DNA and graphene as a new efficient platform for ...

Electrochimica Acta 111 (2013) 543–551

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Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta

DNA and graphene as a new efficient platform for entrapment of methylene blue (MB): Studies of the electrocatalytic oxidation of ␤-nicotinamide adenine dinucleotide Grasyelle Maria Mota Ferreira a , Fernando Mota de Oliveira b , Fernando Roberto Figueiredo Leite c , Camila Marchetti Maroneze c , Lauro Tatsuo Kubota c,d , Flavio Santos Damos a,d , Rita de Cássia Silva Luz a,d,∗ Instituto de Ciência e Tecnologia, UFVJM, Rodovia MGT 367, Km 583, n◦ 5000, Alto do Jacuba, 391000-000 Diamantina, MG, Brazil Departamento de Química, UFVJM, Rodovia MGT 367, Km 583, n◦ 5000, Alto do Jacuba, 391000-000 Diamantina, MG, Brazil c Instituto de Química, UNICAMP, P.O. Box 6154, 13084-971 Campinas, SP, Brazil d Instituto Nacional de Ciência e Tecnologia em Bioanalítica, Unicamp, 13083-970 Campinas, SP, Brazil a

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a r t i c l e

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Article history: Received 11 May 2013 Received in revised form 6 August 2013 Accepted 6 August 2013 Available online 21 August 2013 Keywords: DNA Graphene Methylene blue NADH

a b s t r a c t The modification of glassy carbon (GC) electrode with deoxyribonucleic acid (DNA) and graphene is utilized as a new efficient platform for entrapment of methylene blue (MB). Electrochemical and electroanalytical properties of the modified electrode (DNA/graphene/MB) were investigated by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and amperometry techniques. Cyclic voltammetric results indicated the excellent electrocatalytic activity of the resulting electrode toward oxidation of ␤-nicotinamide adenine dinucleotide (NADH) at reduced overpotential (0.1 V vs. Ag/AgCl). It has been found that the DNA/graphene/MB modification has significantly enhanced the effective electrode response toward NADH oxidation. Cyclic voltammetry and rotating disk electrode (RDE) experiments indicated that the NADH oxidation reaction involves two electrons and an electrocatalytic rate constant (kobs ) of 1.75 × 106 mol−1 L s−1 . The electrochemical sensor presented better performance in 0.1 mol L−1 phosphate buffer at pH 7.0. Other experimental parameters, such as the DNA, graphene, MB concentrations and the applied potential were optimized. Under optimized conditions, a linear response range from 10 ␮mol L−1 to 1.50 mmol L−1 was obtained with a sensitivity of 12.75 ␮A L ␮mol−1 . The detection and quantification limits for NADH determination were 1.0 ␮mol L−1 and 3.3 ␮mol L−1 , respectively. © 2013 Elsevier Ltd. All rights reserved.

1. Introduction ␤-Nicotinamide adenine dinucleotide (NADH) is an important cofactor non-protein that is involved more than 300 enzymatic reactions [1] and also in the fine chemicals industry based on NADdependent biocatalysts. The electrochemical oxidation of NADH has thus been the subject of numerous studies related to the development of sensors and enzymes-based biosensors [1–12]. One big challenge to be overcome concerning to these studies is that the direct electron-transfer between NADH and conventional electrode surfaces requires a relatively high overvoltage [13,14]. One way to solve this problem is the use of chemically modified electrodes to

∗ Corresponding author at: Instituto de Ciência e Tecnologia, UFVJM, Rodovia MGT 367, Km 583, n◦ 5000, Alto do Jacuba, 391000-000 Diamantina, MG, Brazil. Tel.: +55 38 3532 1283; fax: +55 38 3532 1223. E-mail addresses: [email protected], rita [email protected] (R.d.C.S. Luz). 0013-4686/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.electacta.2013.08.037

decrease the activation energy of this reaction, enhancing the speed of electron-transfer. The use of carbon-based nanomaterials for such purposes has shown to be a promising alternative, clearly because these materials exhibit many unusual and remarkable physicochemical properties [3–6,12]. Among the carbon-based nanomaterials such as carbon nanotubes, fullerenes, graphene and nanodiamonds, graphene have received recently widespread attention owing to its excellent electrical conductivity, mainly originated from the delocalized ␲ bonds above and below the basal plane, its large surface area and low production costs [15,16]. Due to these excellent physical and chemical properties, graphene has become an interesting alternative for the development of electronic devices [17] and electrochemical sensors [15,16]. Graphene-based electrochemical sensors present a better performance compared to glassy carbon, graphite and even carbon nanotubes-based sensors, mainly due to sp2 -like planes and edge defects that are more exposed on the graphene nanosheets than on other carbon materials [15,18,19].

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G.M.M. Ferreira et al. / Electrochimica Acta 111 (2013) 543–551

However, a number of known forms of graphene materials are not well dispersible or soluble in most common solvents. This limitation deters to explore the chemistry of graphene at the molecular level and its sensor and biosensors applications. Graphene sheets, which have a high specific surface area, unless well separated from each other, tend to form irreversible agglomerates or even restack to form graphite through van der Waals interactions [20–23]. One known solution to this problem is the use of dispersing agents such as polymers, biopolymers, or surfactants in conjunction with the appropriate experimental conditions. Among the various biomolecules, deoxyribonucleic acid (DNA) has emerged as an appealing biomacromolecule for functional materials due to its biocompatibility and renewability in addition to its very interesting double helix structure, which guarantees a range of unique properties that are difficult to detect in other molecules and polymers [24]. Deoxyribonucleic acid (DNA) as an important biological macromolecule has been paid much attention in the recent years. The base pairs stacked within double helical DNA can provide an effective platform for fabrication of electrochemical sensors [25]. A number of phenoxazines and phenothiazines can be intercalated into DNA duplexes, while maintaining its catalytic activity toward NADH oxidation [26,27]. DNA conjugates have been developed to provide unique functions for various studies such as designing DNA hybridization sensors and evaluating photoinduced DNA damage [28]. In addition, hybrid materials formed by the combination of other species with DNA have been demonstrated as an efficient way to produce nanostructured materials that can respond to a specific stimulus with a particular signal [28]. It is known in the literature that carbon materials can self-organize with DNA molecules [29], and the resulting DNA/carbon composites can be used as new electronic devices, based on theoretical prediction [30] and experimental confirmation [31]. Since DNA solution can gelatinize, the mixed DNA/graphene layer can be kept stable on the electrode surface, and then be used as a support for the immobilization of innumerous electrocatalysts on many kinds of electrode materials. Methylene blue (MB) is a well-known redox indicator in analytical chemistry, because of its electron mediating characteristic as well as its electrocatalytic properties toward NADH oxidation at low potentials. However, such low molecular weight soluble mediator is disadvantageous as it can leach out of the electrode, which may lead to a significant signal loss and affect the stability of sensors. In order to overcome these shortcomings, we have exploited the organic–inorganic nanocomposite material composed of DNA and graphene in immobilizing of MB. In this sense, this work presents a new platform for NADH detection at low potential employing a glassy carbon (GC) electrode modified with DNA/graphene/MB. The efficient immobilization of MB on DNA/graphene was of fundamental importance to obtain a stable and sensitive system. The excellent characteristic of the proposed sensor may be associated to the high conductivity and the large surface area to volume ratio of the graphene as well as the ability of the DNA in immobilizing MB.

2. Experimental

were prepared daily with appropriate dilution of the stock solutions with deionized water. All solutions were prepared with water purified in a Milli-Q Millipore system and the actual pH of the buffer solutions was determined with a Corning pH/Ion Analyser 350 model. Phosphate buffer solutions (0.1 mol L−1 ) were prepared from 0.1 mol L−1 H3 PO4 –NaH2 PO4 , and the pH was adjusted with 0.1 mol L−1 H3 PO4 or 2.0 mol L−1 NaOH. 2.2. Graphite oxide (GO) and DNA/graphene GO was prepared using a modification of Hummers and Offeman’s method from graphite powders (Bay carbon, SP-1) [32–34]. In a typical reaction, 0.5 g of graphite, 0.5 g of NaNO3 , and 23 mL of H2 SO4 were stirred together in an ice bath. Next, 3 g of KMnO4 was slowly added. All chemicals were purchased from Sigma–Aldrich and were used as received. Once mixed, the solution is transferred to a 35 ± 5 ◦ C water bath and stirred for about 1 h, forming a thick paste. Next, 40 mL of water was added, and the solution was stirred for 30 min while the temperature was raised to 90 ± 5 ◦ C. Finally, 100 mL of water was added, followed by the slow addition of 3 mL of H2 O2 (30%), turning the color of the solution from dark brown to yellow. The warm solution was then filtered and washed with 100 mL of water. The filter cake was then dispersed in water by mechanical agitation. Low-speed centrifugation was done at 1000 rpm for 2 min. It was repeated until all visible particles were removed (about 3–5 times) from the precipitates. The supernatant then underwent two more high-speed centrifugation steps at 8000 rpm for 15 min to remove small GO pieces and watersoluble byproduct. The final sediment was washed with distilled water, and air-dried overnight to obtain GO. single-stranded DNA/graphene nanocomposite The (DNA/graphene) was synthesized according to previous reported method with a slight modification [33–36]. Briefly, an aqueous solution of ds-DNA was first heated at 95 ◦ C for 2 h to obtain an aqueous solution of ss-DNA. GO was dispersed in water, and the mixture was sonicated for 3 h to get a homogeneous yellow brown dispersion. The GO dispersion was then mixed with the aqueous solution of ss-DNA, and the resultant mixture was stirred for 1 h. Subsequently, hydrazine (85 wt%) was added to the mixture. The mixture was then heated to reflux at 100 ◦ C for 5 h to prepare the DNA/Graphene nanocomposite. After cooling to room temperature, the resulting materials were then centrifuged and washed three times with distilled water to remove excess hydrazine and ss-DNA. The as-prepared DNA/graphene nanocomposite was obtained. 2.3. Sensor construction Prior to the electrode modification, the glassy carbon electrode surface of area 0.196 cm2 was polished and then cleaned by sonication to remove any adhesive. After cleaning the electrode, a suspension was prepared by mixing MB and DNA/graphene composite dispersion with the aid of sonication. Then, 20 ␮L of this suspension was placed directly onto the glassy carbon electrode surface and allowed to dry at 80 ◦ C for 10 min to form a DNA/graphene/MB composite at the GC electrode surface. This same volume was utilized in all studies. After 10 min the modified electrode was thoroughly rinsed with distilled water.

2.1. Reagents and solutions 2.4. Electrochemical and microscopic measurements All chemicals used were of analytical grade. The graphite powder, ␤-NADH, double-stranded DNA (ds-DNA) (Type I highly polymerized, from Calf Thymus) and methylene blue (MB) were acquired from Sigma, St. Louis, USA. Disodium and monosodium phosphates (Na2 HPO4 and NaH2 PO4 ) and Na2 H2 EDTA·2H2 O were acquired from Synth, São Paulo, Brazil. Working standard solutions

Electrochemical measurements were performed with an Autolab PGSTAT 128N potentiostat/galvanostat from Eco chemie (Utrecht, Netherlands) coupled to a PC microcomputer with GPES 4.9 software. A three electrode electrochemical cell was employed for all electrochemical measurements. The working electrode was a


glassy carbon mounted in Teflon® . The counter and reference electrodes were a platinum electrode and Ag/AgCl (sat.), respectively. Oxygen was removed by bubbling nitrogen through the solution. Electrochemical impedance spectroscopy has been widely used to characterize the interface properties of surface-modified electrodes. The typical impedance spectrum (presented in the form of the Nyquist plot) includes a semicircle portion at higher frequencies corresponding to the electron-transfer-limited process and a linear part at lower frequency range representing the diffusion-limited process. The semicircle diameter in the impedance spectrum is equal to the electron-transfer resistance, Ret , which is related to the electron-transfer kinetics of the redox probe at the electrode surface. The measurements were performed at the formal potential of [Fe(CN)6 ]4−/3− redox couple in 1.0 mmol L−1 Fe(CN)6 4− + Fe(CN)6 3− (1:1) + 1 mol L−1 KCl solution. A simple program based on the simplex optimization and nonlinear least-square-fit analysis was used for fitting the obtained EIS data. The frequency range was from 0.530 Hz to 100 kHz and the sinusoidal potential amplitude was 5 mV. The Ret value was obtained through the non-linear regression analysis of the semicircle portion on the Nyquist plot (Zim vs. Zre ). All SECM measurements were performed with a CHI 920C microscope (CH Instruments) in a four-electrode configuration at room temperature that uses a combination of stepper motors positioners of resolution of 8 nanometers with 50 mm travel distance and an XYZ piezo block in order to position the tip. The electrochemical cell employed was built in Teflon with a 10 mm diameter aperture. SECM tips were ultramicroelectrodes (UMEs) of Pt with radius, a, of 5 ␮m and RG ∼5 (RG is the ratio of the insulating glass radius, rg, to that of a, so RG = rg/a). Approach curves were recorded by moving the tip toward the modified electrode surface at the speed of 2 ␮m s−1 while the tip was held at a constant potential for a diffusion-limited current of solution-phase electrochemical probe at the tip.

3. Results and discussion 3.1. Electrochemical characterization of the DNA/graphene/MB modified electrode

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Fig. 2a shows the cyclic voltammograms of this modified electrode in PBS (pH 7.0) at various potential scan rates. As can be seen, the cyclic voltammogram exhibits an anodic peak at forward scan related to the oxidation of MB, whereas the reverse scan presents a cathodic peak related to reduction of MB+ . The peak currents of the MB/MB+ couple were directly proportional to the scan rate in the range below 0.2 V s−1 as shown in Fig. 2b. In addition, the formal potential is almost independent of the potential scan rate for sweep rates below 0.2 V s−1 , suggesting a high charge transfer kinetics. At scan rates higher than 0.2 V s−1 , the peak currents become proportional to the square root of the scan rate (Fig. 2c) indicating a diffusion-controlled process, which can be related to the limitation of the diffusion of the counter ions into the composite electrode to keep the electroneutrality. The formal potential, E0 , is about −0.266 V and is independent of the potential scan rate for sweep rates ranging between 0.125 and 0.5 V s−1 . The formal potential (E0 ) was obtained from the equation [38]: 0 Em =

1 (Epa + Epc ) 2

(1)

The surface concentration of electroactive species was calculated by the area under the anodic peaks of cyclic voltammograms at 100 mV/s by the following equation [39]: =

Q nFA

(2)

where Q is the charge, n is the number of electrons (n = 2), F is Faraday’s constant, and A is the area of the gold electrode. The surface concentration of electroactive species was found to be 2.2 × 10−9 mol cm−2 . The solution pH dependence of the DNA/graphene/MB/GC electrode was also investigated. Since the DNA/graphene/MB/GC electrode has a methylene blue moiety, we anticipate that the redox response of MB film would be pH dependent. The formal potential of the surface redox couple, taken as the average of positive and negative peak potentials from Eq. (1), was pH dependent (6.0–8.0) with a equation E = 0.195–0.034 pH with a slope of 0.034 V per unit of pH indicating that the electrode reaction involve two electrons and one proton [40,41]. 3.2. Electrochemical impedance studies

After modifying the electrode with DNA/graphene/MB (Fig. 1a); DNA/MB (Fig. 1b) and graphene/MB (Fig. 1c), cycles in the potential range from −0.6 up to 0.0 V in 0.1 mol L−1 PBS (phosphate buffer solution) at a scan rate of 0.05 V s−1 were performed. In Fig. 1a the DNA/graphene/MB/GC electrode provided a stable electrochemical response for the MB/MB+ redox couple with DNA/graphene. When the electrode was modified only with DNA and MB (Fig. 1b) there was an also stable response for modified electrode with a current twice smaller than the current obtained for the modified electrode with DNA/graphene/MB (Fig. 1a). The better electrochemical response of the DNA/graphene/MB modified electrode may be related to the excellent electric properties, the large surface area to volume ratio, and the extreme sensitivity of the surface atoms of the graphene to many surface reactions [37] as well as the ability of the DNA immobilizing MB. However, when the electrode was modified only with graphene and MB (Fig. 1c) the current for electrode was unstable and three times lower than response obtained for the electrode modified with DNA, graphene and MB (Fig. 1a). This result suggests that the MB is better immobilized when the DNA is used as platform for entrapment of MB on graphene. Fig. 1d shows the amperometric curves for the NADH oxidation at different concentrations. According to this figure a better sensitivity for NADH detection was obtained with the DNA/graphene/MB/GC electrode. This behavior can be assigned to the good immobilization of the methylene blue and graphene on the DNA.

Electrochemical impedance, a widely used and effective way to study the interface properties of modified electrodes, was also used to identify the immobilization of DNA/graphene/MB/GC electrode. Fig. 3 shows the results of electrochemical impedance studies on the GCE electrode during the modification process. At a bare electrode the impedance spectra was composed of a semicircle with a straight tail line (Fig. 3a) characteristic of a diffusion limiting step of the Fe(CN)4−/3− process, and based on Randle’s equivalent circuit [42], solution resistance (Rsol ), charge transfer resistance (Rct ), double layer capacitance (Cdl ) and Warburg impedance (W), resulting from the diffusion of ferro/ferricyanide from the bulk of the electrolyte to the electrode. The values of Rct , Cdl and W obtained were 40.5 , 400 , 0.025 ␮F and 1.48 × 10−3 respectively. However, a quite small semicircle spectrum (Fig. 3b) was obtained after graphene electrode modification. The Rct decreases from 400 to 200 , which shows that modification of the electrode with graphene is obviously beneficial for the electron transfer of the redox probe to the electrode surface. This was probably related to the good electrical conductivity and large surface area of graphene. On the other hand, the semicircle diameter increased significantly to 2500 k after the adsorption of DNA layer (Fig. 3c). The DNA layer has introduced a barrier for electron transfer between the redox probe in the electrolyte solution and the electrode. The negatively charged phosphate groups on DNA can also decrease the access

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Fig. 1. Cyclic voltamograms of the: (a) DNA/graphene/MB/GC electrode; (b) DNA/MB/GC electrode; (c) graphene/MB/GC electrode; (d) amperograms of (a); (b) and (c) in the presence of NADH. Experimental conditions: 0.1 mol L−1 phosphate buffer solution at pH 7. Eapp = 0 V vs Ag/AgCl. [DNA] = 0.7 mg mL−1 . [graphene] = 1.0 mg mL−1 and [MB] = 1.0 mmol L−1 .

of the Fe(CN)6 3− /Fe(CN)6 4− redox couple to the electrode surface [43]. On the other hand, for DNA/graphene/MB modified electrode, a linear Nyquist plot (Fig. 3d), which indicates a diffusion controlled process over all range of frequencies, can be observed. This implies that the film modified electrode has high conductivity and the redox behavior of Fe(CN)6 3− /Fe(CN)6 4− couple

90

is controlled by diffusion process. This result suggests that the charge transfer process on modified electrode for [Fe(CN)6 ]3−/4− is easier than in a bare electrode or only modified with DNA or graphene. In order to explain this behavior, it was considered that the DNA/graphene/MB coating is divided in two regions [44]. One represents the region where counter ions are held by electrostatic 90

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Fig. 2. (a) Cyclic voltammograms of the DNA/graphene/MB/GC electrode in 0.1 mol L−1 PBS (pH 7.0) at potential scan rates of 0.01–0.20 V s−1 ; (b) plot of Ip − v obtained from (a) and (c) plot of Ip − v1/2 at potential scan rates of 0.2–0.9 V s−1 . Experiment performed in 0.1 mol L−1 phosphate buffer (pH 7.0). [DNA] = 0.7 mg mL−1 ; [graphene] = 1.0 mg mL−1 and [MB] = 1.0 mmol L−1 .


4000

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0 0

Bare electrode Graphene DNA Graphene/MB/DNA

1000 2000 3000 4000 5000 6000 7000 8000

Zreal / kΩ Fig. 3. Electrochemical impedance spectroscopy referring to: (a) bare electrode. (b) graphene modified electrode. (c) DNA modified electrode and (d) DNA/graphene/MB/GC electrode. [DNA] = 0.7 mg mL−1 ; [graphene] = 1.0 mg mL−1 and [MB] = 1.0 mmol L−1 .

forces (Donnan domain). The second phase comprises the volume of the coating filled by the supporting electrolyte. Here, the counter ions represent the electroactive species as well anionic species from the supporting electrolyte. In this model the polyelectrolyte chains with their accompanying counter ions are separated by a second region. In the Donnan domain the charge can propagate by physical displacement of the ions or electron exchange without their physical displacement [44]. Thus, these charge transfer processes and their coupling could be one point to explain the better charge transfer for the modified electrode. 3.3. SECM images of the DNA/graphene and DNA/graphene/MB/GC electrode The Scanning Electrochemical Microscopy (SECM) is a scanning probe technique in which the diffusive interactions of electroactive species between a microelectrode (the SECM probe or tip) and a substrate electrode are measured in the form of an electrochemical current. Both the topography and conductive nature of the substrate can be probed [45], so the SECM can be used to examine differences in electrochemical activity at surfaces at high resolution [46]. SECM experiments were carried out using a 5 ␮m-radius Pt tip held at ET = −0.1 V vs. Ag/AgCl, a potential selected by tip-cyclic voltammogram with the Pt tip, at d > 200 ␮m. The SECM images of the DNA/graphene and DNA/graphene/MB are shown in Fig. 4a and b, which showed the results of a scan over a 100 ␮m × 100 ␮m region of the film as a Pt tip was scanned in close proximity to the modified electrode surface with the tip potential held at −0.1 V vs. Ag/AgCl, and the substrate potential held at +0.5 V, vs. Ag/AgCl, respectively. The flat response in Fig. 4a indicates that the surface of DNA/graphene is basically uniformly active with no topographic features. In contrast, the bright regions arisen in Fig. 4b represent higher tip currents at the electroactive sites. As a result of this heterogeneous composition, there is a great deal of topographical variation in the conductivity of the DNA/graphene/MB film. When the tip approaches the conductive substrate, the oxidation of [Fe(CN)6 ]4− (reduction product at the tip) occurs, namely, the regeneration of [Fe(CN)6 ]3− takes place, leading to the positive feedback currents at the tip. The rate of [Fe(CN)6 ]3− regeneration at the electroactive sites is faster than that at the DNA/graphene due to the facilitated electron communication properties of the immobilized mediator, resulting in the higher tip currents. The comparison of Fig. 4a and b implies

Fig. 4. (a) SECM images (x–y scans) of the surface of graphene/DNA/GC and (b) DNA/graphene/MB/GC using a 10 ␮m diameter Pt microelectrode in 0.1 mol L−1 phosphate buffer solution containing 1 mmol L−1 [Fe(CN)6 ]3− as redox mediator. Inset in panel (a) and (b) shows the 3D graphics. [DNA] = 0.7 mg mL−1 ; [graphene] = 1.0 mg mL−1 and [MB] = 1.0 mmol L−1 .

that there are more active sites at DNA/graphene/MB surface than at DNA/graphene surface owing to the uniform incorporation of mediator molecules. 3.4. Electrocatalytic oxidation of NADH at the DNA/graphene/MB/GC electrode Due to the stability of MB/MB+ redox couple with DNA/graphene/MB/GC electrode, it can be used as a mediator to shuttle electrons between electrode and NADH. In order to test the electrocatalytic activity of DNA/graphene/MB/GC electrode, cyclic voltammograms were obtained in the absence (Fig. 5, curve 1) and presence of 1.0 mmol L−1 NADH (Fig. 5, curve 2) and compared with the voltammogram recorded with the unmodified GC electrode (Fig. 5, curve 3) in presence of NADH. In Fig. 5, curve 1, a redox couple DNA/graphene/MB/GC electrode can be observed due to the reduction and oxidation process of the couple MB+ /MB. Upon addition of 1.0 mmol L−1 NADH, there is a dramatic decrease in the overpotential of NADH oxidation, which is very characteristic of an electrocatalytic oxidation process. The immobilization of MB on the DNA/graphene and the interaction between these compounds is not explained only as the electrostatic attraction between the positively charged O+ group in methylene blue dye with that of the negatively charged phosphates of the DNA backbone [47,48], since the oxidized form of MB is cationic and the reduced form is neutral [49]. Therefore, the MB reduction produces a neutral dye form, which does not electrostatically interact with DNA. In

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I/μA

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Table 1 Effects of the concentration of: (a) DNA with [graphene] = 1 mg mL−1 and [MB] = 1 mmol L−1 ;(b) graphene with [DNA] = 1.2 mg mL−1 and [MB] = 1 mmol L−1 ; (c) MB with [DNA] = 1.2 mg mL−1 and [graphene] = 3.0 mg mL−1 . Experiments carried in phosphate buffer solution, pH 7.0, containing 1.0 mmol L−1 NADH.

curve 1 curve 2 curve 3 curve 4 curve 5

Ipa (␮A)

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-0.3

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E/V vs Ag/AgCl Fig. 5. Cyclic voltammograms of the DNA/graphene/MB/GC electrode in the absence (curve 1) and the presence of NADH (curve 2); cyclic voltammograms of the unmodified GC electrode in the presence of NADH (curve 3); cyclic voltammograms of the graphene/GC electrode in the presence of NADH (curve 4); cyclic voltammograms of the graphene/MB/GC electrode in the presence of NADH (curve 5). The voltammograms were obtained using a concentration of 1.0 mmol L−1 NADH with 0.1 mol L−1 phosphate buffer (pH 7.0). [DNA] = 0.7 mg mL−1 ; [graphene] = 1.0 mg mL−1 and [MB] = 1.0 mmol L−1 .

this sense, the graphene is of high importance in improvement of the MB adsorption, since it can interact with graphene through charge-transfer and hydrophobic interactions as well as assemble on the surface of graphene by non-covalent ␲–␲ stacking. On the other hand, the anodic peak potential for NADH oxidation on the graphene/GC electrode shows a poor increase of peak current and a small shift in the potential for oxidation of NADH (Fig. 5, curve 4). Finally, for the graphene/MB/GC electrode (Fig. 5, curve 5) was observed a decrease in the response for NADH due probably to the absence of DNA in comparison to DNA/graphene/MB/GC electrode (Fig. 5, curve 2). In this sense, was concluded that the DNA is also essential in this system for obtaining a film with good electrochemical response. Additionally, the scan rate can influence the current responses of NADH on the response of the DNA/graphene/MB/GC electrode and the corresponding electrochemical parameters could be deduced from the relationship between the scan rate of potential sweep and current responses of NADH oxidation. The plot of the peak current Ip vs. the square root of the potential scan rate ␯1/2 obtained from voltammograms of the DNA/graphene/MB/GC electrode in 0.1 mol L−1 phosphate buffer solution (pH 7.0) containing 1.0 mmol L−1 NADH showed that the oxidation currents for NADH increase linearly with the square root of the scan rate in the range studied with the equation Ipa (A) = 7.56(±1.10) + 1/2

270.13(±4.42)v1/2 (V s−1 ) , for scan rate of 0.01–0.1 V s−1 . These results show that the overall electrochemical oxidation of NADH at this electrode might be controlled by the diffusion of NADH from solution to the redox sites of DNA/graphene/MB/GC electrode. From this equation a NADH diffusion coefficient of 3.28 × 10−6 cm2 s−1 was calculated. This value is closed with literature [50]. 3.5. Effect of DNA, graphene and MB concentrations on the response of NADH Current response of the DNA/graphene/MB/GC electrode is also affected by the DNA, graphene and MB concentrations. The amount of DNA on the surface of DNA/graphene/MB/GC electrode was controlled by using the same volume of the suspension with different concentrations of DNA maintaining the graphene and MB

(a) Concentration of DNA (mg mL−1 ) 35.15 (±0.15) 0.4 30.74 (±0.56) 0.7 50.35 (±0.25) 1.2 48.50 (±0.45) 1.5 2.0 40.75 (±0.34) (b) Concentration of graphene (mg mL−1 ) 1.0 30.12 (±0.38) 41.45 (±0.22) 2.0 60.29 (±0.25) 3.0 65.65 (±0.78) 4.0 5.0 70.43 (±0.80) (c) Concentration of MB (mmol L−1 ) 33.33 (±0.40) 1.0 40.29 (±0.52) 2.0 3.0 75.95 (±0.49) 70.43 (±0.51) 4.0 5.0 65.69 (±0.68)

concentrations. The relationship between the current response and the DNA concentration is shown in Table 1a. A moderate DNA concentration of 1.2 mg mL−1 was selected for the fabrication of the DNA/graphene/MB/GC electrode once concentrations higher than 1.2 mg mL−1 result in a lower response of the modified electrode due to the higher charge transfer resistance of the DNA film. The amount of graphene on the surface of GC electrode was controlled by the same volume of the suspension with different concentrations of graphene, maintaining the DNA and MB concentrations. The relationship between the current response and the graphene concentration is shown in Table 1b. A moderate graphene concentration of 3 mg mL−1 was selected for the fabrication of the DNA/graphene/MB/GC electrode as concentrations higher that 3 mg mL−1 present a lower stability and higher potentials, which did not facility the NADH detection. Current response of the DNA/graphene/MB/GC electrode is also affected by the MB concentration. The relationship is shown in Table 1c. With the increment of MB concentration, the response current increased gradually up to 3.0 mmol L−1 and decreases from this concentration. This suggests that for these concentrations a greater amount of DNA would be necessary. In this sense, a moderate MB concentration of 3 mmol L−1 was selected for the future studies. 3.6. Rotating disk electrode (RDE) experiments The catalytic currents for NADH oxidation on a rotating disk DNA/graphene/MB/GC electrode were measured in 0.1 mol L−1 phosphate buffer solutions (pH 7.0) employing linear sweep voltammetry for two different concentrations of NADH (Fig. 6). The limit currents used in the study were obtained at 0.1 V vs Ag/AgCl. As can be seen in Fig. 6, the peak current shows a deviation of the linearity in the Levich plots suggesting kinetic limitations when high scan rates were used. Under these conditions the Koutecky–Levich equation was used to determine the number of electrons and the heterogeneous rate constant. The limiting current is given by Eq. (3): 1 1 1 = + Ilim nFACo kobs 0.62nFAD2/3 v−1/6 ω1/2 Co

(3)

where n is the number of total electrons transferred to the electrode during the oxidation of NADH, A, the area of the electrode (cm2 ), D (cm2 s−1 ) and (0.01 cm2 s−1 ) are the diffusion coefficient and the kinematic viscosity of the aqueous solution while ω (rad s−1 ), Co


-1

0.0024

5 mmol L -1 6 mmol L

549

(13)

20

15

0.0016 0.0012

I/μA

ILim/ A

0.0020

0.0008 0.0004

10

5

(a)

0.0000 -2

0

2

4

6

8

10

12

14

16

(1)

(a)

0

18

200

1/2 -1 1/2 /(rad s )

225

ω -1

1200

250

275

300

325

Time/s 20

−1/2

I =2423.66 + 15801.67 x ω -1 −1/2 I =1730.70 + 13252.49.67 x ω

∆I/μΑ

-1

ILim /A

-1

15

1000 10

800 5

600

(b)

400 0.08

0.12 -1/2

0.16

0.20

-1 -1/2

ω /(rad s )

Fig. 6. (a) Levich plots constructed from linear sweep voltammograms of a DNA/graphene/MB/GC RDE of solutions with 5.0 and 6.0 mmol L−1 NADH in 0.1 mol L−1 phosphate buffer (pH 7.0) using a scan rate of 0.005 V s−1 at various rotation rates (23.56, 34.03, 41.89, 52.36, 65.45, 94.25, 167.57 and 261.78 rad s−1 ). (b) Koutecky–Levich plots obtained from Levich plots shown in Fig. 6a. [DNA] = 1.2 mg mL−1 ; [graphene] = 3.0 mg mL−1 and [MB] = 3.0 mmol L−1 .

(mol cm−3 ), kobs (mol−1 L s−1 ) and (mol cm−2 ) represent the rotation speed, the bulk concentration of the reactant in the solution, the heterogeneous rate constant for the reaction between the catalyst and the NADH molecule and the amount of the active catalyst on the electrode surface, respectively. The diffusion coefficient used was 3.28 × 10−6 cm2 vs−1 . All other parameters have their usual meanings. According to Eq. (4), at a given potential, a plot of I−1 vs. ω−1/2 should be linear (as shown in Fig. 6b). The number of total electrons calculated, n, involved in the oxidation process of NADH on the DNA/graphene/MB/GC electrode can be obtained from the slope of the Koutecky–Levich plot. The value found for the n was 2.06, which is in agreement to others works also based on the catalytic oxidation of NADH [51]. Therefore, the NADH oxidation reaction catalyzed by the DNA/graphene/MB/GC electrode proceeds via a 2-electron mechanism with formation of NAD+ as the main reaction product. In addition, the rate constant kobs could be calculated from the intercept of the Koutecky–Levich plot (Eq. of Fig. 6b). The values of kobs decreased from 1.05 × 106 to 0.91 × 106 mol−1 L s−1 as the bulk concentration of NADH increased from 5.0 mmol L−1 to 6.0 mmol L−1 . From of the slope and intercept of the plot kobs vs [NADH] a straight line was obtained according

(b)

0 0

400

800

[NADH]/μmol L

1200

1600

-1

Fig. 7. (a) Amperometric response for the oxidation of NADH in 0.1 mol L−1 phosphate buffer at pH 7.0 on the DNA/graphene/MB/GC electrode under optimized conditions at concentrations of: (1) 10, (2) 150, (3) 250, (4) 450, (5) 550, (6) 650, (7) 750, (8) 850, (9) 950, (10) 1050, (11) 1150, (12) 1300 and (13) 1500 ␮mol L−1 . (b) Calibration plot. Applied potential of 0.1 V. [DNA] = 1.2 mg mL−1 ; [graphene] = 3.0 mg mL−1 and [MB] = 3.0 mmol L−1 .

to equation kobs = 1.75 × 106 − 1.40 × 108 [NADH]. The extrapolated value of kobs to zero NADH concentration was estimated at 1.75 × 106 mol−1 L s−1 . The kobs , at [NADH] = 0 was higher than those reported previously for NADH electrocatalytic oxidation on electrodes modified with other mediators such as Variamine blue [52], phenothiazine derivatives [6,51,53], and o-Phenylenediamine [54].

3.7. Analytical characterization of the modified electrode The analytical characteristics of the DNA/graphene/MB/GC electrode were verified by amperometry. As the applied potential in the amperometric measurements contributes to the sensitivity of the system, an initial study was performed in order to determine the best potential to be applied to the electrode (0.05–0.25 V). The potential 0.10 V showed a higher current, and was chosen for the amperometric curve. Fig. 7a shows the amperogram recorded for the DNA/graphene/MB/GC electrode at a working potential of 0.1 V with successive additions of NADH into the phosphate buffer solution. Fig. 7b shows the plot of the analytical curve. A wide

550


Table 2 Comparison of some characteristics of the different modified electrodes for the detection of NADH. Linear range (␮mol L−1 )

Sensing surface

Eoxidation vs. (Ag/AgCl) (V)

ERGO/PTH/GC electrode PEDOTSDS–AgNP–MB/GCE Graphene paste electrode Pencil graphite electrode modified with quercetin – PGE/QH2 Fe3 O4 magnetic nanoparticles/multiwalled carbon nanotubes (Fe3 O4/ MWCNTs) diphenylalanine peptide/MWCNTs Graphene/GC Co3 O4 nanosheet/carbon ink Meldola blue/ZnO Aryl diazonium cations of Azure A NiONPs/ADH-Nafion/GC Multilayered all-MWNTs films DNA/graphene/MB/GCE

0.3 −0.05 0.47 0.3

10–3900 10–560 5–200 0.5–100

0.0

1–70

0.6 0.5 0.1 0.0 0.0 0.35 0.18 0.1

20–800 50–1400 10–100 50–300 0.66–6.25 0.11–1000 2–1234 10–1500

Sensitivity (␮A L ␮mol−1 )

DL (␮mol L−1 )

Reference

0.01 0.0023 NR 0.034

0.1 0.1 NR 0.15

[1] [2] [3] [4]

0.0070–0.0035

0.3

[5]

0.00287 0.013 0.0028 NR 9.48 0.052 0.016 12.75

10 20 4.25 10 0.57 0.11 1.5 1.0

[6] [7] [8] [9] [10] [11] [12] This work

Electroreduced graphene oxide (ERGO) and polythionine (PTH); NiONPs/ADH-Nafion, nickel oxide nanoparticles/alcohol dehydrogenase enzyme; PEDOTSDS–AgNP–MB, silver nanoparticles incorporated poly(3,4-ethylene dioxythiophene-sodium dodecyl sulfate)-Meldola blue; Fe3 O4 , iron oxide; Co3 O4 , cobalt oxide; ZnO, zinc oxide; NiONPs, nickel oxide nanoparticles.

linear response range from 10 ␮mol L−1 up to 1.50 mmol L−1 was observed, which can be expressed according to the equation: Ip (A) = 0.26(±0.08) + 12.75(±0.17) [NADH]/mmol L−1

(4)

with a correlation coefficient of 0.997. The detection and quantification limits were estimated as 1.0 and 3.3 ␮mol L−1 , respectively, using 3 × SDb /slope and 10 × SDb /slope ratios, respectively, where SDb is the standard deviation calculated from the ten background current values (blank measurements), determined according to the IUPAC recommendations [55]. The sensitivity was better when compared to many works reported in the literature (Table 2). Such good limit of detection can be attributed to the efficiency of the electron transfer between the DNA/graphene/MB/GC electrode and NADH, favored by a high reaction rate, kobs , and the low charge transfer resistance of the composite, as a consequence of a highly dispersed MB on the graphene on the GC electrode. 3.8. Stability studies of the DNA/graphene/MB/GC electrode in presence of NADH The stability of the DNA/graphene/MB/GC electrode was checked in the presence of 0.5 mmol L−1 NADH performing successive amperometric measurements in 0.1 mol L−1 phosphate buffer solution at pH 7.0. After 100 amperometric measurements no change was observed in the response of the modified electrode. When the modified electrode was stored at room temperature no significant change in the response was observed in the period of 2 months. In order to study the repeatability of the electrode preparation procedure, five independent electrodes were modified with DNA/graphene/MB. Amperometric measurements of prepared modified electrode in the buffer solution were recorded. The RSD values of measured cathodic and anodic peak currents were 5%. 4. Conclusion This work shows that a glassy carbon electrode modified with DNA/graphene/MB is a feasible alternative for analytical determination of NADH. Due to the observed chemical stability, electrochemical reversibility and high electron transfer rate constant of MB/MB+ redox couple immobilized on graphene, it can be used in electrocatalysis as an electron transfer mediator to shuttle electrons between NADH and substrate electrodes.

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