Spatially Resolved Electrochemiluminescence on an Array of

Jul 25, 2003 - An array of electrode tips with 6-μm center-to-center spacing, fabricated through chemical etching of an optical fiber bundle, and coa...
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Anal. Chem. 2003, 75, 4382-4388

Spatially Resolved Electrochemiluminescence on an Array of Electrode Tips S. Szunerits,†,§ J. M. Tam,† L. Thouin,‡ C. Amatore,‡ and David R. Walt*,†

The Max Tishler Laboratory for Organic Chemistry, Department of Chemistry, Tufts University, Medford, Massachusetts, 02155, Ecole Normale Supe´ rieure, De´ partement de Chimie, UMR CNRS 8640 Pasteur, 24 rue Lhomond, 75231 Paris Cedex 05, France

An array of electrode tips with 6-µm center-to-center spacing, fabricated through chemical etching of an optical fiber bundle, and coated with gold, was used for initiating electrochemiluminescence (ECL) in an aqueous solution of Ru(bpy)32+ and tri-n-propylamine (TPrA). ECL generated at the tips of the electrodes in the array was detected with a CCD camera and exhibited both high sensitivity and high resolution. In the case in which the ECL signal could not be distinguished from the background, ECL signals could be obtained by pulsing the array and summing multiple CCD images. The behavior of this array was compared to a second array that consisted of individual electrodes insulated with an electrophoretic paint. There is burgeoning interest in arrays, and a variety of fabrication methods have been devised to construct electrode arrays ranging from microlithography,1 deposition of metals using membrane pores as templates,2,3 cyanide etching of hexadecanethiol monolayers confined to Cu-underpotential deposited (UPD)-modified Au(111),4 and mechanical assembly of conducting wires into a random array.4,5 We have previously fabricated an array of electrode tips using an optical fiber bundle template coated with gold.6-8 The chemical etching of the fiber bundle’s distal end results in arrays of either tips or wells, depending on the composition of the core relative to the cladding. These etched arrays have been used to fabricate optical nanoarrays. It was shown recently that individual nanotips could be generated by coating the base of the fiber array with an electrophoretic paint.6,7 In this paper, we report the electrochemical behavior of electrode arrays prepared from tapered and gold covered optical

fiber bundles. By connecting the array to an optical microscope fitted with a high sensitivity CCD camera, we obtain ECL images. The purpose of this study is to demonstrate the technique and to characterize the spatially resolved ECL images obtained from the focal plane of a microscope. In addition, we present details regarding the electrochemical behavior of the tapered surface of an electrode tip array. The ultimate application of such arrays is to simultaneously acquire both spatially resolved electrochemical and optical information from analytical samples. ECL at microelectrodes has been the subject of many studies,9-14 and the use of ECL at ultramicroelectrodes as well as at nanometer tips as a light source for scanning optical microscopy has been demonstrated recently.15-17 In this work, arrays of nanotips were formed through chemical wet etching of a commercially available optical fiber bundle (Figure 1A). The electrode dimension, r, is imposed by the optical fiber size and cannot be varied significantly. Similarly, d, the distance between adjacent electrodes in a hexagonal array, is imposed by the optical fiber bundle construction (d ) 6 µm and r ) 1.75 µm in the optical fiber bundle discussed here). Interelectrode distances (d) can be artificially varied by connecting only a fraction of the individual electrodes.18,19 Similarly, the use of insulating paint to decrease the dimension of individual electrodes in such arrays produces the same result as increasing the d/r ratio, with the advantage that the hexagonal symmetry arrangement of optical fibers in the array is retained.7 The disadvantage of the insulating method, however, is that the relatively low electrochemical stability of the paint insulation requires that the potential window be rather limited (e.g., E > -0.2 V/Ag/AgCl for reductions) to avoid irreversible dissolution of the paint, which leads to exposure of the full electrode surface.

†

Tufts University. Ecole Normale Supe´rieure. § Current address: CEA Grenoble, UMR SPrAM 5819, 17 avenue des Martyrs, 38054 Grenoble, France. (1) Scharifker, B. J. In Microelectrodes: Theory and Application; Montenegro, M. I., Queiros, M. A., Daschbach, J. L., Ed.; NATO ASI Series; Kluwer Academic Publishers: Dordrecht, 1991; Vol. 4, pp 227-239. (2) Martin, C. R. Science 1994, 266, 1961. (3) Ugo, P.; Moretto, L. M.; Bellomi, S.; Menon, V. P.; R., M. C. Anal. Chem. 1996, 68, 4160. (4) Baker, W. S.; Crooks, R. M. J. Phys. Chem. B 1998, 102, 10041. (5) Fletcher, S.; Horne, M. D. Electrochem. Commun. 1999, 1, 502-512. (6) Pantano, P.; Walt, D. R. Rev. Sci. Instrum. 1997, 68, 1357-1359. (7) Szunerits, S.; Garrigue, P.; Bruneel, J.-L.; Servant, L.; Sojic, N. Electroanalysis 2003, in press. (8) Tam, J. M.; Szunerits, S.; Walt, D. R. In Encyclipedia of Nanoscience and Technology; Nalwa, H. S., Ed.; American Scientific Publishers: Stevenson Ranch, 2003. ‡

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(9) Engstrom, R. C.; Johnson, K. W.; Desjarlais, S. Anal. Chem. 1987, 59, 670673. (10) Bartelt, J. E.; Drew, S. M.; Wightman, R. M. J. Electrochem. Soc. 1992, 139, 70-74. (11) Amatore, C.; Fosset, B.; Maness, K. M.; Wightman, R. M. Anal. Chem. 1993, 65, 2311. (12) Collinson, M. M.; Wightman, R. M. Anal. Chem. 1993, 65, 2576-2582. (13) Maness, K. M.; Terrill, R. H.; Meyer, T. J.; Murray, R. W.; Wightman, R. M. J. Am. Chem. Soc. 1996, 118, 10609-10616. (14) Wightman, R. M.; Curtis, C.; Flowers, P. A.; Maus, R. G.; McDonald, E. M. J. Phys. Chem. B 1998, 102, 9991-9996. (15) Fan, F. F.; Cliffel, D.; Bard, A. J. Anal. Chem. 1998, 70, 2941-2948. (16) Zu, Y. B.; Ding, Z. F.; Zhou, J. F.; Lee, Y. M.; Bard, A. J. Anal. Chem. 2001, 73, 2153-2156. (17) Maus, R. G.; Wightman, R. M. Anal. Chem. 2001, 73, 3993-3998. (18) Szunerits, S.; Walt, D. R. Anal. Chem. 2002, 74, 1718-1723. (19) Szunerits, S.; Walt, D. R. ChemPhysChem 2002, 3, 101. 10.1021/ac034370s CCC: $25.00

© 2003 American Chemical Society Published on Web 07/25/2003

Figure 1. SEM images of (A) an etched and gold-coated fiber array (array 1) and (B) of an etched, gold-coated, and resin-insulated electrode array (array 2).

Figure 2. Schematic of the tip array used and the experimental set up of focusing only on the end of the tips. The diffusion layer is represented in light gray and the part of the diffusion layer which is analyzed in the focal plane of the microscope is represented in dark gray. (A) From left to right: Detection of the ECL signal in the focal plane at different increasing times of electrode polarization. The ECL spots, which are monitored, are displayed on the top of each figure (dark gray circles). (B) Evaluation of the ECL spot size dECL according to the geometry of the tip; δ is the thickness of the diffusion layer, ∆ is the focal depth, r and h are the radius and the height of the etched tip, respectively and θ is the cone angle.

To characterize the electrochemical behavior of the array at the apex of the electrodes via ECL, insulation between the tips is not necessary. Indeed, we demonstrate here that by focusing on the ends of the tips, diffusional decoupling between individual electrodes may be achieved simply by controlling the time scale of the experiment to adjust the size of the ECL zone. Only the ECL generated within the focal plane, which included only the electrode apexes (not the base of the electrode tip array), was detected by the CCD camera with proper focusing (Figure 2A). The novelty of this approach is that it is possible to collect spatially

resolved light generated through ECL and to correlate it with the electrochemical behavior of the array at the electrode tips. ECL can be generated in the presence of oxygen with good quantum yield via oxidation of Ru(bpy)32+ with aliphatic amines as coreactants.20,21 ECL generated in the presence of tri-npropylamine (TPrA) is well-documented,22-25 and significant effort (20) Noffsinger, J. B.; Danielson, N. D. Anal. Chem. 1987, 59, 865-868. (21) Leland, J. K.; Powell, M. J. J. Electrochem. Soc. 1990, 137, 3127-3131. (22) Knight, A. W.; Greenway, G. M. Analyst 1994, 119, 879. (23) Lee, W.-Y. Microchim. Acta 1997, 127, 19.

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has been put into understanding the mechanism of this reaction.20,21,26-29 It is now accepted that the reaction proceeds via oxidation of TPrA, which generates a strongly reducing species after deprotonation of the initial cation radical.30 Several routes have been investigated,26-29 but studies at Au and Pt surfaces26,27 have shown that a catalytic oxidation of TPrA is the dominant process at high concentrations of Ru(bpy)32+ (>0.1 mM) where Ru(bpy)33+ is reduced to Ru(bpy)32+* and emits light upon relaxation to its ground state (eqs 1-5).

Ru(bpy)32+ f Ru(bpy)33+ + e-

(1)

Ru(bpy)33+ + TPrA f Ru(bpy)32+ + TPrA+ kcat (2) TPrA+ f TPrA• + H+

(3)

Ru(bpy)33+ + TPrA• f Ru(bpy)32+* + products

(4)

Ru(bpy)32+* f Ru(bpy)32+ + hv λ ) 610 nm

(5)

We report the electrochemical luminescence of the Ru(bpy)32+/TPrA system at a nanotip array based on tapered and gold-covered optical fiber bundles. In this paper, we demonstrate that both redox and spectral information can be obtained from a substrate with the spatial resolution afforded by nanoelectrode tips. EXPERIMENTAL SECTION Materials. Ammonium fluoride, (NH4F, 99.99%), hydrofluoric acid (HF, 48 wt % in water, 99.99%) glacial acetic acid, potassium hexacyanoferrocyanide (K4Fe[CN]6), tris(2,2′-bipyridyl)ruthenium(II) chloride hexahydrate [Ru(bpy)32+], and tri-n-propylamine (TPrA) were obtained from Aldrich (Milwaukee, WI). Insulating epoxy (Araldite AM 136/Hy 994) was obtained from Ciba Geigy (East Lansing, MI). Conducting epoxy (epo-tek H20E) was purchased from Epoxy Technology (Billerica, MA). Tip array insulation was achieved using a cathodic electrophoretic paint (PPG, ZQ84-3225) and diluted with water (1:50) before use. The contact wires used were Kynar Insulated 30 awg wrapping wires (Allied Electronics, Texas). Silica imaging fibers of 5 cm length with a total diameter of 350 µm comprising 6000 individually clad 3-4-µm-diameter optical fibers were purchased from Sumitomo Electric Industries (IGN035/06). This bundle was coated with a silica jacket and a silicone resin in order to preserve mechanical strength. The fiber bundle was polished before use with 30-15-3-0.3-µm lapping films (General Fiber Optics, Fairfield, NJ). Instrumentation. The sputter coater used was a Fisons Instruments (Beverly, MA) Polaron SC502. The potentiostat used was a PGSTAT 30 Autolab (Ecochemie, Netherlands). All experi(24) Gerardi, R. D.; Bernett, N. W.; Lewis, S. W. Anal. Chim. Acta 1999, 378, 1. (25) Fa¨hnrich, K. A.; Pravda, M.; Guilbault, G. G. Talanta 2001, 54, 531-559. (26) Kanoufi, F.; Zu, Y. B.; Bard, A. J. J. Phys. Chem. B 2001, 105, 210-216. (27) Zu, Y. B.; Bard, A. J. Anal. Chem. 2000, 72, 3223-3232. (28) Gross, E. M.; Pastore, P.; Wightman, R. M. J. Phys. Chem. B 2001, 105, 8732-8738. (29) Miao, W. J.; Choi, J. P.; Bard, A. J. J. Am. Chem. Soc. 2002, 124, 1447814485. (30) Rubinstein, I.; Bard, A. J. J. Am. Chem. Soc. 1981, 103, 512.

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ments were performed using a Ag/AgCl reference electrode immersed in 1 M KCl solutions. The counter electrode was a platinum wire. For cyclic voltammetry studies, a low-current amplifier and a Faraday cage were used (Bioanalytical Systems, model C2). The instrumental set up for imaging and fluorescence measurements was a vertical arrangement of the system described in detail previously.31 The electrode array was fixed in a cell (V ) 1 mL) containing the counter and reference electrodes and mounted vertically on the stage of the imaging system (Olympus Microscope BX61 with a Prior-ProScan stage controller and Olympus camera U-CMAD3, Japan). The objective used was a 50× LMPlanFl with NA ) 0.50 and a working distance of 0.85 µm. No excitation wavelength was needed because Ru(bpy)32+ was excited electrochemically. The ECL at 610 nm generated by the electrode array was transmitted through a dichroic mirror and detected by a CCD camera (ORCA-C4742-95-12ER, Digital Camera, Hamamatsu). The potential was applied to the array in steps at 1.2 V/Ag/AgCl with a pulse duration varying between 0.1 and 1 s. Between pulses, rather than opening the circuit, a potential of 0.0 V/Ag/AgCl was applied to the array where no ECL reaction takes place, thereby allowing the system to relax diffusionally to its initial stage. Images were collected every 50 ms for 200 ms. One to twenty cycles (pulsing and reposing) were recorded. Images from multiple pulses were averaged to increase the optical resolution. SEM measurements were performed on a field emission scanning electron microscope FE-SEM (LEO 982, Thornwood, NY) located at Harvard University. Electrode Array Fabrication. Before etching, the imaging fiber was polished on both sides. The insulating jacket of the bundle was removed with dichloromethane before etching, and the top of the bundle was sonicated in water for 30 s to remove any residuals. The fiber bundle was etched by placing it into a mixture of 600 µL of 40% (w/w) aqueous NH4F solution, 100 µL of 48% HF solution, and 100 µL of deionized water. The side of the fiber to be etched was placed horizontal into the HF etching solution and left for 12 h. The etched bundle was sonicated and washed with water. Two series of arrays were prepared. In the first series (array 1), the etched bundle was covered on its etched end and sides with a gold layer ∼1 µm thick via sputter coating. Its SEM image is seen in Figure 1A. The height of the tips formed under these conditions is ∼4 µm. The gold on the side of the array was connected electrically to a copper wire through a silver paste, and the bundle was further insulated on the side with a fast drying insulating epoxy. Array 2 was designed and fabricated in the same way as array 1, but the tips were further insulated with an electrophoretic paint to obtain an array of submicroelectrodes.7,32 A potential of 2 V was applied for this purpose for two periods of 1 min each between the etched fiber bundle and a Pt coil, in which the fiber was centrally positioned. After each deposition step, the coated fibers were cured for 3 min at 180 °C to induce film shrinkage and expose the gold surface at the apex of each individual electrode (31) Bronk, K. S.; Michael, K. L.; Pantano, P.; Walt, D. R. Anal. Chem. 1995, 67, 2750. (32) Slevin, C. J.; Gray, N. J.; Macpherson, J. V.; Webb, M. A.; Unwin, P. R. Electrochem. Commun. 1999, 1, 282-288.

Figure 3. (A) Cyclic voltammogram of the gold-coated electrode tip array (array 1) and (B) of the resin insulated electrode array (array 2); 10 mM Fe(CN)64-/1 M KCl/water, scan rate v ) 0.1 V s-1.

Figure 4. ECL images monitored at an etched, gold-deposited and partially polymer-insulated electrode array (array 2). White circles indicate identified ECL spots. E ) 1.2 V/Ag/AgCl for 60 s, acquisition time 2 s, 2 mM Ru(bpy)32+/100 mM TPrA/phosphate buffer pH 7. The red color corresponds to the most intense ECL signal; green and blue correspond to little or no ECL signal.

(Figure 1B). Such arrays have been characterized in detail by SEM, AFM, and Raman confocal studies,7,32 and for further details, one is referred to these papers. RESULTS AND DISCUSSION Electrochemical Characterization. In the arrays, all electrodes are connected and operate at an identical potential. The electrochemical response depends on the relative sizes of the array’s characteristic dimensions, as compared to the thickness of the diffusion layer δ that develops at each element of the array. Distinct limiting situations can be encountered, depending on the geometry of the arrays and the duration of the experiment.33 Figure 3A shows a voltammogram recorded at 0.1 V s-1 using the electrode array 1 in an aqueous solution of ferrocyanide (10 mM). This array was obtained through chemical wet etching of a fused bundle of 6000 individual optical fibers followed by coating the distal end of the etched surface with a continuous layer of gold via sputter coating. Within this long time scale, such an array behaves as a macroelectrode, producing a voltammogram shape characteristic of planar diffusion. Indeed, at low scan rates, the diffusion length δ is large enough so that the interelectrode distance d is small compared to δ ) (DRT/Fv)1/2, where D is the diffusion coefficient and v is the voltammetric scan rate. The individual diffusion layers developing at adjacent tips interpen-

etrate, and this overlap creates an apparent planar diffusion layer that extends over the entire array.33,34 Under this condition, the array behaves like a large electrode whose surface area corresponds to the nominal area of the bundle before etching (i.e., an array without any tips). Figure 3B, on the other hand, shows a voltammogram obtained at the same scan rate but using array 2 (Figure 1B), in which the electrode elements were insulated from each other with an electrophoretic paint so as to produce an array of submicrometer electrodes. Diffusional overlap between these electrodes is minimized, as compared to array 1, and a steady-state cyclic voltammogram is thus observed. Indeed, the diffusion layer δ at each electrode of the array is sufficiently small vis-a`-vis the distance d between electrodes, but is large with respect to the electrode dimensions so that steady-state diffusion occurs at each electrode and depends on their shape.33,34 The total current is the arithmetic sum of each electrode’s individual current. The current thus depends both on the shape and number of active electrodes. By insulating the array, we observe a decrease in the overall electroactive surface area by ∼70%, as compared to array 1 (compare parts A and B of Figure 3). Electrochemiluminescence. Both electrode arrays were placed independently into a voltammetric cell consisting of Ru(bpy)32+/TPrA. A platinum plate counter electrode was placed

(33) Amatore, C. In Physical Electrochemistry; Rubinstein, I., Ed.; M. Dekker: New York, 1995; pp 131-208.

(34) Amatore, C.; Saveant, J. M.; Tessier, D. J. Electroanal. Chem. 1983, 147, 39-51.

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Figure 5. ECL images monitored at a gold-covered electrode array (array 1) at different polarization times θ by focusing at the apex of the tips. (A1-F1) images obtained from θ ) 1 to 0.1 s. (A2-F2), wider view of the areas shown in A1-F1, by white rectangles. White circles indicate identified ECL spots. E ) 1.2 V/Ag/AgCl; objective, 50× LWD; exposure time 200 ms (for 0.1 pulses 100 ms); 2 mM Ru(bpy)32+/100 mM TPrA/phosphate buffer, pH 7. The red color corresponds to the most intense ECL signal; green and blue correspond to little or no ECL signal.

parallel to the device to ensure that no reflected light from the platinum surface would be collected by the CCD camera.4 Ru(bpy)32+ was oxidized by stepping the potential from 0 V/Ag/ AgCl, at which point no electrochemical reaction takes place, to 1.20 V/Ag/AgCl at which oxidation of Ru(bpy)32+ and the catalytic oxidation of TPrA occur. After a potential of 1.2 V/Ag/AgCl had been applied for 60 s, the ensuing ECL emission (550-650 nm) generated at the tips of the array was collected at 610 nm by the high-sensitivity CCD camera by focusing on the tip plane (compare Figure 2A). Figure 4 shows the images at two representative regions of array 2. Bright spots (highlighted by the white circles) correspond to the regions where electroactive tips are located. From Figure 4, it is observed that the zones giving rise to intense light emission remain confined around each tip, even after a long polarization time of 60 s. These results demonstrate further that the interelectrode distance d is large relative to the size of the electrodes to ensure that no significant diffusional overlap occurs, 4386

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even at times as long as 60 s. This result is in accordance with the steady-state voltammogram of Figure 3B, which corresponds to a lower characteristic time scale of about RT/Fv ≈ 0.25 s.35 In addition, these images provide information about the actual size of the electrodes. Indeed, the ECL generating zone is necessarily restricted to a zone that is comparable to the electrode dimension as a result of the spherical/hemispherical nature of diffusion at a microelectrode.36 If the active tips are considered to be equivalent to hemispherical electrodes, the specific diffusion length δ of Ru(bpy)32+ can be determined at steady state by the following equation,26,37,38

δ ) [1/r + (2kcat[TPrA]0/D)1/2]-1

(6)

where r is the electrode equivalent radius, kcat is the apparent rate constant of reaction 2 and D is the diffusion coefficient of

Ru(bpy)32+/3+. On one hand, the rate constant kcat, which is related to the overall concentration [TPrA]0 of TPrA, can be evaluated from knowledge of the acidity constant of TPrA (pKa ) 10.4) and from the intrinsic value of the rate constant, k ) 1.3 × 107 M-1 s-1, determined for unprotonated TPrA.26 Since in our experimental conditions, tri-n-propylamine is nearly completely protonated (i.e., TPrA is almost totally in its inactive form TPrAH+, which does not participate in reaction 2, kcat is evaluated as kcat ) k/(1 + 10-pH+pKa) which leads to kcat ) 5 × 103 M-1 s-1 at pH ) 7 under our conditions. On the other hand, δ can be estimated from the size of the ECL spots if one assumes to a first approximation (compare Figure 2A) that the spot size is equivalent to 2δ (i.e., 0.5-1 µm; see Figure 4). Thus, taking D ) 5 × 10-6 cm2 s-1, eq 6 affords an estimate of the equivalent radius of the tip electrodes in the array which is r ) (1 ( 0.5) µm. This value appears reasonable according to the SEM image of the partially insulated array (compare Figure 1B) and validates a posteriori the approximations made in deriving eq 6 and estimating kcat. One observes from Figure 4 that not all tips are active, because there are regions where no ECL is observable. This feature is an important limitation in the use of electrophoretic paints for generating nanotips,7,32 since the process cannot be easily controlled and results in parts of the array being completely covered with paint. Under our experimental conditions, a small tilt angle between the surface of the array and the focal plane of the microscope may also partially explain these results, because it is possible that not all of the electrode tips were coplanar because of imperfect etching (compare Figure 1B). The same type of experiment was carried out with array 1, in which the gold-covered array surface was not further insulated. In this experiment, we focused on the very apex of the tip electrodes (Figure 2A). ECL collected by the CCD camera at the focal plane is then discriminated from ECL emitted at the base of the tips. This experiment showed that after 60 s, the entire surface of the array displayed an ECL signal (data not shown) where no individual ECL sites could be visualized. With such a long polarization time, this result is in accordance with the planar diffusion behavior observed voltammetrically in Figure 3A. To create the situation in which an ECL generation zone could be distinguished around each electroactive zone at the tips, short potential pulses were applied. Indeed, if the time scale θ of the experiment is sufficiently short such that the diffusion length δ is extremely small with respect to the dimension of the electrode tips and if the interelectrode spacing d is large compared to δ developed, no diffusional overlap between the ECL spots is expected33,34 (Figure 2A). Short potential pulses θ were applied with θ ranging from 0.1 s to 1 s. After each pulse, a potential plateau at 0.0 V/Ag/AgCl was applied for 1.2 s, during which no ECL reaction took place and during which the system had time to relax diffusionally before a new cycle ensued. The CCD camera was used to record images of the ECL reaction on the array by opening the shutter every 50 ms for 200 ms for pulses of 0.2-1-s duration and every 50 ms for 0.1-s pulse durations. Figure 5 shows a series of images obtained on the same zone of the electrode (35) Bard, A. J.; Faulkner, L. R. Electrochemical Methods, 2nd ed.; Wiley: New York, 2001. (36) Amatore, C.; Fosset, B. Anal. Chem. 1996, 68, 4377-4388. (37) Delmastro, J. R.; Smith, D. E. J. Phys. Chem. 1967, 71, 2138. (38) Diao, G. W.; Zhang, Z. X. J. Electroanal. Chem. 1997, 429, 67.

Figure 6. Simulated propagation of the diffusion layer thickness δ of Ru(bpy)32+ versus time for the catalytic scheme defined by reactions 1 and 2 under planar diffusion; 2 mM Ru(bpy)32+/100 mM TPrA/phosphate buffer, pH 7. See text for the other parameters used.

array surface for pulse durations θ between 0.1 and 1 s with array 1. The red color corresponds to the most intense ECL signal; green and blue correspond to the areas where little or no ECL signal was observed. Decreasing the pulsing time from 1 s (Figure 5A1), to 0.8 s (Figure 5B1), 0.4 s (Figure 5C1), 0.3 s (Figure 5D1), 0.2 s (Figure 5E1), and finally, to 0.1 s (Figure 5F1) shows that the sizes of the diffusion layers are decreasing, as evidenced by the noticeable decrease of ECL spreading. For shorter pulses, one reaches the situation when the size of the diffusion layer δ is comparable to the interelectrode distance d (i.e., δ ≈ d, Figure 5E1) and then even smaller than d (i.e., δ < d, Figure 5F1). In these images, one can easily identify the hexagonal structure of the fiber bundle where the active tips are highlighted by white circles. Furthermore, the interelectrode distance d matches very well with the 6-µm center-to-center spacing of the fiber bundle before chemical etching. Accordingly, these results validate the idea that one can characterize an array of active tips at the micrometer scale with spatially resolved ECL images by decoupling the diffusion layers around each element using short time scale measurements. The diffusion length δ of Ru(bpy)32+ in the presence of TPrA can be evaluated by digital simulation using Digisim software. Assuming at short times diffusion above the tip surface is mainly planar (Figure 2B), simulation of δ versus time was performed according to the catalytic scheme defined by reactions 1 and 2 using values of kcat and D discussed above. The heterogeneous rate constant of reaction 1 was adjusted to ks1 ) 0.01 cms-1.29 Simulated data in Figure 6 show that after an initial fast rise of a few milliseconds, δ increases with time and approaches a nearby constant slope up to the point where δ becomes comparable to one-half the center-to-center spacing d between the individual tips (∼ 3 µm). In such a situation, the calculations are no longer relevant, since individual diffusion layers around each tip necessarily overlap, and diffusion is no longer planar locally, so Figure 6 is only indicative at longer times. A more quantitative discussion should take into account the specificity of diffusion at the conical electrodes;39 however, for our purposes, Figure 6 is sufficient to illustrate qualitatively the phenomenon. The size of the ECL spots (39) Zoski, C. G.; Mirkin, M. V. Anal. Chem. 2002, 74, 1986-1992.

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Figure 7. Accumulation of ECL images at a gold-covered array (array 1) by pulsing the potential from E1 ) 1.2 V/Ag/AgCl for 50 ms to E2 ) 0 V/Ag/AgCl for 1 s. (A) 1 frame, (B) 3 frames, (C) 20 frames. Objective, 50× LWD; exposure time 50 ms; 2 mM Ru(bpy)32+/100 mM TPrA/ phosphate buffer, pH 7. The red color corresponds to the most intense ECL signal; green and blue correspond to little or no ECL signal.

recorded on the CCD camera can be estimated from this simulation by considering the tip geometry described in Figure 2B. To a first approximation, this size dECL can be estimated by dECL ) 2(∆ tan θ + δ/cos θ), where ∆ is the focus depth at the level of the electrode tips where light is collected. According to our experimental conditions and considering that ∆ ) (2 µm, a calculation of dECL shows that no overlap between ECL spots is expected as long as θ is lower than 0.4 s (i.e., as long as dECL < d; compare Figure 2A). At this time scale, convection can neither alter nor control significantly the development of the diffusion layer, including diffusion of TPrA.40 This result is in perfect agreement with our experimental observations, where individual spots can be identified for θ ) 0.1-0.2 s (Figs. 5D1-F1). Figure 5A2-F2 shows a wider view of the areas shown in A1F1 with the white rectangle on each image corresponding to Figure 5A1-F1. It can be seen that ECL is not observed at all in the tips over this larger surface, especially on the right-hand side of each image. Even when the tips were not insulated with an electrophoretic paint, there still may not be an observable ECL signal. This result may be due to the array’s being incompletely covered with gold or that the gold film thickness is not homogeneous, leading to a local increase in the ohmic drop and no ECL generation. In addition, part of the array may be passivated by an adsorbed organic layer. If one considers that there is a tilt angle between the array surface and the focal plane, part of the array may be out of focus in our experiments. Finally, if the array tips are not located in the focal plane because of variability in the efficiency of the etching procedure, smaller tips may be ECLactive, but their activity will not be observable. Image Addition of Pulse Experiments. At very short pulse times (e.g., 50 ms), the signal of the ECL was not strong enough to be clearly identified above the noise on the surrounding surface. Signal averaging was therefore necessary to obtain information about the diffusion layers generated around the electrode tips of the array. A potential of 1.2 V/Ag/AgCl was applied to the array for 50 ms to initiate the ECL reaction followed by a potential step at 0.0 V for 1 s, during which no ECL reaction takes place. This cycle was repeated 20 times, and the ECL pattern was recorded separately for each cycle by the CCD camera and stored as a (40) Amatore, C.; Szunerits, S.; Thouin, L.; Warkocz, J.-S. J. Electroanal. Chem. 2001, 500, 62-70.

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separate image file. The CCD images were then backgroundcorrected individually, and the intensities of each of these corrected frames were added, pixel by pixel. As seen in Figure 7, the accumulation of only three frames (Figure 7B) was sufficient to improve the signal-to-noise ratio significantly. To obtain a sufficiently good S/N and monitor the signals on the tip of the array, it was necessary to sum 20 frames (Figure 7C) to obtain an image with acceptable resolution. CONCLUSION We prepared nanoelectrode arrays of 350-µm total diameter via chemical etching of bundles consisting of 6000 individual optical fibers and used them to investigate the electrochemical behavior at the apex of the electrodes via ECL. The ECL images obtained for the partially insulated array 2 provided information about the equivalent radius, r, of the electrochemically active tips, which was determined to be ∼0.5-1.5 µm. It was further demonstrated that diffusional decoupling between individual electrodes in an array could be achieved without the need to insulate the array. The diffusion length δ could be adjusted simply by controlling the time scale of the experiment. As evidenced by pulsing experiments with different pulse lengths, the size of the diffusion layer could be varied from the regime where diffusion layers interpenetrate and the array behaved like a continuous planar macroelectrode to the regime where the response of the array was the sum of the individual responses of the electrodes. These results demonstrate that an electrochemical array based on metallized nanotips can be used to obtain spatially resolved information at the tip level as long as diffusional decoupling between the electrochemical processes induced around each tip are achieved. Since optical fiber bundles were used as the array template, it should be possible to simultaneously acquire both electrochemical and optical information from analytical samples over short time scales. Research in this direction is currently underway. ACKNOWLEDGMENT We thank J. V. Macpherson for providing us with a sample of the electrophoretic paint. Received for review April 10, 2003. Accepted May 30, 2003. AC034370S