Experimental and Theoretical Investigations on the Adsorption of 2

Anal. Chem. 2007, 79, 3741-3746

Experimental and Theoretical Investigations on the Adsorption of 2′-deoxyguanosine Oxidation Products at Oxidized Boron-Doped Diamond Electrodes Elodie Fortin,† Eric Vieil,*,‡ Pascal Mailley,† Sabine Szunerits,‡ and Thierry Livache†

Groupe Chimie de la Reconnaissance et Etude des Assemblages Biologiques - SprAM (UMR 5819 CEA/CNRS/UJF), DRFMC/SPrAM/CREAB, CEA Grenoble, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France, and Laboratoire d’Electrochimie et de Physico-chimie des Mate´ riaux et des Interfaces (LEPMI-UMR 5631 CNRS/INPG/UJF), ENSEEG, BP 75, 38402 Saint Martin d’He` res Cedex, France

Electrochemical oxidation of 2′-deoxyguanosine has been performed on boron-doped diamond (BDD) electrodes, resulting in a strong adsorption of the formed oxidized products onto the BDD surface. The adsorption behavior has been investigated by studying the electrochemical behavior of a redox probe ([IrCl6]3-) using cyclic voltammetry. The most probable situations are the formation of (A) an insulating adsorbed film resulting in a partially blocked electrode behavior, (B) a porous film, or (C) an overall conductive film. Different parameters such as the standard rate constant, the charge-transfer coefficient, the electrode/adsorbed products/solution interface resistance, and the formal potential of the redox couple were determined. Through comparison of theoretical currentpotential curves obtained by analytical calculations with experimental cyclic voltammograms, we found that the oxidized products of 2′-deoxyguanosine form a continuous conductive film on BDD. DNA detection has attracted considerable interest in the last years. The standard method for the detection of hybridization is so far confocal fluorescence imaging using fluorescence-labeled target DNA. One of the major drawbacks of fluorescence detection is, beside size and price, the quenching of the fluorescent dyes with time. Electrochemical hybridization methods are an interesting alternative to optical readouts and have been demonstrated as being rapid, sensitive, and selective means for the study of DNA oxidative damage, for trace DNA analysis, and for detection of hybridization events. For label-free analysis, guanine oxidation is a commonly utilized route. Two variants are normally encountered, direct guanine oxidation at relatively high positive oxidation potential (Eguanine) 0.81 V vs SCE) or redox-mediated oxidation.1 * Corresponding author. Tel: +33 476 826 698, Fax: +33 476 826 777, E-mail: [email protected]. † Groupe Chimie de la Reconnaissance et Etude des Assemblages Biologiques. ‡ Laboratoire d’Electrochimie et de Physico-chimie des Mate´riaux et des Interfaces. (1) Chiti, G.; Marrazza, G.; Mascini, M. Anal. Chim. Acta 2001, 427, 155164. 10.1021/ac061765d CCC: $37.00 Published on Web 04/06/2007

© 2007 American Chemical Society

The electrochemical detection requires a judicious choice of the electrode material. Boron-doped diamond (BDD) thin films appear as a promising electrode material in order to detect the oxidation of these electroactive species occurring at high positive potentials. Indeed, this material has attracted considerable interest in various fields ranging from electroanalysis,2 electrocatalysis,3 electrosynthesis,4 to electrochemical water treatment,5 thanks to its specific chemical and physical properties. BDD electrodes show excellent mechanical properties, extreme chemical stability, good biocompatibility, good electrical conductivity, low background current densities, and a large potential window in aqueous electrolytes (about -1.35 to 2.3 V/NHE). This latter characteristic permits us to detect species that are not easily electrochemically detectable on other substrates as their detection is masked by the electrochemical decomposition of the used solvent or by surface reactions on classical carbon electrodes. Thanks to the enlarged potential window of BDD in aqueous media, the direct nucleoside oxidation can be detected, which occurs at potentials of 1.1 and 1.4 V versus Ag/AgCl for 2′-deoxyguanosine and 2′-deoxyadenosine, respectively. Moreover, BDD electrodes present a low background-tosignal ratio, enabling the detection of small nucleoside concentrations. However, as recently shown by us using differential pulse voltammetry and scanning electrochemical microscopy (SECM), the nucleoside oxidation results in a fast and irreversible blocking of the BDD electrode due to the formed oxidized products on the surface for each nucleoside oxidation.6 The observed adsorption mechanism may proceed along three different ways (Figure 1). One situation resembles that of a partially blocking electrode with a preferential adsorption on different sites of the surface (2) Zhang, J.; Oyama, M. Microchem. J. 2004, 78, 217-222. (3) Marken, F.; Bhambra, A. S.; Kim, D.-H.; Mortimer, R. J.; Stott, S. J. Electrochem. Commun. 2004, 6 (11), 1153-1158. (4) Serrano, K.; Michaud, P. A.; Comninellis, C.; Savall, A. Electrochim. Acta 2002, 48, 431-436. (5) Tro¨ster, I.; Fryda, M.; Herrmann, D.; Scha¨fer, L.; Ha¨nni, W.; Perret, A.; Blaschke, M.; Kraft, A.; Stadelmann, M. Diamond Relat. Mater. 2002, 11, 640-645. (6) Fortin, E.; Chane-Tune, J.; Mailley, P.; Szunerits, S.; Marcus, B.; Petit, J.-P.; Mermoux, M.; Vieil, E. Bioelectrochemistry 2004, 63, 303-306.

Analytical Chemistry, Vol. 79, No. 10, May 15, 2007 3741

concern the parts of the surface that are active and therefore lead to electrochemical responses.

EXPERIMENTAL SECTION

Figure 1. Different adsorption models of dG products on BDD: (A) partially blocking electrode, (B) continuous conductive film, (C) continuous porous film.

behaving like inactive areas.7 In this case, the electrical interface contains isolated electrochemically active sites. As a result, the density of these active sites was found to increase with the boron doping level.8 Furthermore, the oxidized products could form a continuous conductive film permitting electron transfer at the film/ solution interface. The last hypothesis is that of the formation of a continuous porous film where the redox probe is transported through the film. We want to show in this paper how cyclic voltammetry can be used to distinguish between the three adsorption routes of the oxidation products of guanosine on BDD. Experimental data were compared to theoretical potential-current curves by using the Butler-Volmer model coupled with an electrical electrode/ adsorbed products/solution resistance. The rate constant k°, the charge-transfer coefficient R, the BDD/adsorbed products/solution interface resistance RΩ, and the formal potential E1/2 of the redox couple were determined, allowing us to distinguish among the proposed mechanism. The distinction was made on the following basis. If the situation is thus of a partially blocking electrode, the ratio of the active areas/inactive ones would change with increasing conditioning time, varying the formal potential E1/2. To discriminate between the situations of the formation of a continuous conductive film or a continuous porous one BDD, the change of the rate constant and of the resistance will be used as a criterion. BDD rather then glassy carbon electrodes were chosen as electrical interface as they showed to be able to detect 2′-deoxyguanosine (dG) oxidation with high sensitivity. Indeed, due to the favored background/signal ratio, the more complicated BDD interface was investigated. The known problem using BDD electrodes is that these carbon interfaces are not active over the whole surface. Thus, any conclusions made in the following only (7) Brookes, B. A.; Davies, T. J.; Fisher, A. C.; Evans, R. G.; Wilkins, S. J.; Yunus, K.; Wadhawan, J. D.; Compton, R. G. J. Phys. Chem. B 2003, 107, 16161627. (8) Holt, K. B.; Bard, A. J.; Show, Y.; Swain, G. M. J. Phys. Chem. B 2004, 108, 15117-15127.

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Analytical Chemistry, Vol. 79, No. 10, May 15, 2007

Materials. Potassium hexachloroiridate(III) (K3IrCl6), hydroxymethylferrocene, and lithium perchlorate (LiClO4) were purchased from Aldrich. 2′-Deoxyguanosine was obtained from Aldrich and used without purification. Stock solutions of the nucleoside (10 mM) were prepared in 0.1 M phosphate buffer pH 7. OH-Terminated Diamond Samples. BDD films were synthesized on p-type silicon wafers by microwave plasma-assisted CVD, as described elsewhere.9 The films were doped with boron during the deposition at a concentration of ∼1020 atom cm-3.10 The hydrogen-terminated BDD electrodes were electrochemically oxidized by applying a potential of 2.4 V in KH2PO4 (0.1 M) for 1 h. Between each electrochemical measurement, the oxidized diamond surface was regenerated by applying the same electrochemical treatment during 15 min. Electrochemical Measurements. Cyclic voltammetric (CV) measurements were performed with a PAR 283 potentiostat (EGG) at room temperature at different scan rates between 0.02 and 0.5 V s-1. An aqueous solution of K3IrCl6 (2 mM) in LiClO4 (0.1 M) was used for the electrochemical studies. The diamond working electrode (electroactive area A ≈ 0.21 cm2, determined using CV with the hexachloroiridate solution) was sealed against the bottom of a single-compartment electrochemical cell (600 µL) by means of a rubber O-ring. The electrical contact was made to a copper plate, through the bottom of the silicon substance where the diamond was deposited. This ohmic contact introduced an important contact resistance, which has been taken into consideration in our calculations. The counter electrode was a platinum wire and the reference electrode a KCl saturated Ag/AgCl electrode. All potentials are referred to this reference electrode in this work. The diffusion coefficient used for [IrCl6]2-/3- is D ) 6.8 × 10-6 cm2 s-1.11 The electrochemical oxidation of an aqueous solution of dG (2.5 mM) in phosphate buffer (0.1 M pH 7) was achieved at 1.2 V at different time lengths (tcond ) 0, 300, 600, or 900 s) in order to form the oxidized product, which is adsorbed on the surface. The following differential pulse voltammetry (DPV) parameters were applied: pulse amplitude of 25 mV, pulse width of 60 ms, and scan rate of 22.2 mV s-1. Theoretical Model. Two models based on analytical calculations were performed to describe the electrochemical behavior of BDD electrodes in K3IrCl6. The electrochemical reaction can be decomposed into three steps: two matter transfers for the oxidation and the reduction of the redox species with the same nature (semi-infinite diffusion) but difference in diffusion coefficients embedded in E1/2 value, and a charge transfer according to the Butler-Volmer model. It is assumed that the electrolysis time is small enough with no consumption of the reactive. In the (9) Mermoux, M.; Fayette, L.; Marcus, B.; Rosman, N.; Abello, L.; Lucazeau, G. Diamond Relat. Mater. 1995, 4, 745-749. (10) Marcus, B., unpublished work, Grenoble, France, 2003. (11) Fischer, A. E.; Show, Y.; Swain, G. M. Anal. Chem. 2004, 76, 2553-2560.

case of semi-infinite diffusion, using i(-1/2) for the semi-integral of the current as explicited by eq 1

i(-1/2) ) (d-1/2/dt-1/2)i

(1)

the current can be expressed by

i(-1/2) i + )1 1/2 i τDA ilAVAB AB

(2)

X ) (nF/R T)(E - E°)

(3)

1 1 + e [X - X1/2]

(4)

VAB )

-

iAB ) nF Ak°cA* eRAX

(5)

ilA ) nFAcA*(DA/lA)

(6)

τDi ) li2/Di

(7)

with X the adimensional potential, VAB the Nernstian wave function, iAB the anodic irreversibility current, ilA the anodic stationary limiting current,τDi the diffusion time constant, E the imposed potential, k° the standard electrochemical rate constant, A the geometric area of the electrode, c* the concentration of oxidized (subscript A) and reduced (subscript B) form, R the charge-transfer coefficient, D the diffusion coefficient, and E° the formal potential of the redox couple. The characteristic distance l for the transport process is shown here for the generality of the model, but is not relevant in the case of semi-infinite diffusion. Homemade software written in Visual Basic (VB6) has been used for computations of this model because of implicit equations and noninteger integration. The software provides basic curve treatments issued from the potentiostat and runs with Windows XP on an ordinary PC. The second model considers the BDD/adsorbed products/ solution resistance. Indeed, the potential at the electrode/solution interface can be expressed by

E)E h - RΩi

(8)

where E h is the imposed potential and RΩ the ohmic drop that, in addition to the film or interface resistance, includes the electrolyte resistivity and the connecting resistance. RESULTS AND DISCUSSION Figure 2A shows the DPV curves obtained in a solution of hydroxymethylferrocene prior to and after positively conditionning the BDD electrode at 1.2 V for varying times in a dG solution. The peak intensity decreases with increasing conditioning time (Figure 2B) while the peak shape and potential position remains unchanged. The peak intensity is lowered by 78% after 900 s of conditioning in the dG solution, while for longer times, the peak intensity is stabilized at ∼80% of the response. This behavior is characteristic of an adsorption of the dG oxidation products.6 The electrochemical response of a redox probe (K3IrCl6) on the oxidized BDD surface and after positive bias (tcond ) 0, 300,

Figure 2. Influence of the conditioning time (tcond) at 1.2 V vs Ag/ AgCl: (A) DPVs on BDD for different tcond in dG (2.5 mM)/phosphate buffer (pH 7, 0.1 M); (B) evolution of the current density of the oxidation peak ipeak/A with tcond. Solution, in K3IrCl6 (2 mM)/LiClO4 (0.1 M); DPV parameters, pulse amplitude 25 mV, pulse width 60 ms, scan rate 22.2 mV s-1, tcond ) 0, 10, 30, 60, 120, 300, 450, 600, and 900 s.

Figure 3. Cyclic voltammograms of a BDD electrode after conditioning at 1.2 V vs Ag/AgCl in dG (2.5 mM)/phosphate buffer (pH 7, 0.1 M) for (a) 0, (b) 300, (c) 600, and (d) 900 s. Solution, K3IrCl6 (2 mM)/LiClO4 (0.1 M); scan rate, 0.2 V s-1.

600, or 900 s) in the presence of dG was studied using cyclic voltammetry at five scan rates (0.02, 0.05, 0.1, 0.2, 0.5 V s-1) to discriminate between the three possible film formations (A blocking electrode, B conductive film, or C porous film) as outlined in Figure 1. Figure 3 shows cyclic voltammetric curves obtained at 0.2 V s-1 for the different conditioning times. The response of the oxidized BDD electrode without polarization in the presence of dG (Figure 3, curve a) is significantly different from the others (Figure 3, curves b-d). Indeed, when conditioning the BDD electrode, the potential separation between the anodic and cathodic peaks is increasing and remains the same for the different polarization times. This indicates that a conditioning of 300 s in a dG solution is sufficient to totally modify the reversibility of the redox couple. Figure 4 presents the normalized cyclic voltammograms without conditioning (Figure 4A) and after 900-s conditioning in a dG solution (Figure 4B). The voltammograms (not shown) obtained for conditioning times of 300 and 600 s present an Analytical Chemistry, Vol. 79, No. 10, May 15, 2007

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Figure 5. Plots of log(v) vs E1/2 on a pretreated BDD without conditioning in dG solution at different scan rates: 0.02, 0.05, 0.1, 0.2, and 0.5 V s-1 (solid lines are obtained from least-square regression). Solution: K3IrCl6 (2 mM)/LiClO4 (0.1 M).

values of the anodic (Ra) and cathodic (Rc) transfer coefficients can be obtained through eqs 9 and 10,

RT m ln nFR k°

(9)

a ) (nF/R T)v

(10)

E1/2 ) E° + m ) xaD,

Figure 4. Cyclic voltammograms of a BDD electrode at different scan rates (a) 0.02, (b) 0.05, (c) 0.1, (d) 0.2, and (e) 0.5 V s-1(A) without conditioning, (B) after conditioning at 1.2 V vs Ag/AgCl in dG (2.5 mM)/phosphate buffer (pH 7, 0.1 M), tcond ) 900 s, and (C) dependence of ipeak/A with the square root of v, Solution: K3IrCl6 (2 mM)/LiClO4 (0.1 M).

intermediate behavior between these two curves. Figure 4C shows the dependence of the peak density (ipeak/A) with the square root of the scan rate in order to characterize the dominant mass transfer. When cyclic voltammetries were performed directly on a pretreated BDD film without conditioning, the correlation coefficient for the linear regression of ipeak/A ) f (v1/2) is very close to 1, indicating that the evolution of these two factors is strongly correlated as is the case when semi-infinite diffusion is the rate-limiting step. In the other case represented in Figure 4C, (tcond ) 900 s), the CV recorded at a scan rate of 0.5 V s-1 has a particular shape and is different from the others. This curve is not taken into consideration when determining the correlation coefficient of the linear regression. Moreover, as will be shown later, this experimental curve does not match well with theoretical CV curves, contrary to those at other scan rates. The influence of the background current, which is more significant for higher scan rates, could be the reason for this observation. Considering thus only four scan rates, the evolution of the peak intensity with the square root of the scan rate is quasi linear. Moreover, no adsorption effect was observed by cycling the BDD electrode for various times in the pure redox probe solution. These observations confirm that mass transfer occurs by semi-infinite diffusion. The 3744


where n is the number of electrons transferred and v the scan rate. The others symbols have their usual meaning. From Figure 5, we can observe that, in the absence of dG, the anodic and cathodic peak potentials vary linearly with log v and values of Ra ) 0.22 and Rc ) 0.18 are obtained. The sum of the anodic and cathodic transfer coefficients is significantly less than 1. This means that rate-determining electron transfers are different between the forward and backward reactions, indicating that the mechanism is not the same for oxidation and reduction on such BDD electrodes. This behavior has also been recorded by Pleskov et al. on BDD electrodes for Fe(CN)63-/4- and quinone/hydroquinone,12 and they concluded that the BDD electrode has an electrochemical behavior resembling that of a semimetal. After positive biasing the BDD electrode, the linear dependence between the peaks potentials and log v is lost, and eq 8 cannot be applied to determine the transfer coefficients. As we shall discuss later, this is due to a significant increase of the interfacial resistance (RΩ). Furthermore, the standard rate constant k° and the transfer coefficient Ra of the theoretical CVs were varied in such a way as to match with the experimental cyclic voltammograms. Two different mechanisms in the anodic and cathodic waves were observed. To conclude about the adsorption mechanism in place, we have chosen to focus only on the anodic behavior of the redox probe. Figure 6 presents experimental CV curves (black lines) for five different scan rates. of a BDD electrode after conditioning in a dG solution during 600 s and the theoretical (gray lines) calculated with the first model (i.e., without any uncompensated resistance RΩ ) 0). The anodic peaks change differently with increasing scan rates for the experimental curves compared to the theoretical ones. The experimental curves present a larger range of peak potential values, which is characteristic of an uncompensated resistance effect on the CV curves. Thus, RΩ cannot be neglected and has been added to our model (2). (12) Modestov, A. D.; Evstefeeva, Y. A.; Pleskov, Y. V.; Mazin, V. M.; Varnin, V. P.; Teremetskaya, I. G. J. Electroanal. Chem. 1997, 431, 211-218.

Table 1. Parameters Used To Fit Experimental Cyclic Voltammograms Recorded in K3IrCl6 (2 mM)/LiClO4 (0.1 M) after Conditioning the BDD Electrode at 1.1 V vs Ag/AgCl in dG (2.5 mM)/Phosphate Buffer pH 7 (0.1 M) for 0, 300, 600, and 900 sa

Figure 6. Experimental (black lines) and theoretical (gray lines) cyclic voltammograms on BDD after conditioning at 1.2 V vs Ag/AgCl in dG (2.5 mM)/phosphate buffer(pH 7, 0.1 M) for tcond ) 600 s. Calculation parameters: k° ) 0.001 cm s-1, R ) 0.5, and E° ) 797 mV. Solution: K3IrCl6 (2 mM)/LiClO4 (0.1 M).

Considering the second model and taking into consideration the resistance effect, although the anodic peaks of the calculated curves correspond approximately to the experimental ones in terms of intensity and potential, the current after the anodic peak is largely different (curves are not shown). This is characteristic of an additional phenomenon attributed to residual current. It is thus preferable to correct the theoretical curves of the second model by the background current. As it was not possible to determine experimentally the background current for each conditioning time, the value was obtained through approximation. The best approximation was obtained by a combination of an exponential and a linear shape, and it is further adjusted by multiplying it by each scan rate, according to the observed variations of experimental residual currents with scan rates. The quality of the comparison must be judged in accordance with the constraints imposed for the fit to keep exactly the same parameters, except the applied potential (and the resistance to be evaluated). It can be observed that this method leads to good

conditioning time (s)

Ra

k0 (cm s-1)

E0 (mV)

RΩ (Ω cm-2)

0 300 600 900

0.28 0.28 0.28 0.28

0.001 0.001 0.001 0.001

925 797 797 771

1740 2440 8700 12900

a k° is the standard rate constant, R the charge-transfer coefficient, Rω the BDD/adsorbed products/solution interface resistance, and E° the formal potential of the redox couple.

agreements only for not too high potentials. This is however not a limitation, as the main characteristics of the mechanism appear in the potential range before and up to the peaks. Using this approach in connection with model 2, calculations have been made by searching identical values for Ra and k° and allowing E1/2 and RΩ to be adjusted. Figure 7 shows that the fitting between theoretical curves and experimental cyclic voltammograms is always good up to the peak potential. Table 1 summarizes the different parameters used in these analytical calculations for the different conditioning times. In the case of tcond ) 900 s, the use of a high ohmic drop of RΩ ) 12.9 kΩ cm-2 improves the fit. The determined E1/2 values fall in two groups depending on the application of a conditioning time or not: a value of 0.925 V at tcond ) 0 s and a value 0.780 V for longer polarization times. The remaining question to answer is which one of the three models prevails under these conditions. In the case of a blocking electrode, the ratio of blocking and active sites would evolve with tcond. It can be expected that a decrease in the number of active sites would result in a decrease in potentials for obtaining the same current density. However, E° is constant for various

Figure 7. Experimental (black lines) and theoretical (gray lines) cyclic voltammograms on BDD after conditioning at 1.2 V vs Ag/AgCl in 2 dG (2.5 mM)/phosphate buffer (pH 7, 0.1 M) for (A) tcond ) 0, (B) 300, (C) 600, and (D) 900 s. Calculation parameters: k° ) 0.001 cm s-1, R ) 0.5, and E° ) 797 mV. Solution: K3IrCl6 (2 mM)/LiClO4 (0.1 M). The calculation parameters used are summarized in Table 1.


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conditioning durations of the electrode in a dG solution, indicating that the adsorption of nucleoside oxidation products does not occur in accordance with the model of a blocking electrode. Moreover, the standard rate constant remains identical. For a porous film, one could expect a decrease of k0. Thus, the model where a continuous conductive film is formed on the BDD surface seems to fit mostly the experimental data. These results are also in accordance with the results of an analogous compound (2′deoxyadenosine) using SECM.13 Initially, before any nucleoside oxidation and consequently without oxidized products adsorption, the 2D image reveals inhomogeneities in the surface reactivity. After conditioning of the BDD electrode in a 2′-deoxyadenosine solution, the measured feedback current is lowered by a factor of 15% homogeneously over the entire scanned surface (50 × 50 µm), indicating that the adsorbed oxidized product forms a continuous conductive film on the BDD surface.

three models was possible by fitting theoretical cyclic voltammetry curves from analytical calculations to experimental ones for different conditioning times. Several parameters such as the standard rate constant, the charge-transfer coefficient, the ohmic drop, and the formal potential of the redox couple were determined as a function of the positive polarization time of the BDD interface in the nucleoside solution. These parameters and in particular the variation of the rate constant and the formal potential provide evidence that the adsorbed products form a continuous conductive film on the BDD surface enabling electron transfer. This study allows understanding better this new material, and more particularly its surface, which is interesting for its use for other applications in the future. While the question regarding the adsorption of products of nucleoside oxidation on BDD is addressed here, the proposed strategy can be applied to other reactions and surfaces where adsorption might occur.

CONCLUSION During the electrochemical detection of dG on pretreated oxygenated BDD electrodes, an adsorption phenomenon of the formed oxidized products on the surface was observed. This adsorption can result in a blocked electrode as well as in the formation of conductive or porous film. The differentiation of the

ACKNOWLEDGMENT The authors warmly thank Bernadette Marcus and Michel Mermoux from LEPMI for the synthesis and characterization of the BDD substrates.

(13) Fortin, E.; Chane-Tune, J.; Delabouglise, D.; Bouvier, P.; Livache, T.; Mailley, P.; Marcus, B.; Mermoux, M.; Petit, J.-P.; Szunerits, S.; Vieil, E. Electroanalysis 2005, 17, 517-526.

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Received for review September 19, 2006. Accepted March 7, 2007. AC061765D

Experimental and Theoretical Investigations on the Adsorption of 2

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