Development of an Ordered Array of Optoelectrochemical Individually

Dec 3, 2003 - a new “electroptode” immunosensor to detect cholera antitoxin antibodies.19,20 They have modified an optical fiber with indium tin o...
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Anal. Chem. 2004, 76, 357-364

Development of an Ordered Array of Optoelectrochemical Individually Readable Sensors with Submicrometer Dimensions: Application to Remote Electrochemiluminescence Imaging Arnaud Chovin,† Patrick Garrigue,† Philippe Vinatier,‡ and Neso Sojic*,†

Laboratoire d’Analyse Chimique par Reconnaissance Mole´ culaire, Universite´ Bordeaux I, ENSCPB, 16 avenue Pey-Berland, 33607 Pessac, France, and Institut de Chimie de la Matie` re Condense´ e de Bordeaux, CNRS and ENSCPB, 16 avenue Pey-Berland, 33607 Pessac, France

A novel array of optoelectrochemical submicrometer sensors for remote electrochemiluminescence (ECL) imaging is presented. This device was fabricated by chemical etching of a coherent optical fiber bundle to produce a nanotip array. The surface of the etched bundle was sputter-coated with a thin layer of indium tin oxide in order to create a transparent and electrically conductive surface that is insulated eventually by a new electrophoretic paint except for the apex of the tip. These fabrication steps produced an ordered array of optoelectrochemical sensors with submicrometer dimensions that retains the optical fiber bundle architecture. The electrochemical behavior of the sensor array was independently characterized by cyclic voltammetry and ECL experiments. The steady-state current indicates that the sensors are diffusively independent. This sensor array was further studied with a co-reactant ECL model system, such as Ru(bpy)32+/TPrA. We clearly observed an ordered array of individual ECL micrometer spots, which corresponds to the sensor array structure. While the sensors of the array are not individually addressable electrochemically, we could establish that the sensors are optically independent and individually readable. Finally, we show that remote ECL imaging is performed quantitatively through the optoelectrochemical sensor array itself. Ultramicrolectrodes and optical fibers are well-established tools in analytical and bioanalytical chemistry.1,2 Both provide high spatial resolution and allow us to probe concentrations and concentration profiles on micrometer to nanometer scales with good selectivity depending on the detection scheme.3-7 The ability to intimately merge these tools, which are based on different basic * Corresponding author. e-mail: [email protected]. † Laboratoire d’Analyse Chimique par Reconnaissance Mole ´ culaire. ‡ Institut de Chimie de la Matie ` re Condense´e de Bordeaux. (1) Amatore, C. In Physical electrochemistry; Rubinstein, I., Ed.; M. Dekker: New York, 1995; p 131. (2) Walt, D. R. Acc. Chem. Res. 1998, 31, 267-278. (3) Zu, Y.; Ding, Z.; Zhou, J.; Lee, Y.; Bard, A. J. Anal. Chem. 2001, 73, 21532156. (4) Maus, R. G.; Wightman, R. M. Anal. Chem. 2001, 73, 3993-3998. 10.1021/ac034974w CCC: $27.50 Published on Web 12/03/2003

© 2004 American Chemical Society

principles, offers the opportunity to acquire various and complementary information on complex dynamic microenvironments. Furthermore, this approach allows the development of miniaturized probes for ECL, which is a sensitive and selective detection method having important applications in bioanalytical science. A variety of optoelectrochemical devices based on optical fibers has thus been developed. Ring microelectrodes were fabricated initially by coating a single optical fiber with gold or other electrically conductive materials.8-12 Similar photoelectrochemical devices have been applied to study reactions at the surface of a TiO2/Ti disk13 andtocombinescanningelectrochemical/opticalmicroscopy.14-16 Single optical fibers embedded into a “cage”17 or modified with a minigrid18 have also been reported. Marks et al. have developed a new “electroptode” immunosensor to detect cholera antitoxin antibodies.19,20 They have modified an optical fiber with indium tin oxide (ITO) in order to electropolymerize biotin-pyrrole monomers and to be able to detect the chemiluminescence emitted by the luminol/hydrogen peroxide/peroxidase system through the modified optical fiber.19,20 Szunerits and Walt have fabricated a random assembly of optoelectrochemical microring electrodes.21 Individually gold-coated optical fibers have been randomly arranged, embedded in an insulating resin and then a fraction of the fibers have been connected. Pantano et al. have (5) Amatore, C.; Szunerits, S.; Thouin, L.; Warkocz, J.-S. Electroanalysis 2001, 13, 646-652. (6) Rosenzweig, Z.; Kopelman, R. Anal. Chem. 1995, 67, 2650-2654. (7) Tan, W.; Shi, Z.-Y.; Kopelman, R. Anal. Chem. 1992, 64, 2985-2990. (8) Cohen, C. B.; Weber, S. G. Anal. Chem. 1993, 65, 169-175. (9) Kuhn, L. S.; Weber, A.; Weber, S. G. Anal. Chem. 1990, 62, 1631-1636. (10) Pennarun, G. I.; Boxall, C.; O’Hare, D. Analyst 1996, 121, 1779-1788. (11) Van Dyke, D. A.; Cheng, H. Y. Anal. Chem. 1988, 60, 1256-1260. (12) Smith, P. J. S.; Haydon, P. G.; Hengstenberg, A.; Jung, S.-K. Electrochim. Acta 2001, 47, 283-292. (13) Casillas, N.; James, P.; Smyrl, W. H. J. Electrochem. Soc. 1995, 142, L16L18. (14) Lee, Y.; Amemiya, S.; Bard, A. J. Anal. Chem. 2001, 73, 2261-2267. (15) Lee, Y.; Bard, A. J. Anal. Chem. 2002, 74, 3626-3633. (16) Lee, Y.; Ding, Z.; Bard, A. J. Anal. Chem. 2002, 74, 3634-3643. (17) VanDyke, D. A.; Cheng, H. Y. Anal. Chem. 1989, 61, 633-636. (18) Wang, H.; Xu, G.; Dong, S. Electrochem. Commun. 2002, 4, 214-217. (19) Marks, R. S.; Novoa, A.; Konry, T.; Krais, R.; Cosnier, S. Mater. Sci. Eng. C 2002, 21, 189-194. (20) Konry, T.; Novoa, A.; Cosnier, S.; Marks, R. S. Anal. Chem. 2003, 75, 26332639.

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demonstrated the electrochemical modulation of remote fluorescence22 and ECL23 at an imaging fiber electrode chemical sensor. In brief, a coherent optical fiber bundle was first gold-coated to serve as a macroelectrode and then modified with a thin layer of Nafion doped with a redox fluorophore22 or with an ECL reagent.23 Very recently, we have described the fabrication and the characterization by confocal Raman microspectroscopy of a submicrometer electrode array.24 Such an array was prepared through the etching of an optical fiber bundle, coating it with a gold layer of ∼1-µm thickness and eventually insulating the fiber tips with an electrophoretic paint. This approach, however, presented a major drawback because light could not be transmitted through the gold layer, i.e., via the imaging fiber. This obviously prevented the development of an optoelectrochemical sensor array. In this work, we continue and extend our previous approach24 by developing an ordered array of optoelectrochemical submicrometer sensors that is applied to remote ECL imaging. The first fabrication step also involves the chemical etching of an optical fiber bundle comprising 6000 individually cladded 3-4-µmdiameter optical fibers. Etching of the glass core produces the typical conical shape of the tip apex. Conductive coating is essential to create the electrode surface. To overcome the problem related to light transmission through the electrically conductive surface, we substituted the gold layer with a transparent conductive ITO film. This oxide is a good electrode material for this application since it is stable at the relatively high potential required for ECL generation with the tris(2,2′-bipyridine)ruthenium(II)/ tri-n-propylamine (TPrA) system. Furthermore, ITO films offer high transmission in the visible region and low electrical resistivity. Thus they have been extensively used in a variety of optoelectronic and spectroelectrochemical applications.25,26 Numerous techniques have been reported for ITO deposition such as radio frequency (rf) sputtering, dc field sputtering, electron or ion beam deposition, chemical vapor deposition, and chemical solution deposition.25,26 ITO electrochemical properties are highly dependent on the deposition and postdeposition conditions.27 In this study, we have chosen to use rf magnetron sputtering since it is a well-known method for preparing high-quality ITO films. After ITO deposition, we have been able to reproducibly insulate the surface of the etched bundle with a new electrophoretic paint except for the apex of the tip (Figure 1). This produced a regularly spaced array of submicrometer transparent electrodes that retains the optical fiber bundle architecture and allows transmission of the electrochemically generated light at the tip through the corresponding core. Figure 1A represents a part of the modified imaging fiber. Furthermore, it illustrates the high density and very large number of electrodes (6000) in the array that enables the parallel acquisition of large amounts of data. The array is characterized independently by cyclic voltammetry and ECL experiments. Finally, we show that this ordered array of optoelectrochemical (21) Szunerits, S.; Walt, D. R. Anal. Chem. 2002, 74, 1718-1723. (22) Khan, S. S.; Jin, E. S.; Sojic, N.; Pantano, P. Anal. Chim. Acta 2000, 404, 213-221. (23) Jin, E. S.; Norris, B. J.; Pantano, P. Electroanalysis 2001, 13, 1287-1290. (24) Szunerits, S.; Garrigue, P.; Bruneel, J.-L.; Servant, L.; Sojic, N. Electroanalysis 2003, 15, 548-555. (25) Wu, W.-F.; Chiou, B.-S. Thin Solid Films 1994, 247, 201-207. (26) Lewis, B. G.; Paine, D. C. MRS Bull. 2000, 25, 22-27. (27) Popovich, N. D.; Wong, S.-S.; Yen, B. K. H.; Yeom, H.-Y.; Paine, D. C. Anal. Chem. 2002, 74, 3127-3133.

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Figure 1. (A) Scanning electron micrograph and (B) schematic illustration (side view) of the sensor array.

sensors is able to generate ECL, to transmit it via the optical fiber bundle, and to detect the remote ECL radiation with a micrometer resolution. EXPERIMENTAL SECTION Materials. Ammonium fluoride (99.99%), hydrofluoric acid (99.99%), potassium chloride, hexaamineruthenium(III) chloride, tris(2,2′-bipyridine)ruthenium(II) chloride, tri-n-propylamine, phosphate-buffered saline (PBS; pH ) 7.5) were obtained from Aldrich. Sodium sulfate (99%) and acetic acid were from Prolabo. The insulating varnish (Monoliss’ Satin Blanc 01) was obtained from ICI Dulux Valentine. The cathodic electrophoretic paint (BASF FT83-0250) was a gift from BASF. Contacts were made with High Purity Silver paint (SPI, West Chester, PA). The contact wires used were Kynar Insulated 30 awg wrapping wires (RS). The epoxy resin used was Plexcil C6 and A6 purchased from Escil. All aqueous solutions were prepared with Milli-Q purified water (Millipore). Instrumentation. The potentiostat used was a PGSTAT 12 Autolab (Eco Chemie). All experiments were performed using a Ag/AgCl reference electrode or a Ag quasi-reference electrode. The counter electrode was a platinum wire. The spectroelectrochemical cell used to acquire images of Figure 3 was similar to the one previously described.24 The distal face of the array immersed into the Ru(bpy)32+/TPrA solution was

positioned in front of the microscope/CCD camera. The cell consisted of a circular Kel-F sheet. The quasi-reference and counter electrodes, Ag and Pt, respectively, were mounted in a linear arrangement and the assembly was held in position by casting it into an epoxy resin, which was centered vertically in the middle of the cell, and whose top received a screw cap bearing a 100-µm-thick glass window, parallel to the rod surface and the microscope objective. In the middle of these electrodes, a 1-mm hole was drilled, where the sensor array was introduced in such a way that the connections were on one side and the active surface was in the same plane as the other electrodes. The varnish and the copper wire (see below) seal the gap around the imaging bundle. The array is positioned in the cell with a three-axis submicron manipulator (MDT616, Thorlabs) and a goniometer (Melles Griot). Images of Figure 4 were acquired through the optical fiber bundle. In other words, ECL is generated at the distal face of the bundle and is detected at the proximal face of the bundle with a microscope/CCD camera setup. The instrument used for ECL imaging was a modified epifluorescence microscope (BX-30, Olympus) similar to the one described previously.28 ECL light transmitted through the imaging fiber was collected by a 40× microscope objective, filtered by a band-pass filter (605 ( 25 nm) and detected by a two-dimensional detector, such as a charge-coupled device (CCD) camera (Roper Scientific). Therefore, ECL intensities of all the sensors forming the array are captured simultaneously and individually at a given time in a single image. The ECL images were acquired by a CCD camera, which is fitted with a back-illuminated chip (Marconi 4710) that has 1024 × 1024 pixels. Maximum pixel readout rate is 1 MHz at 16 bit. The camera head cools the chip thermoelectrically to -40 °C and has a shutter. To increase the CCD camera dynamics and thus to obtain a better temporal resolution, a region of interest was selected from the complete image. A computer controls the camera and collects and processes all the 16-bit coded images. SEM images were obtained with a scanning electron microscope (JEOL). A magnetron rf-sputter coater (Alcatel SCM 451) was used for ITO deposition on the etched imaging fiber. Sensor Array Fabrication. Silica imaging fibers of 8-cm length with a total diameter of 350 µm comprising 6000 individually cladded 3-4-µm-diameter optical fibers were purchased from Sumitomo Electric Industries (IGN-035/06). The active area and the numerical aperture (NA) of the coherent optical fiber bundle were 270 µm and 0.35, respectively. The distal face of the array was polished before use with 30-15-3-0.3-µm lapping films (Thorlabs). The etching of the fibers was accomplished through the combination of several etching methods reported in the literature.29,30 The HF solution used for etching was prepared by mixing 500 µL of 40% (wt/wt) aqueous NH4F solution, 100 µL of a 48% HF solution, and 100 µL of deionized water. (Caution: HF etching solutions are extremely corrosive!) The insulating jacket of the bundle was removed with chloromethane before etching and sonicated in water for 30 s to remove any residuals. The polished side was placed horizontal into the HF etching solution and left for 5 h. The etched bundle was then washed with water. (28) Bronk, K. S.; Michael, K. L.; Pantano, P.; Walt, D. R. Anal. Chem. 1995, 67, 2750-2757.

The nanotip array was covered on the distal end and the sides with an ITO layer through rf sputtering. The sputtering target was a 100-mm-diameter powder disk with 99.99% purity containing In2O3 and SnO2 90/10 wt % (Cerac). The fiber bundle was placed at a distance of 5 cm from the target. Before deposition, the chamber pressure was reduced to 2 × 10-6 mbar. The samples were deposited in a mixture of argon and oxygen. Oxygen was first introduced in the chamber to a pressure of 10-5 mbar. Argon was then added, and the total pressure was set to 10-2 mbar. A rf power of 120 W was applied to the target. Before deposition, the target was presputtered for 30 min to remove any contaminants. The nanotip array was exposed to the sputter beam for 1 h. The ITO-coated nanotip array was electrically connected to a copper wire with a silver paste and mechanically insulated on the side with a varnish. The only electroactive surface was then the etched surface of the fiber bundle. We adapted a procedure described in the literature31,32 to insulate the etched imaging fiber except the tip apex by deposition of a cathodic electrophoretic paint. The initial paint solution was diluted in a 1:1 (v/v) ratio with a 3 mM acetic acid aqueous solution. The deposition of the cathodic electrophoretic paint occurs by depletion of protons at the cathode by applying a potential between the ITO-coated nanotip array and anode (Cu coil). The resulting increase in pH destabilizes the paint emulsion, and it induces the precipitation of an insulating film onto the cathode surface. To ensure complete wetting of the probe’s surface, the sample was immersed and withdrawn at least three times before the coating procedure was started. The deposition of a single insulating layer was carried out by scanning the potential from 0 to -2 V/Ag/AgCl at 50 mV/s. At this stage, the fibers were completely insulated. However, curing the fibers at 180 °C for 1 h induces the shrinking of the deposited film. The whole surface of the sensor array is then insulated except the tip apex (Figure 1). RESULTS AND DISCUSSION Steady-State Voltammetric Characterization of the Sensor Array. Electrochemical properties of the sensor array can be studied by cyclic voltammetry in a qualitative and quantitative way.1,33 Figure 2 shows the reduction of hexaamineruthenium(III), Ru(NH3)63+, at the array before (A) and after (B) insulating the nanotips. Ru(NH3)63+/2+ was selected as a redox probe since it is a nonadsorbing species with a very high electron-transfer rate.34 The electrochemical behavior of the ITO-coated nanotip array shows a classical macroelectrode transient response (Figure 2A). Therefore, the sputtered ITO film demonstrates an excellent electrochemical performance (Figure 2). The surface of the etched and ITO-coated bundle was then insulated with an electrophoretic paint31 except for the apex of the tip (Figure 1B). We used a new cathodic paint32 with optimized deposition conditions. With our previous procedure,24 a part of the array was totally covered with the electrophoretic paint, while the array was homogeneously (29) Pantano, P.; Walt, D. R. Rev. Sci. Instrum. 1997, 68, 1357-1359. (30) Liu, Y.-H.; Dam, T. H.; Pantano, P. Anal. Chim. Acta 2000, 419, 215-225. (31) Slevin, C. J.; Gray, N. J.; Macpherson, J. V.; Webb, M. A.; Unwin, P. R. Electrochem. Commun. 1999, 1, 282-288. (32) Abbou, J.; Demaille, C.; Druet, M.; Moiroux, J. Anal. Chem. 2002, 74, 63556363. (33) Lee, H. J.; Beriet, C.; Ferrigno, R.; Girault, H. H. J. Electroanal. Chem. 2001, 502, 138-145. (34) Conyers, J. L.; White, H. S. Anal. Chem. 2000, 72, 4441-4446.

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Figure 2. Cyclic voltammograms of the nanotip array coated with ITO before (A) and after (B) the insulation step with an electrophoretic paint in an aqueous 5 mM Ru(NH3)63+ solution containing 1 M Na2SO4; scan rate 50 (A) and 5 mV s-1 (B).

coated in this work (Figure 1A). This new procedure leads to better reproducibility and uniformity of the paint deposition on the tips. Figure 2B shows the voltammogram recorded with the electrophoretically coated array. The shape and the amplitude of the electrochemical signal are modified. The voltammetric response is sigmoidal in shape, indicating that nearly pure radial diffusion conditions were established. This feature implies that the ultramicroelectrodes forming the array have sufficiently small dimensions and are sufficiently spaced that the individual diffusion layers do not overlap.1,33 This result is in agreement with previous work where we have demonstrated by confocal Raman microspectroscopy that the submicrometer electrodes were diffusively independent.24 Moreover, the magnitude of the faradaic current is decreased since the electroactive area was decreased. Assuming that the exposed tips have all the same hemispherical shape (Figure 1), the plateau current, ilim, is related to the average apparent radius of a single ultramicroelectrode of the array, rav, by eq 1, with n the number of electrons transferred per molecule

ilim ) 2πnFDC*ravNtips

(1)

(n ) 1), F the Faraday constant, D is the diffusion coefficient of Ru(NH3)63+ (D ) 7.1 × 10-6 cm2 s-1),35 C* its known bulk concentration (C* ) 5 mM), and Ntips the number of active ultramicroelectrodes in the array (Ntips ) 6000). From the typical steady-state voltammogram represented in Figure 2B, an apparent average electrode radius in the range of 300 nm could be estimated using the hemispherical approximation. Recently, Zoski and Mirkin simulated the influence of conical microelectrode geometry surrounded by a finite insulating layer on the steady(35) Chailapakul, O.; Crooks, R. M. Langmuir 1995, 11, 1329-1340.

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state diffusion-limiting current.36 From their model, we calculated that the underestimation of the tip radius resulting from the hemispherical geometry is less than 12%. This size is consistent with the diffusively independent behavior of the submicrometer electrodes forming the array.1,33 With the electrophoretic paint used in our previous work,24 a stable steady-state limiting current could not be achieved at potentials more negative than -0.3 V/Ag. Indeed, the insulating paint dissolves in this negative potential window, and it leads to progressive exposure of the full electrode surface. In this study, we insulated the array with a new cathodic electrophoretic paint known in the literature for its very large potential range.32 Indeed, continuous cycling of the array in the negative potential range does not affect the shape and the magnitude of the steady-state response. The coating is much more stable and allows us to exploit, in the future, electrochemical and ECL systems in the negative potential domain. ECL Imaging of the Sensor Array. In this part, we want to characterize our sensor array by ECL imaging. Luminescence imaging has been widely used in electrochemistry. For example, gold minigrid electrodes37 and arrays of band ultramicroelectrodes38 have been visualized by fluorescence imaging. ECL imaging has been applied to provide valuable information at the submicrometer scale about various electrochemical phenomena and very small electrodes.39-48 During the preparation of this paper, Walt et al. reported spatially resolved ECL on gold-coated optical fiber bundles.49 Using ECL imaging of the etched bundle’s distal face, they demonstrated that diffusional decoupling between individual electrodes of the array may be achieved by adjusting the size of the diffusion layer through pulsing experiments. ECL has also been used in order to create submicrometer light sources for scanning optical microscopy.3,4,50 An aqueous co-reactant ECL model system, i.e., Ru(bpy)32+/TPrA, was selected to further characterize our optoelectrochemical array. One important feature for this application is that it provides high light intensity. At high concentrations of Ru(bpy)32+ (>0.1 mM), the “catalytic route” (also called EC′ route) is the dominant process for ECL.51-53 Along this path, the catalytic oxidation of TPrA occurs by a reaction with (36) Zoski, C. G.; Mirkin, M. V. Anal. Chem. 2002, 74, 1986-1992. (37) Engstrom, R. C.; Ghaffari, S.; Qu, H. Anal. Chem. 1992, 64, 2525-2529. (38) Fiedler, S.; Hagedorn, R.; Schelle, T.; Richter, E.; Wagner, B.; Fuhr, G. Anal. Chem. 1995, 67, 820-828. (39) Bowling, R. J.; McCreery, R. L.; Pharr, C. M.; Engstrom, R. C. Anal. Chem. 1989, 61, 2763-2766. (40) Engstrom, R. C.; Johnson, K. W.; DesJarlais, S. Anal. Chem. 1987, 59, 670673. (41) Petersen, S. L.; Weisshaar, D. E.; Tallman, D. E.; Schulze, R. K.; Evans, J. F.; DesJarlais, S. E.; Engstrom, R. C. Anal. Chem. 1988, 60, 2385-2392. (42) Pharr, C. M.; Engstrom, R. C.; Tople, R. A.; Bee, T. K.; Unzelman, P. L. J. Electroanal. Chem. 1990, 278, 119-128. (43) Pharr, C. M.; Engstrom, R. C.; Klancke, J.; Unzelman, P. L. Electroanalysis 1990, 2, 217-221. (44) Engstrom, R. C.; Pharr, C. M.; Koppang, M. D. J. Electroanal. Chem. 1987, 221, 251-255. (45) Pantano, P.; Kuhr, W. G. Anal. Chem. 1993, 65, 2452-2458. (46) Hopper, P.; Kuhr, W. G. Anal. Chem. 1994, 66, 1996-2004. (47) Wightman, R. M.; Curtis, C. L.; Flowers, P. A.; Maus, R. G.; McDonald, E. M. J. Phys. Chem. B 1998, 102, 9991-9996. (48) Maus, R. G.; McDonald, E. M.; Wightman, R. M. Anal. Chem. 1999, 71, 4944-4950. (49) Szunerits, S.; Tam, J. M.; Thouin, L.; Amatore, C.; Walt, D. R. Anal. Chem., in press. (50) Fan, F.-R. F.; Cliffel, D.; Bard, A. J. Anal. Chem. 1998, 70, 2941-2948. (51) Zu, Y.; Bard, A. J. Anal. Chem. 2000, 72, 3223-3232. (52) Kanoufi, F.; Zu, Y.; Bard, A. J. J. Phys. Chem. B 2001, 105, 210-216.

Figure 3. Images acquired with the sensor array positioned in front of the microscope objective (40×) and immersed in a 0.15 M PBS (pH ) 7.5) solution containing 1 mM Ru(bpy)32+ and 100 mM TPrA. (A) Reflection visible light image focused on a region of interest of the array surface before applying the potential. (B) ECL image of the same region of interest after application of 1.2 V/Ag. The CCD exposure time was 5 s. Black represents low light intensity.

electrogenerated Ru(bpy)33+. The ECL route can be described by the following reaction scheme:51,52

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

(2)

Ru(bpy)33+ + TPrA f Ru(bpy)32+ + TPrA+

(3)

TPrA+ f TPrA• + H+

(4)

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

(5)

Ru(bpy)32+* f Ru(bpy)32+ + hν

(6)

We used ECL imaging to visualize the spatial distribution of the electroactive spots of the array surface. The images of Figure 3 were captured with the distal face of the array positioned in front of the microscope/CCD camera. Figure 3A displays a whitelight image focused on a region of interest of the array surface before applying an adequate potential. On this image, one can observe ∼280 tips. We generated ECL by stepping the potential from 0 V/Ag, where no faradaic reaction takes place, to + 1.2 V/Ag. This potential is high enough to be within the diffusionlimited current range for generation of Ru(bpy)33+. When the array was polarized, ECL was generated at each electroactive tip, and Figure 3B shows the corresponding false color ECL image of the array surface. Blue represents higher light intensity. The ECL spots reveal the electroactive ultramicroelectrodes over the region of interest depicted in Figure 3A. We clearly observed an ordered array of well-separated individual ECL spots of micrometer dimensions. Such very small light sources could find a number of applications in photonic devices or electrooptical sensors. The spatial distribution of ECL emission follows a pattern resembling

imaging fiber architecture. A total of 260-270 ECL spots were observable in Figure 3B. The slight difference between the number of tips in Figure 3A and ECL spots in Figure 3B could be partially explained if all the tips were not in the same focal plane (i.e., coplanar) because of imperfect etching.49 Furthermore, it gives information about the size of the ultramicroelectrodes. Indeed, the average size of ECL spots determined from this figure is ∼1-1.5 µm whereas the average apparent diameter of a single ultramicroelectrode determined from eq 1 is 0.6 µm. To extract valuable information from this image and to explain this relative discrepancy, we have to consider the spatial resolution. The spatial resolution is governed by various effects related to the electrochemical process and also to the optical detection, such as diffusional broadening or microscope resolution. Two factors associated with the electrochemical phenomenon can contribute to expand the ECL-emitting region around the electrode (i.e., the site of photon emission does not coincide virtually with the site of electron transfer at the electrode surface).42 The first one is related to the thickness of the reaction layer since the mechanism via the “catalytic route” allows the generation of ECL light at some distance from the electrode surface. From the theory of catalytic processes at a hemispherical ultramicroelectrode,54 it is possible to deduce the catalytic reaction layer thickness where the homogeneous oxidation of TPrA by electrogenerated Ru(bpy)33+ occurs. The size of the catalytic reaction layer is determined by the overall rate of ECL reaction sequence:3,42,49,54

µ)

[ (

)]

2k3[TPrA] 1 + rav D

1/2 -1

(7)

where rav is the average apparent radius of a single electroactive (53) Gross, E. M.; Pastore, P.; Wightman, R. M. J. Phys. Chem. B 2001, 105, 8732-8738.

(54) Diao, G.; Zhang, Z. J. Electroanal. Chem. 1997, 429, 67-74.

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tip of the array, k3 is the apparent rate constant of reaction 3 between Ru(bpy)33+ and TPrA, and D is the diffusion coefficient of Ru(bpy)32+/3+. D is 5.9 × 10-6 cm2 s-1.3,52 The variation of k3 with pH is related to the acid-base behavior of TPrA and is given by k3 ) k/(1 + 10-pH+pKa), where k is the intrinsic value of the rate constant.49,52 Values of k and pKa are respectively 1.3 × 107 M-1 s-1 and 10.4.49,52 For our experimental conditions at pH ) 7.5, k3 is estimated as 1.6 × 104 M-1 s-1. Therefore, for an average radius of 300 nm, the thickness of the reaction layer is ∼2OO nm under conditions where the “catalytic route” is the dominant process for ECL.51,52 It means that the rate of the global ECL reaction sequence is fast compared to the diffusional time scale.42,44 In other words, the catalytic reaction confines the ECL-emitting region close to the electrode surface. Therefore, the individual ECL-emitting layers developing at adjacent electrodes cannot overlap and discrete ECL sites are created on the array by the catalytic mechanism. The second factor that can affect the global size of the ECL-emitting region depends on the diffusion length of the excited state of Ru(bpy)32+. Indeed, during its lifetime, Ru(bpy)32+* can diffuse away from the generation site (i.e., reaction layer) to the photon emission site. Therefore, the lifetime τ of Ru(bpy)32+* has to be taken into account and translates into a diffusional distance (∼(Dτ)1/2). The excited state is relatively longlived in solution (τ ) 0.64 µs at 25 °C in water in the absence of oxygen).55 Assuming that its diffusion coefficient is equal to the value at the ground state,3 the excited state can diffuse by ∼20 nm before emitting light. Thus, the light-emitting region is not considerably broadened by diffusion of the reactants or of the excited state away from the tip.3,49 In other words, the sites of ECL photon emission are confined within ∼2µ (i.e., ∼400 nm) around the electrode. Finally, we also have to take into account the optical resolution. Theoretical optical resolution is limited by the Abbe´ diffraction barrier to approximately half the wavelength of the illuminating light (∼200-300 nm). Experimental optical resolution is governed by the pixel size of the CCD camera and by the magnification of the objective used to acquire the image. The experimental limit equals the size of the pixels in the CCD camera (13 µm) divided by the magnification. In other words, with a 40× objective, each pixel corresponds to ∼0.3 µm. This value is very close to the theoretical limit and comparable to the diffusional broadening calculated above. Therefore, spatial resolution is limited by diffusional broadening, by optical resolution of our microscope/ CCD camera setup, and eventually by the precision of the focusing over such a large area. If we consider the different limits affecting the spatial resolution of ECL imaging, the micrometric dimension of the ECL spots (∼1-1.5 µm) is in very good agreement with the value obtained from steady-state measurement (∼2rav ∼0.6 µm). Finally, this value validates the approximation made on the tip geometry and mode of transport. In addition, Figure 3B reveals a quite uniform statistic distribution of the size and intensity of the ECL spots on the array. This result suggests that the size of the individual ultramicroelectrodes varies slightly around the average apparent radius estimated by cyclic voltammetry and that the electrophoretic coating leads to fairly uniform and homogeneous results. (55) Van Houten, J.; Watts, R. J. J. Am. Chem. Soc. 1976, 98, 4853-4858.

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In brief, a single ECL image reveals the size within the resolution limits and the spatial distribution of the electroactive sites of the array. These results show the interest of ECL imaging to visualize and to characterize with a single ECL image the electrochemical activity of the sensor array in a fast and reliable way. Application of the Sensor Array to Remote ECL Imaging. Ru(bpy)32+/TPrA is an aqueous co-reactant ECL model system. It offers many intrinsic advantages over other detection systems, and it has been commercially developed for clinical diagnostics (e.g., immunoassays and DNA analysis). We applied the optoelectrochemical sensor array to concomitantly generate and detect ECL through the imaging fiber. Figure 4 summarizes the array response characteristics. Figure 4A presents a false color ECL image of a region of interest when the array is polarized. We want to stress that this image is obtained through the optical fiber bundle and not with the sensor array positioned in front of the CCD camera as in Figure 3. Indeed, ECL is generated at the electroactive apex of the tip and is transmitted through the transparent ITO film whereas thick gold coating prevents light transmission through the optical fiber bundle.24,49 A fraction of the electrochemically generated luminescence is then collected by the corresponding core, conducted via the same core and finally acquired with the CCD camera (Figure 4A and B). ECL intensities of all the sensors forming the array are acquired simultaneously and individually at a given time in a single image. In other words, remote ECL imaging is performed through the sensor array itself. At a higher TPrA concentration (Figure 4B), ECL intensity detected by the CCD camera is increased. Calibration curves were established to test the quantitative validity of this strategy. A linear dynamic range of 3 orders of magnitude between ECL intensity and concentration of TPrA was found from 100 mM to 100 µM. The line that passes through the data points in Figure 4C is the linear regression fit. It shows that the ECL intensity is proportional to the TPrA concentration. Figure 4D shows the influence of Ru(bpy)32+ concentration on the ECL signal initiated and detected through the sensor array. The plot is linear (r2 ) 0.996) from 50 µM, the detection limit, to 1 mM. ECL is induced at the apex of each etched core. Since this electrochemically stimulated emission generates photons traveling in all directions (4π sr), only a fraction is collected and transmitted by the corresponding core. ECL generated by this core could also be collected by neighboring cores. This latter fraction depends on the acceptance cone of the optical fiber, on the size of the ECLemitting layer, on the distance between adjacent tips, and on a term proportional to the square of the distance, which is related to the isotropically expanding sphere of emitted ECL. We assumed to a first approximation that the acceptance cone of the etched core is similar to the original fiber. A fiber of 0.35 NA has a halfacceptance angle of 20°. Since the size of the ECL-emitting region is ∼2µ (i.e., ∼0.4 µm; vide supra) and the distance between two adjacent tips is ∼4 µm, ECL generated at a tip cannot be collected by neighboring tips. The distance between adjacent tips is sufficiently large compared to the ECL-emitting layer. Furthermore, an acceptance cone corresponding to an individual tip does not overlap with adjacent ECL-emitting layer. Therefore, a tip collects only the ECL light generated at its apex. In other words, the sensors forming the array can be considered as optically independent. This important result is further established experi-

Figure 4. ECL images acquired through the same sensor array immersed in 1 mM Ru(bpy)32+ and (A) 10 or (B) 100 mM TPrA. Both images were coded according to the same color scale (right). White represents high ECL intensities. (C) Plot of normalized ECL intensity vs TPrA concentration in solutions containing 1 mM Ru(bpy)32+. (D) Plot of normalized ECL intensity vs Ru(bpy)32+ concentration in solutions containing 100 mM TPrA. ECL calibration curves were obtained from the same ROI after application of 1.2 V/Ag to the sensor array. Solutions were 0.15 M PBS (pH ) 7.5).

mentally. In some experiments, a part of the array was completely covered by the insulating paint and no ECL was detectable in this region even if it was immediately adjacent to ECL-emitting spots (data not shown). Therefore, ECL generated at each tip is optically wired to the corresponding core and each optically independent sensor of the array probes a different microenvironment. The sensors in this array are not individually addressable (i.e., all the sensors operate at the same potential),24,49 which is often a crucial requirement for practical devices.56-58 However, the fabrication procedure produced an ordered array of optoelectrochemical sensors that retains the optical fiber bundle architecture. The coherent structure of the fiber array used in this study allows transmission of an image through the imaging fiber.59 ECL light generated at the tip apex and collected by the corresponding core propagates under guided conditions in the fiber core by total internal reflection. In fact, the core diameter varies slightly to (56) Nagale, M. P.; Fritsch, I. Anal. Chem. 1998, 70, 2902-2907. (57) Nagale, M. P.; Fritsch, I. Anal. Chem. 1998, 70, 2908-2913. (58) Liu, C.-Y.; Bard, A. J. J. Am. Chem. Soc. 2002, 124, 4190-4191. (59) Pantano, P.; Walt, D. R. Anal. Chem. 1995, 67, 481A-487A.

minimize cross-talk between the cores and to improve spatial resolution when used as imaging fibers.60 Since ECL is optically wired to the corresponding core and the imaging fiber is coherent, the sensors are individually readable with a two-dimensional detector, such as a CCD camera. Therefore, ECL intensity of each sensor can be monitored individually and the sensor array probes different microenvironments covering a large area. Since the sensors are optically independent and individually readable, Panels A and B of Figure 4 reveal that all the individual sensors generate and transmit light and are actually electroactive in the array. This result is in complete agreement with the deductions based on Figure 3. Finally, it validates the Ntips value used in eq 1 to determine the average apparent radius of a single sensor in the array. CONCLUSION We prepared and characterized an ordered array of 6000 optoelectrochemical submicrometer-sized sensors. The sigmoidal (60) Mogi, M.; Yoshimura, K. Proc. SPIE Int. Soc. Opt. Eng. 1989, 1067, 172181.

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shape of the steady-state voltammogram indicates that the sensors are diffusively independent. Furthermore, ECL imaging provides precious information on the sensor array. ECL was generated at the tip apex coated with ITO. Well-separated individual ECL spots of micrometer dimensions reveal the spatial distribution of electroactive sensors, which forms a pattern matching the imaging fiber structure. It shows also that all the tips are electroactive in the array. Eventually, ECL imaging allows us to estimate the size of the sensors, which is in agreement with the value (rav ∼300 nm) determined by cyclic voltammetry. Considering the size of the ECL-emitting region, numerical aperture, and distance between neighboring cores, it was further demonstrated that ECL sensors are optically independent. Since each ECL spot is optically wired to the corresponding core of the imaging fiber, the sensors are individually readable. Therefore, the new sensor array is able to perform remote ECL imaging with the advantages of ultramicroelectrode properties, and it also allows the parallel acquisition

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of large amounts of data. Our approach should allow concomitantly ECL sensing within microenvironments and direct imaging of the sample. Finally, such micrometer ECL sources could find a wide range of applications in photonic devices or electrooptical sensors. ACKNOWLEDGMENT This work has been supported in part by the French Ministry of Research (Programme Action Concerte´e Incitative Jeunes Chercheurs) and by the Conseil Re´gional d′Aquitaine. The authors gratefully acknowledge Laurent Servant (Laboratoire de PhysicoChimie Mole´culaire, Universite´ Bordeaux I) for helpful discussions and Gilles Lovo (BASF) for the gift of the electrophoretic paint.

Received for review August 21, 2003. Accepted October 17, 2003. AC034974W