shows the Nyquist plot of the Faradaic impedance spectra recorded in [Fe(CN)6]4−/3− solution upon the stepwise modification process. The carboxyphenyl modified

graphene electrode (blue curve) exhibited an increase in the semicircle compared with the bare graphene electrode (black curve), corresponding to an increase in the electron-transfer resistance RCT of the redox probe as explained above. Subsequently, the covalent immobilization of the ovalbumin antibodies caused a significant drop in the RCT (green curve). This decrease could be attributed to the

Fig. 5 Nyquist plot of bare GE; 4-CP/ GE, Ab/4-CP/ GE, and after blocking with 0.1% BSA for 30 min. in the presence of 10 mM [Fe(CN)6]4−/3− probe in PBS, pH 7.4.

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Fig. 6 (A) Nyquist plot of OVA immunosensor before (black) and after (red) incubation with 0.1 ng/ml OVA. (B) Dependence of the immunosensor response on OVA concentration (plot of (R-Ro)/Ro % (the percent change in the charge transfer resistance) vs. COVA). Inset: calibration curve for OVA, plot of (R-Ro)/Ro % vs. log COA.

Fig. 7 (A) Nyquist plot of OVA immunosensor incubated with 1µg/ml Lysozyme (green), and β-lactoglobulin (red), (B) Comparison of response of the immunosensor to 100 ng ml-1 ovalbumin, 1µg/ml Lysozyme, and β-lactoglobulin. neutralization of the negative charge of the carboxylic groups with the positively charged antibodies [36,37] facilitating access of the [Fe(CN)6]4−/3− redox couple to the graphene surface. Finally, RCT was subsequently increased after treatment with BSA solution (red curve) due to the blocking effect of this bulky protein. Fig. 6A shows the increase in RCT upon ovalbumin binding that is the basis of ovalbumin detection. This further increase in RCT is likewise due to the bulky size of the ovalbumin protein as well as its negative charge at pH 7.4, leading to an enhancement in blocking of the immunosensor surface that retards the access of the [Fe(CN)6]4−/3− redox couple to the surface and slows

electron transfer kinetics. A calibration plot based on the percentage change in the RCT following ovalbumin binding is shown in Fig. 6B. A good linear relationship was obtained between the percentage change in the RCT of the immunosensor versus the logarithm of the ovalbumin concentrations in the range from 1 pg mL-1 to 100 ng mL−1. The standard deviations of the measurements were ranging from 3.0 % to 7.0 % indicating good reproducibility of the immunosensor. The linear regression equation was (R-Ro)/Ro % = 39.0 + 4.29×log C [ng mL-1], R = 0.98, with a calculated detection limit (LOD) of 0.9 pg mL−1 (S/N = 3). The result confirms that the CVD monolayer graphene


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platform offers good sensitivity and exhibits comparable detection limit to that reported with graphene modified screen printed carbon electrodes [36]. The selectivity of the proposed immunosensor was investigated in order to confirm that the observed impedimetric response is due to specific ovalbumin binding. The immunosensor was incubated with β-lactoglobulin and lysozyme as two non-specific proteins. As shown in Fig. 7A, there was no significant change in the impedimetric response upon incubation of the immunosensor with Lysozyme and β-LG. Fig. 7B shows the response of the impedimetric immunosensor to β-lactoglobulin and lysozyme binding which was much lower than that of the ovalbumin, illustrating good selectivity and negligible non-specific adsorption on the graphene platform. 4. Conclusions A controlled, easy and fast electrochemical modification method for CVD monolayer graphene with carboxyphenyl diazonium salt has been demonstrated. We have also investigated for the first time the behaviour of the functionalized monolayer graphene for impedimetric immunosensing of protein. Our results show that carboxyphenyl functionalized CVD monolayer graphene can be used as a sensitive and selective platform for protein biosensing. Our work can be extended to further biosensing applications of CVD graphene.

Acknowledgements M. Siaj is grateful to NSERC individual Discovery Grants. We acknowledge funding from FRQNT through a team grant. This research has been enabled by the use of NanoQAM and CM2 facilities. Electronic Supplementary Material: Supplementary material (The equivalent circuit model used to fit the impedance data, Optical microscopy images, Raman spectra and XPS of CVD monolayer graphene film before and after transfer, AFM imaging of bare and functionalized graphene and estimation of the length of the carboxyphenyl moiety using ChemDraw) is

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Nano Res.

Electronic Supplementary Material

Functionalized CVD Monolayer Graphene for Label-Free Impedimetric Biosensing

Shimaa Eissa1,2, Gaston Contreras Jimenez2, Farzaneh Mahvash2,3, Abdeladim Guermoune2, Chaker Tlili1, Thomas Szkopek3, Mohammed Zourob4, and Mohamed Siaj2 () 1

Supporting information to DOI 10.1007/s12274-****-****-* (automatically inserted by the publisher)


Nano Res.

Fig. S1 (A) Nyquist plot of bare graphene electrode in 10 mM [Fe(CN)6]4−/3− solution at 0.2 V over a frequency range between 0.1 Hz and 100 Hz. The symbols represent the experimental data and the red curve represent the fitted data using the equivalent circuit (B) The equivalent circuit model used to fit the experimental data. C, is the double layer capacitance ; Rct , is the charge transfer resistance; Rs, is the solution resistance.


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Fig. S2 Optical microscopy images of CVD monolayer graphene film before (A) and after (B) the transfer process; (C) Raman spectra of CVD monolayer graphene film before (bottom) and after (top) the transfer process; (D) XPS C1s core level spectra for CVD monolayer graphene film before (bottom) and after (top) the transfer process.


Nano Res.

Fig. S3 (A) AFM images of bare graphene and (B) carboxyphenyl modified graphene. (C) The height profiles with two selected line scans. Green line indicates the bare graphene, and blue line indicates the modified graphene. The average height of the carboxyphenyl groups is around 6.9 nm.

Fig. S4 Theoretical estimation of the length of the carboxyphenyl moiety using ChemDraw Ultra 13.0 software.

Address correspondence to [email protected]

functionalized cvd monolayer graphene for label-free

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