ARTICLE pubs.acs.org/Langmuir
Scanning Electrochemical Microscopy Studies of Glutathione-Modified Surfaces. An Erasable and Sensitive-to-Reactive Oxygen Species Surface Alina Latus,† Jean-Marc No€el,‡ Elena Volanschi,† Corinne Lagrost,‡ and Philippe Hapiot*,‡ † ‡
Department of Physical Chemistry, University of Bucharest, Boulevard Elisabeta 4-12, Bucharest 030018, Romania Sciences Chimiques de Rennes (Equipe MaCSE), Universite de Rennes 1, CNRS, UMR No. 6226, Campus de Beaulieu, 35042 Rennes Cedex, France ABSTRACT:
A surface sensitive to reactive oxygen species (ROS) was prepared by reduction of a diazonium salt on glassy carbon electrode followed by the chemical coupling of glutathione (GSH) playing the role of an antioxidant species. The presence of active GSH was characterized through spectroscopic studies and electrochemical analysis after labeling of the SH group with ferrocene moieties. The specific reactivity of GSH vs ROS was evaluated with scanning electrochemical microscopy (SECM) using the reduction of O2 to superoxide, O2•, near the GSH-modified surface. Approach curves show a considerable decrease of the blocking properties of the layer due to reaction of the immobilized GSH with O2• and the passage of GSH to the glutathione disulfide (GSSG). The initial surface could be regenerated several times with no significant variations of its antioxidant capacity by simply using the biological system glutathione reductase (GR)/NADPH that reduces GSSG back to GSH. SECM imaging shows also the possibility of writing local and erasable micropatterns on the GSH surface by production of O2• at the tip probe electrode.
’ INTRODUCTION Glutathione (GSH), a well-known biological tripeptide, is a key reactant of intracellular reductiveoxidative metabolic cycles.1 GSH plays important physiological functions notably concerning the detoxification and protection against reactive oxygen species (ROS), which are thought to participate to the development of many diseases including cancer, heart attack, and arthritis.2 In these processes, glutathione is oxidized to glutathione disulfide (GSSG) that is produced by reaction between two GSH molecules and the formation of a disulfide bond. GSSG could be reduced back to the initial GSH by the glutathione reductase (GR) enzyme in the presence of nicotinamide adenine dinucleotide phosphate, NADPH, as cofactor.1,3 In view of the general properties of GSH in solution, immobilization of active GSH appears as an attractive way for sensor preparation or for the design of new protected interfaces able to resist against ROS attack (for a general review about electrochemical investigations and protection against ROS and oxidative stress, see, for example, ref 4). In this connection, an interesting platform for immobilizing biomolecules such as GSH has been previously presented.5 The method follows a general strategy in which a first anchoring layer r 2011 American Chemical Society
is deposited by electrografting of a diazonium salt, followed by the postfunctionalization of the deposited layer.6,7 Advantages of such a two-step procedure have been demonstrated before as it allows an independent optimization of the molecular platform and the use of specific and milder conditions to attach the molecule of interest.6,7 In this work, we have followed the same approach5 for the preparation of functionalized surfaces with radical scavenging action. An aminobenzylamine-modified glassy carbon electrode (GC) functionalized with GSH was used in the study of the reactivity of superoxide radical anions, O2•, generated in an organic solvent. O2• was selected because it could be easily electrogenerated.8 Its redox properties have been studied in different conditions by electrochemical methods over these past decades, and this radical is a good example of reactive oxygen species.8 For example, O2• was proposed as a standard for evaluating the antioxidant capacity of polyphenols in solution by using an electrochemical method.9 In solution, superoxide is Received: May 30, 2011 Revised: July 14, 2011 Published: July 14, 2011 11206
dx.doi.org/10.1021/la2020034 | Langmuir 2011, 27, 11206–11211
Langmuir
ARTICLE
Scheme 1. Immobilization of the GSH on a Glassy Carbon Surface (GC)
reported to oxidize GSH molecule to GSSG.10 Several mechanisms have been envisaged on the basis of a radical scavenging of an H atom of the SH group of the GSH by superoxide or transient formation of hydroperoxyl radicals. All reactions lead to the formation of GSSG and H2O2 or H2O.10b For investigating the redox properties of the modified surface, scanning electrochemical microscopy (SECM)11 in feedback mode was used for both generating superoxide at local scale and examining the modification of the surface using a nonreactive probe (hydroxymethylferrocene, FcMeOH). All of these experiments will help to design a surface with high activity against ROS usable for sensing applications or radical attack protection.
’ EXPERIMENTAL PROCEDURES Chemicals. 4-Aminobenzylamine (98%) was purchased from Acros. Hydrochloric acid from Panseac and sodium nitrite (97%) and L-glutathione reduced (99%) received from Aldrich were used in surface modification without further purification. Tetrabutylammonium hexafluorophosphate, NBu4PF6, was obtained from Fluka. N-Ethyl-N0 -(3(dimethylamino)propyl)carbodiimide hydrochloride (EDC; ∼98%), N-hydroxysuccinimide (NHS; 98%), and 2-(N-morpholino)ethanesulfonic acid, monohydrate 98% (MES), used for COOH activation, and potassium hydrogen phosphate and potassium dihydrogen phosphate, used for buffer solutions (PBS) preparation, were purchased from Alfa Aesar. Electrochemical mediators, ferrocene methanol (FcCH2OH; 98%) and tetrathiafulvalene (TTF; 97%), were from Alfa Aesar. Glutathione reductase (GR) from baker yeast (Sigma) and tetrasodium nicotinamide adenine dinucleotide phosphate salt (NADPH; Roth), used for simulation of the biological system, were used as received. Acetonitrile (ACN), dimethylformamide (DMF), trifluoroacetic acid (TFA), and acetone were obtained from Sigma-Aldrich, and absolute ethanol (EtOH) was from Carlo Erba. Substrate Preparation before Modification. The substrates consist of a 3 mm diameter glassy carbon disk electrode (CH Instruments, Austin, TX) or a platinum plate (1 cm 1 cm). Between each series of experiments, the surfaces were successively polished using 5 μm SiC paper (Struers) and 1 μm DP-Nap paper (Struers) with 0.3 μm Al2O3 slurry (Struers). The platinum plate was electrochemically polished by cycling the electrode potential between 0 and 1.6 V (vs SCE) in 0.5 mol L1 H2SO4 solution at a scan rate of 0.2 V s1 for 50 cycles. After each polishing step, surfaces were thoroughly washed with ultrapure water (18.2 MΩ cm). Prior to the electrografting of the aryl diazonium salts, the surface state was checked by recording a cyclic voltammogram of aqueous potassium ferrocyanide, K4Fe(CN)6 (Acros). Substrate Modification. A thin phenylmethylamine layer was grafted onto the surface of the electrode (glassy carbon or Pt plate) by electroreduction of 4-(aminomethyl)benzenediazonium salt5 following the general in situ procedure proposed by Baraton and Belanger at room
temperature.12 Two successive voltametric cycles at 0.2 V s1 between +0.6 and 1.0 V (vs SCE) were performed on a freshly polished electrode in a solution containing 2 102 mol L1 of 4-aminobenzylaniline in 0.5 mol L1 HCl with 4 102 mol L1 of sodium nitrite. The obtained electrode was rinsed several times with large amounts of ultrapure water. Covalent attachment of GSH to an amino-derivatized electrode was done following the procedure described in the literature.5 A 3.3 102 mol L1 glutathione solution containing 3 102 mol L1 EDC and 4 103 mol L1 NHS was prepared in MES buffer (pH 6.8) and allowed to react under argon atmosphere for 3 h, to activate the carboxyl groups of glutathione. All amino-derivative electrodes were then immersed in this solution for 12 h under argon and in the dark to give the peptide-modified glassy carbon electrodes. The modified electrodes were rinsed thoroughly with ultrapure water and high-purity ethanol prior to use. Thiol groups of GSH molecules grafted at amino-derivative electrodes were marked with a ferrocenyl group, chosen as a convenient reagent for thiol quantization and analysis. Glutathione reacts with FcCH2OH in aqueous medium in the presence of a catalytic amount of TFA. The labeling of the GSH layer with the ferrocene electroactive probe was realized according to the method describe in the literature.13 The introduction of a ferrocene group into GSH layer was achieved by immersion of the modified electrodes in a 1/1 water/acetone mixture containing 2 102 mol L1 of FcCH2OH and two drops of TFA. The mixture was heated to 45 °C and stirred for 2 h and then left to stand overnight at room temperature. The modified electrodes were then rinsed several times with acetone, ultrapure water, and ACN. Cyclic Voltammetry Measurements. Electrochemical measurements were performed in a one-compartment cell using a platinum wire as a counter electrode and a SCE electrode (Radiometer Analytical) as a reference electrode. Cyclic voltamograms were performed with an Autolab electrochemical analyzer (PGSTAT 30 potentiostat galvanostat from Eco Chemie B.V. equipped with GPES software). SECM Experiments. SECM experiments were performed with a CHI 900B instrument from CH Instruments equipped with an adjustable stage for tilt angle correction. The substrate was not connected (unbiased mode). The electrochemical cell was the original one purchased with the SECM and used in a three-electrode configuration. The gold tip electrode had a 5 μm radius. The reference electrode was Ag/AgNO3, and Pt electrode was used as a counter electrode. Approach curves in feedback mode and imageries were recorded in DMF. When possible, these curves were characterized by the estimation of the apparent charge-transfer rate constant, kel, taking for the diffusion coefficients of FcCH2OH a value of 8.7 106 cm2 s1 in DMF.14 Two redox mediators (FcCH2OH and TTF) were used at a concentration of 103 mol L1. Supporting electrolyte was 0.1 mol L1 NBu4PF6. The approach scan rate was 5 μm s1. All approach curves in the same set of experiments are plotted using the same origin value in such a way that they could be compared and determined as the touching point. 11207
dx.doi.org/10.1021/la2020034 |Langmuir 2011, 27, 11206–11211
Langmuir IR Experiments. IRRAS measurements were done using a Vertex 70 FT-IR spectrometer with a Gratzing angle unit A513/3 from Bruker. The angle of the Pt-modified surface was 75°. The spectra were recorded with a 2 cm1 resolution and an averaging of 32 scans.
’ RESULTS AND DISCUSSION Characterization of GSH-Modified Surfaces. The functionalization procedure is a classical two-step procedure in which we first prepare the molecular platform by electroreduction of a functional diazonium salt (arylmethylamine diazonium)57 that is followed by the attachment of GSH on the linking group (Scheme 1). A first point to address concerns the presence of active GSH on the surface after final treatment. Indeed, it is important not only to show the presence of the GSH on the surface but also to evaluate the amount of SH groups that are chemically active. In this purpose, we performed a series of IRRAS spectroscopic analyses combined with electrochemical studies in which SH groups were marked with a redox label (ferrocene groups). Because IRAAS experiments are difficult to perform on glassy carbon substrate, note that the spectroscopic analyses were performed with (4-aminomethyl)benzyl layers prepared on a Pt substrate. Figure 1 displays the IRRAS spectra obtained for a Pt surface modified by a (4-aminomethyl)benzyl layer before and after the immobilization of GSH. The GSH molecule has two carboxylic acid groups, one thiol group, and one amino group, resulting in four possibilities for acidic dissociation with pKa values of 2.12 (COOH of glu), 3.59 (COOH of gly), 8.75 (SH) and 9.2 (NH3+
Figure 1. IRRAS spectra in the 6002000 cm1 region recorded on a platinum surface modified by a (4-aminomethyl)benzyl layer before (black line) and after (red line) GSH coupling. The sample was washed several times with ultrapure water before each analysis.
ARTICLE
of glu), respectively. Depending on the pH value, there are then five possible ionic forms of GSH that display different patterns of their characteristic IR absorption bands.15,16 Since the introduction of the GSH moiety is carried out in a pH 6.8 medium (MES buffer), all spectra in this work refer to IR absorption analysis corresponding to GSH at pH 6.8 (zwiterrionic form, NH3+/ COO/SH/COO). The IR absorption spectrum of the initial surface modified only with the (4-aminomethyl)benzyl layer (Figure 1, black line) shows a strong characteristic band at 841 cm1, which corresponds to the out-of-plane vibration of the substituted benzene ring.17 After chemical coupling of GSH to the (4-aminomethyl)benzyl layer, this peak remains present, but with a significant decrease in intensity while new IR absorption bands clearly appear in the 13001800 cm1 region (Figure 1) and could be interpreted as characteristic of immobilized GSH by reference to literature data.16 The intense bands centered at 1650 and at 1554 cm1 represent the contribution of the amide I (CdO stretching) and II (δ(NH) and ν(CN)) deformation modes.16,17 Two less intense bands, at 1636 and 1525 cm1, are attributed to the deformation modes of the ammonium groups, respectively δasym (NH3+) and δsym (NH3+).16,17 The bands at 1618 and 1392 cm1 could be ascribed to the antisymmetric and symmetric stretching of COO, respectively.16,17 Finally, the band at 1461 cm1 is assigned to the deformation mode of δ(CH2) of the alkyl chain. In the 20004000 cm1 region, bands are observed at 2930 and 2853 cm1 and correspond to the antisymmetric and symmetric vibration of CH2 groups, respectively. A weak absorption band corresponding to the SH stretching is observed at 2531 cm1.16,18 Besides the IR study, to prove the presence and the accessibility of the free SH groups, we used a reaction of thiol groups that allows the anchoring of FcCH2OH in aqueous medium in the presence of a catalytic amount of TFA (Scheme 2).13 In this reaction, one coupled ferrocene molecule (Fc) corresponds to one free SH group. Cyclic voltammetry of the modified electrode was then performed in a blank acetonitrile solution containing only the supporting electrolyte (0.1 mol L1 NBu4PF6). Figure 2 displays the reversible electrochemical response of the attached ferrocene. One could notice a high value of the peak to peak potential separation around 0.19 V characteristic of a slow charge-transfer rate,19 certainly due to a long-distance charge transfer between the carbon substrate and the ferrocene moiety. A plot of anodic peak currents (ipa) against the scan rates between 0.05 and 0.5 V s1 shows a linear variation (correlation
Scheme 2. Labeling of GSH-Modified Glassy Carbon Surface with FcCH2OH
11208
dx.doi.org/10.1021/la2020034 |Langmuir 2011, 27, 11206–11211
Langmuir coefficient of 0.995) confirming that Fc groups were surfaceconfined.19 A Fc surface coverage of Γ0 = 6.0 1010 mol cm2 was derived from the charge integration. This value corresponds approximately to a monolayer of ferrocene (4.5 1010 mol cm2)20 grafted on a surface showing that at least the same concentration of active GSH was initially immobilized on the carbon substrate. Reactivity of the GSH-Modified Surface with Superoxide. Superoxide ion (O2•) is a common reactive oxygen species that could be easily produced by electrochemical methods such as SECM.8 In our experiments, SECM in feedback mode11 was used both to induce the reaction of superoxide with GSH-modified surface and to monitor the resulting modifications of the interface (Scheme 3). Superoxide ion is considered as a standard radical for estimating antioxidant capacity in solution,9 and thus SECM investigations provide a view of the antioxidant capacity of the surface. O2 is reduced to O2• at a 5 μm radius gold disk ultramicroelectrode that is maintained at a constant distance (45 μm) from the surface in a DMF solution (+ 0.1 mol L1 NBu4PF6) under air. Superoxide diffuses from the tip to the surface where it could locally react with the immobilized species. The evolution of the surface properties before and after reaction with O2• is detected by recording a series of SECM approach curves in feedback mode under unbiased conditions (the surface is not electrically connected) with a different electrochemical probe (103 mol L1 FcMeOH in DMF; Figure 3). Approach curves consist of recording the normalized current I = I/Iinf vs the normalized distance L = d/a (I, Iinf: steady-state currents at the
Figure 2. Cyclic voltammogram after the immobilization of the FcCH2OH on GSH-modified surface recorded in 0.1 mol L1 ACN/ NBu4PF6 at the scan rate of 100 mV s1.
ARTICLE
microelectrode (UME) at a distance d from the substrate and at an infinite distance).11 In our conditions, analysis of the curves provides information about the permeation of the probe through the deposited layer.11 On a freshly GSH-modified-electrode, the curve (black open triangles) presents a negative feedback, corresponding to a slow charge transfer between FcMeOH+ and the modified surface11 and showing that the layer has a strong blocking character but not sufficient for totally inhibiting permeation of the mediator. After 10 s of reaction with superoxide, a new approach curve (red open circles) is recorded without moving the tip electrode. A positive feedback is now observed, meaning that FcMeOH+ could now pass through the layer and thus that new charge-transfer pathways have been opened in the layer likely due to the transformation of GSH to the corresponding disulfide GSSG (see Scheme 3). To check that FcMeOH acts as a nonspecific probe, SECM experiments were repeated using another redox probe, the tetrathiafulvalene/tetrathifulvalene radical cation couple (TTF/TTF•+) that presents a standard potential close to the ferrocene one. The observed responses of the surface upon the different modifications were similar, showing that there was no special behavior due to the use of ferrocene as a SECM probe. Finally, the same experiments were also performed with an electrode that was only modified by reduction of the aryl methylamine diazonium, i.e., without the postfunctionalization with GSH. Approach curves (not shown) were not modified after exposition to superoxide, demonstrating that the reaction is specific to GSH. In the mechanism proposed in Scheme 3, it is noticeable that the process leads to the formation of H2O2 that is a potentially oxidizing agent vs SH groups. For highlighting the specific reactivity of our GSH surface toward O2•, we added 2 103 mol L1 of phenol (around 2 equiv) in the solution. Indeed, O2• reacts rapidly with phenol to form H2O2 through a DISP2 mechanism involving protons and electron transfers. In these conditions, superoxide is rapidly converted to H2O2 that only reaches the GSH surface.8b After treatment with O2 reduction, the approach curve recorded with FcMeOH did not evidence any modifications of the surface (data not shown) confirming that O2• is the only species to react with the GSH surface at the time scale of the experiment. Glutathione is a well-known antioxidant compound that presents a general protection against many radicals in solution.1,2 Our experiments with O2• indicate that this activity is retained when GSH is immobilized on the surface and that this surface could be used as a ROS-sensitive surface.
Scheme 3. Reversible Mechanism of GSH-Modified Electrode Oxidation vs. O2•
11209
dx.doi.org/10.1021/la2020034 |Langmuir 2011, 27, 11206–11211
Langmuir
ARTICLE
Figure 3. SECM approach curves (Au UME, a = 5 μm) on GSHmodified surfaces of GSH in DMF. FcMeOH (103 mol L1) is used as a redox probe. Cycle 1: (black open triangles) before O2 reduction, (red open circles) after 10 s of O2 reduction, and (green open triangles) after 5 min in GR/NADPH solution. Cycle 2: (black open triangles) before O2 reduction, (red open circles) after 10 s of O2 reduction, (blue open circles) after 10 s of O2 reduction and 10 min in PBS pH 7. Lines are the simulated curves for irreversible electron-transfer kinetics. kel = 7.0 103 cm s1 (black line). Diffusion coefficient D = 8.7 106 cm2 s1. d is the tipsubstrate distance and a the radius of the tip.
Surface Regeneration and Micropatterning. It is reported that a solution of GSSG could be reduced back to GSH by the glutathione reductase (GR) in the presence of NADPH.1 To evaluate if our GSH surface could be regenerated (erased), a solution containing GR (10 units) and NADPH (5 104 mol L1) in PBS pH 7 was added in the SECM cell and allowed to react for 5 min at 40 °C (Scheme 2). Then, the cell was rinsed several times with ultrapure water and DMF. During this treatment, we maintained the SECM tip at a fixed position for examining the same area of the modified surface. A new approach curve was then recorded using the FcMeOH solution in DMF. The approach curve (Figure 3, green open triangles) displays a negative feedback and is almost undistinguishable from the initial curve, indicating that the layer has been regenerated. In a subsequent step, superoxide was produced at the tip and the surface reexamined with the FcMeOH solution, leading again to the positive feedback behavior (black open circles). The full procedure where the surface is exposed to O2• followed by the regeneration with GR/NADPH, was performed 45 times. We did not observe any considerable change of the SECM responses, demonstrating that the surface could be oxidized by O2• and then regenerated with negligible degradations several times. The same cycle of experiments was repeated but using a blank solution without GR. As expected, the SECM studies show that the GSH-modified electrode could not be regenerated after the exposure to O2•. Finally, the reversible oxidation of the GSH-modified surface was tested to form a reversible micropatterned surface. Indeed, SECM is a convenient technique for inducing localized reactions with radicals and in a highly controllable manner in order to make micropatterned surfaces.21 Using superoxide, a series of localized modification were performed to produce a recognizable square (O2 was reduced for 10 s in eight different points, each spaced 20 μm and forming a square). Figure 4 and Figure 5 display the SECM image recorded with TTF as redox probe, showing the patterning due to the localized reaction between GSH and O2• and for two different tips radii. All of the circular area arranged in a square show the localization of the formed diffusing channel. The diameter of each circular area corresponds to about twice the diameter of the microelectrode due to the broadening of the diffusional pathways. 11 When the modified surface is
Figure 4. SECM image acquired with a 7.4 μm radius Au UME using a solution of 103 mol L1 TTF in 0.1 mol L1 DMF/NBu4PF6 as redox mediator and applying a potential of 0.6 V vs. Ag/AgNO3 (where, for example, 4.02e-9 represents 4.02 109). Images recorded after interaction with O2• (A), after interaction with GR/NADPH solution (B), and after the second interaction with O2• (C). The tip velocity was 20 μm s1, and the tipsubstrate distance was 5 μm.
soaked in the GR/NADPH solution, the micropatterns are erased according to the behavior shown on the approach curves (Figure 3). Then, a new pattern could be drawn on the surface. As seen on Figure 5, using a smaller tip electrode allows us to decrease the size of the modification in agreement with literature reports. 11210
dx.doi.org/10.1021/la2020034 |Langmuir 2011, 27, 11206–11211
Langmuir
ARTICLE
’ REFERENCES
Figure 5. SECM image acquired with a 5 μm radius gold ultra-microelectrode using a solution of 103 mol L1 TTF in DMF/NBu4PF6 0.1 mol L1 as redox mediator and applying a potential of 0.6 V vs Ag/ AgNO3 (where, for example, 4.20e-10 represents 4.20 1010). The image was recorded after repeated interactions of the surface with the superoxide. The tip velocity was 20 μm s1, and the tipsubstrate distance was 5 μm.
’ CONCLUSION The electrografing of aryl methylamine diazonium salt followed by the coupling of an antioxidant species (GSH) is a convenient strategy for preparing surfaces sensitive to ROS. On the basis of our experiments and the fact that gluthatione is a wellknown antioxidant reported to react with many radicals in solution, we could propose such modified surfaces as a good candidate for preparing sensitive-to-ROS surfaces. IR analysis and cyclic voltammetry studies (after labeling of the active SH of GSH with a redox group) reveal a high surface concentration of immobilized active SH groups (Γ0 > 6 1010 mol cm2). SECM technique allows the electrochemical evaluation of the specific reactivity of the modified surface versus ROS through the passage of GSH to its dimeric form GSSG after reaction with O2• (produced at the SECM tip electrode). It results in a local decrease of the blocking properties of the surface that could be evidenced from the SECM approach curves using indifferent probes (ferrocene, TTF). The regeneration of the initial GSH surface is easy by using the enzymatic system GR/NADPH that reduces GSSG back to GSH and “erases” the surface. Besides the use of such surfaces for radical sensing purpose, modified-GSH surface appears as an interesting way to protect an interface from ROS attack. Concerning the methodology, the procedure used in this work based on SECM for evaluating the antioxidant properties of the surface should certainly be adaptable to the investigations of many other modified interfaces for which antioxidant properties are desired. SECM in feedback mode evidences changes that could be difficult to detect by other methods. The large choice of redox probe could be adjusted to follow the properties under investigation. ’ AUTHOR INFORMATION
(1) (a) Dringen, R.; Gutterer, J. M.; Hirrlinger, J. Eur. J. Biochem. 2000, 267, 4912. (b) Shan, X.; Aw, T. Y.; Jones, D. P. Pharmacol. Ther. 1990, 47, 61. (c) Munday, R.; Winterbourn, C. C. Biochem. Pharmacol. 1989, 38, 4349. (2) (a) Cerutti, P. A. Science 1985, 227, 375. (b) Meister, A. Cancer Res. 1994, 54, 1969s. (3) Gilliland, G. L. Curr. Opin. Struct. Biol. 1993, 3, 875. (4) Hillard, E. A.; de Abreu, F. C.; Ferreira, D. C. M.; Jaouen, G.; Goulart, M. O. F.; Amatore, C. Chem. Commun. (Cambridge, U. K.) 2008, 2612. (5) Griveau, S.; Mercier, D.; Vautrin-Ul, C.; Chaussee, A. Electrochem. Commun. 2007, 9, 2768. (6) Pinson, J.; Podvorica, F. Chem. Soc. Rev. 2005, 34, 429. (7) (a) No€el, J.-M.; Sj€oberg, B.; Marsac, R.; Zigah, D.; Bergamini, J. F.; Wang, A.; Rigaut, S.; Hapiot, P.; Lagrost, C. Langmuir 2009, 25, 12742. (b) Malmos, K.; Dong, M.; Pillai, S.; Kingshott, P.; Besenbacher, F.; Pedersen, S. U.; Daasbjerg, K. J. Am. Chem. Soc. 2009, 131, 4928. (c) Leroux, Y. R.; Fei, H.; No€el, J.-M.; Roux, C.; Hapiot, P. J. Am. Chem. Soc. 2010, 132, 14039. (8) (a) Sawyer, D. T.; Valentine, J. S. Acc. Chem. Res. 1981, 14, 393. (b) Andrieux, C. P.; Hapiot, P.; Saveant, J.-M. J. Am. Chem. Soc. 1987, 109, 3768. (9) Rene, A.; Abasq, M.-L.; Hauchard, D.; Hapiot, P. Anal. Chem. 2010, 82, 8598. (10) (a) Dickinson, D. A.; Forman, H. J. Biochem. Pharmacol. 2002, 64, 1019. (b) Winterbourn, C. C.; Metodiewa, D. Free Radical Biol. Med. 1999, 27, 322. (11) (a) Bard, A. J., Mirkin, M. V., Eds. Scanning Electrochemical Microscopy; Dekker: New York, 2001. (b) Amemiya, S.; Bard, A. J.; Fan, F. R. F.; Mirkin, M. V.; Unwin, P. R. Ann. Rev. Anal. Chem. 2008, 1, 95. (c) Wittstock, G.; Burchardt, M.; Pust, S. E.; Shen, Y.; Zhao, C. Angew. Chem., Int. Ed. 2007, 46, 1584. (12) Baraton, S.; Belanger, D. J. Phys. Chem. B 2005, 109, 24401. (13) Misterkiewicz, B.; Salmain, M.; Jaouen, G. Tetrahedron Lett. 2004, 45, 7511. (14) Noel, J.-M.; Zigah, D.; Simonet, J.; Hapiot, P. Langmuir 2010, 26, 7638. (15) Lange, N. A., Dean, J. A., Lange’s Handbook of Chemistry, 13th ed.; McGraw-Hill: New York, 1985; pp 540. (16) (a) Vallee, A.; Humblot, V.; Methivier, C.; Pradier, C.-M. Surf. Interface Anal. 2008, 40, 395. (b) Picquart, M.; Grajcar, L.; Baron, M.-H.; Abedinzadeh, Z. Biospectroscopy 1999, 5, 328. (17) Socrates, G. Infrared and Raman Characteristic Group Frequencies, 3rd ed.; Wiley: New York, 2001. (18) (a) Mateo Marti, E.; Metivier, C.; Pradier, C.-M. Langmuir 2004, 20, 10223. (b) Xu, X; Gimenez, S; Mora-Sero; Abate, A.; Bisquert, J.; Xu, G. Mater. Chem. Phys. 2010, 124, 709. (19) Bard, A. J., Faulkner, L. R. Electrochemical Methods. Fundamentals and Applications; Wiley: New York, 2001. (20) (a) Boland, S.; Barriere, F.; Leech, D. Langmuir 2008, 24, 6351. (b) Vijaikanth, V.; Capon, J.-F.; Gloaguen, F.; Schollhammer, P.; Talarmin, J. Electrochem. Commun. 2005, 7, 427. (c) Buriez, O.; Labbe, E.; Pigeon, P.; Jaouen, G.; Amatore, C. J. Electroanal. Chem. 2008, 619620, 168. (21) (a) Shiku, H.; Uchida, I.; Matsue, T. Langmuir 1997, 13, 7239. (b) Ktari, N.; Poncet, P.; Senechal, H.; Malaquin, L.; Kanoufi, F.; Combellas, C. Langmuir 2010, 26, 17348.
Corresponding Author
*E-mail:
[email protected].
’ ACKNOWLEDGMENT This work was supported by the Universite Europeenne de Bretagne, France and Bilateral ANCS project, Romania- PHC Brancusi, France, No. 215/2009. 11211
dx.doi.org/10.1021/la2020034 |Langmuir 2011, 27, 11206–11211
