Development of a New Device for Ultrasensitive

Jun 25, 2009 - J.; Peterman, J.; Powell, M. J.; Shah, A.; Talley, D. B. Clin. Chem. ... Detection, and Single Cell Analysis; Xiao-Hong, N. X., Ed.; Jo...
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Anal. Chem. 2009, 81, 6234–6241

Development of a New Device for Ultrasensitive Electrochemiluminescence Microscopy Imaging Luisa S. Dolci,†,‡ Simone Zanarini,§ Leopoldo Della Ciana,§ Francesco Paolucci,§ and Aldo Roda*,†,‡ Department of Pharmaceutical Sciences, University of Bologna, Bologna, Italy 40126, Istituto Nazionale Biostrutture e Biosistemi (INBB), Rome, Italy 00136, and Department of Chemistry G. Ciamician, University of Bologna, Bologna, Italy 40126 Electrochemiluminescence (ECL) is widely used in biosensors and immunoassays thanks to the high sensitivity and specificity of the electrochemically triggered luminescence signal. So far, no applications have been reported on the use of ECL as a probe for ultrasensitive lowlightmicroscopeimaging.Thisworkreportsthedevelopment of a new transparent electrochemical cell for ECL imaging suitable for single cell analysis. The system is based on the use of a microscope placed in a dark box equipped with a CCD camera and a potentiostat. Transparent conducting glass coated with fluorine-doped tin oxide (FTO) has been used, and a three electrode configuration has been designed. The electrochemical cell was optimized using 8 µm diameter polystyrene beads coated with a Ru(bpy)32+ complex in order to simulate living cells. The Ru(bpy)32+ immobilized on the microbeads can be imaged and quantified at a concentration as low as 1 × 10-19 mol/µm2. Microscope imaging showed that the ECL signal was detected only in correspondence to the beads present on the electrode surface, and the probe could be accurately localized with a spatial resolution of 0.4 µm. The new ECL imaging device can be used in conjunction with other chemiluminescence-based imaging methods for ultrasensitive multiplex imaging on cells and tissues. Electrochemiluminescence (ECL) is a chemiluminescent electron-transfer reaction in which the reactants are generated electrochemically at the surface of an electrode.1-11 The peculiar analytical performances in terms of high detectability of the * Corresponding author. Laboratory of Analytical and Bioanalytical Chemistry, Department of Pharmaceutical Sciences, Alma Mater Studiorum-University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy. Phone and Fax: +39 051 343398. E-mail: [email protected]. Web site: http://www.anchem.unibo.it. † Department of Pharmaceutical Sciences, University of Bologna. ‡ Istituto Nazionale Biostrutture e Biosistemi. § Department of Chemistry G. Ciamician, University of Bologna. (1) Gerardi, R. D.; Barnett, N. W.; Lewis, S. W. Anal. Chim. Acta 1999, 378, 1–41. (2) Knight, A. W. Trends Anal. Chem. 1999, 18 (1), 47–62. (3) Fahnrich, K. A.; Pravda, M.; Guilbault, G. G. Talanta 2001, 54, 531–559. (4) Bard, A. J., Ed. Electrogenerated Chemiluminescence; Marcel Dekker: New York, 2004. (5) Richter, M. M. Chem. Rev. 2004, 104, 3003–3036. (6) Xue-Bo, Y.; Shaojun, D.; Wang, E. Trends Anal. Chem. 2004, 23 (6), 432– 441. (7) Gorman, B. A.; Francis, P. S.; Barnett, N. W. Analyst 2006, 131, 616–639.

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conventional chemiluminescence (CL) are retained and, in addition, the electrochemical reaction allows the time and position of light emitting reaction to be controlled. These properties make ECL systems particularly attractive as a probe for ultrasensitive detection of immunological, cDNA, or enzyme catalyzed reactions not only in solution12-15 but also for direct imaging in biological tissue cryosections or single cells. Microscope imaging techniques such as in situ hybridization (ISH) and immunohystochemistry (IHC) usually rely on photoluminescence or bio-chemiluminescence for the detection of an antibody or a DNA probe directly or indirectly labeled with a luminescent molecule or an enzyme.16-19 Conventional prompt fluorescence measurements are the most widely used, and multiple localization can be achieved by spectra resolved analysis of the light emission using narrow band photoluminescent probes.20,21 Despite the use of combined techniques such as confocal or pseudoconfocal microscopy which improve the signal-to-noise ratio and the imaging resolution, photoluminescence is still affected by the autofluorescence of the sample matrix, which is often several orders of magnitude higher than the analyte specific signal. The high background signal can be efficiently minimized by the time-resolved fluorescence (TRF) analysis of long-lived fluorescence labels such as lanthanide (8) Xiao-Hong, N. X., Ed. Chapter 11. New Frontiers in Ultrasensitive Bioanalysis; Wiley: Hoboken, NJ, 2007. (9) Miao, W. Chem. Rev. 2008, 108, 2506–2553. (10) Wei, H.; Wang, E. Trends Anal. Chem. 2008, 27 (5), 447–459. (11) Marquette, C. A.; Blum, L. J. Anal. Bioanal. Chem. 2008, 390 (1), 155– 168. (12) Lin, J.; Ju, H. Biosens. Bioelectron. 2005, 20 (8), 1461–1470. (13) Pittman, T. L.; Thomson, B.; Miao, W. Anal. Chim. Acta 2009, 632 (2), 197–202. (14) Lu, Y.; Young, J.; Meng, Y. G. Curr. Opin. Pharmacol. 2007, 7 (5), 541– 546. (15) Bard, A. J.; Li, X.; Zhan, W. Biosens. Bioelectron. 2006, 22 (4), 461–472. (16) Roda, A.; Pasini, P.; Musiani, M.; Girotti, S.; Baraldini, M.; Carrea, G.; Suozzi, A. Anal. Chem. 1996, 68, 1073–1080. (17) Roda, A.; Guardigli, M.; Pasini, P.; Musiani, M.; Baraldini, M. In Luminescence Biotechnology: Instruments and Applications; Van Dyke, K., Van Dyke, C.,Woodfork, K. Eds.; CRC Press: Boca Raton, FL, 2002; pp 481-501. (18) Bonvicini, F.; Mirasoli, M.; Gallinella, G.; Zerbini, M.; Musiani, M.; Roda, A. Analyst 2007, 132, 519–523. (19) Guardigli, M.; Marangi, M.; Casanova, S.; Grigioni, W. F.; Roda, E.; Roda, A. J. Histochem. Cytochem. 2005, 53, 1451–1457. (20) Creton, R.; Jaffe, L. F. Biotechniques 2001, 31, 1098. (21) Martin, H., Rudolf, H., Vlastimil, F., Eds. Fluorescence Spectroscopy in Biology: Advanced Methods and Their Applications to Membranes, Proteins, DNA, and Cells; Springer: Heidelberg, Germany, 2005. 10.1021/ac900756a CCC: $40.75  2009 American Chemical Society Published on Web 06/25/2009

chelates.22-25 Using TRF, the background can be minimized, but the intrinsic principles of the TRF do not allow one to achieve accurate quantitative information. More recently, CL has been proposed successfully for ultrasensitive quantitative imaging on single cells and tissue using horseradish peroxidase (HRP) or alkaline phosphatase (AP) labeled probes with the use of suitable chemiluminescent substrates.16,26-28 Despite the high detectability of CL-based techniques, the achievable resolution is in part compromised by diffusion of the emitting product of the enzymatic reaction that could emit light far away from the reaction site, where the label enzyme is localized. This occurs when the lifetime of the excited state emitting product is significantly longer than its diffusion rate in solution.29 ECL represents an additional potential tool for ultrasensitive microscope imaging that may combine the advantages of CL, in terms of detectability, with improved resolution since, in this case, the label itself emits light and no diffusion phenomena may affect the imaging resolution. The best known ECL systems use the Ru(bpy)32+ complex properly functionalized to label biomolecules and tri-n-propylamine (TPA) as a coreactant.5,6 These complexes are characterized by high quantum efficiency, short response time, and high stability, and this allows an ultrasensitive assay to be setup.30,31 ECL is generated in an oxidative/reduction mechanism by the electrochemical oxidation of Ru(bpy)32+ in the presence of TPA to an active redox state via a series of cyclic oxidation/reduction steps at the electrode surface by applying an electrical potential at the working electrodes of +1.2 V (vs Ag/AgCl). The difference of electrochemical potential between Ru(bpy)33+ and the oxidized TPA is sufficient to produce the Ru(bpy)32+* excited state, which decays releasing light at 620 nm. During the regeneration process, each ECL active label can emit many photons, thereby amplifying the analytical signal. Despite the wide use of ECL in clinical chemistry,32 so far, imaging applications have been essentially limited to microarray (22) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.; Springer: New York, 2006; p 659. (23) Roda, A.; Guardigli, M.; Pasini, M.; Baraldini, M. Development of a timeresolved fluorescence microscope for imaging analysis. In Bioluminescence and Chemiluminescence 2000; Case, J. F., Herring, P. J., Robison, B. H., Haddock, S. H. D., Kricka, L. J., Stanley, P. E., Eds.; World Scientific Publishing Company: Singapore, 2001; pp 493-496. (24) Charbonnie´re, L.; Ziessel, R.; Guardigli, M.; Roda, A.; Sabbatini, N.; Cesario, M. J. Am. Chem. Soc. 2001, 123, 2436–2437. (25) Roda, A.; Guardigli, M.; Ziessel, R.; Mirasoli, M.; Michelini, E.; Musiani, M. Microchem. J. 2007, 85, 5–12. (26) Christenson, M. A. In Luminescence Biotechnology: Instruments and Applications; Van Dyke, K. Van Dyke, C., Woodfork, K., Eds. CRC Press: Boca Raton, FL, 2002. (27) Roda, A.; Musiani, M.; Pasini, P.; Baraldini, M.; Crabtree, J. E. Methods Enzymol. 2000, 305, 577–590. (28) Roda, A.; Guardigli, M.; Michelini, E.; Pasini, P.; Mirasoli, M. Anal. Chem. 2003, 75, 462A–470A. (29) Roda, A.; Pasini, P.; Baraldini, M.; Musiani, M.; Gentilomi, G.; Robert, C. Anal. Biochem. 1998, 257, 53–62. (30) Blackburn, G. F.; Shah, H. P.; Kenten, J. H.; Leland, J.; Kamin, R. A.; Link, J.; Peterman, J.; Powell, M. J.; Shah, A.; Talley, D. B. Clin. Chem. 1991, 37 (9), 1534–1539. (31) Yang, H.; Leland, J. K.; Yost, D.; Massey, R. J. Biotechnology 1994, 12, 193–194. (32) www.roche-diagnostics.it.

or multispot devices4,5,9,11,33-35 and no application for microscope imaging of cells and tissue cryosections has been reported. The main problem of using ECL for cell or tissue imaging is that it is essentially a surface technique, and all the labeled biomolecules need to be located at a limited distance from the electrode; in fact, the electrogenerated species diffusion is usually limited by their gradient of concentration and only can be reached at a discrete distance from the electrode. While this aspect can be easily afforded in a bulk solution, ECL active probes imaging for tissues and cells need a specific cell design and well-optimized experimental conditions. For microscope imaging applied to localize the analyte in an unknown region of the tissue cryosection or cells, the electrode device needs to be transparent for the acquisition of both the ECL signal and the conventional transmitted light image; the overlay of the two images will in fact permit the localization of the probe in the studied structure. This process cannot be performed with the previous electrode used for microarray imaging where only one side of the electrode was transparent. The transparent electrode device can be used not only for ECL but also as a simple microscope slide for multiplex detections using other labels based on CL, TRF, and conventional fluorescence. In this work, a new transparent electrochemical cell using fluorine-doped tin oxide (FTO)-coated glass has been developed. The device presents a transparent field of view (16 mm2) that is compatible with optical microscopy using conventional magnification lenses. The device has a dimension similar to a conventional microscope glass slide and a volume of 20 µL; therefore, it allows one to acquire both the transmitted light image (thanks to the transparency) and the ECL-generated image. A dedicated instrument composed of a microscope in a dark box equipped with an ultrasensitive nitrogen cooled CCD camera and a potentiostat/galvanostat has been set up and optimized. Polystyrene microbeads with a diameter of 8 µm were used to simulate randomly distributed biological cells on the microscope field of view. The microbeads were labeled directly or conjugated using a biotin-streptavidin interaction with a Ru(bpy)32+ complex analogue and used to optimize the system and evaluate the suitability of the device for microscope ECL imaging of cells and tissue. EXPERIMENTAL SECTION Materials. [Ru(4(4′-methyl-2,2′-bipyridin-4-yl)butan-1-aminium(2,2′-bipyridine)2](ClO4)3 (Ru(bpy)32+-NH2), bis(2,2′-bipyridine)-[4-[4′-methyl-2,2′-bipyridin-4-yl)butanoic acid] ruthenium bis(hexafluorophosphate) (Ru(bpy)32+-COOH), 3-sulfo-N-hydroxysuccinimide (s-NHS), and biotin-cadaverine-TFAc were purchased from Cyanagen (Bologna, Italy). Tris(2,2′-bipyridine)dichlororuthenium(II) (Ru(bpy)32+), streptavidin from Streptomyces avidinii, 2-(N-morpholino)ethanesulfonic acid (MES), tripropylamine (TPA), N-(3-dimethylaminopropyl)-N′(33) Xiao-Hong, N. X.; Yanbing, Z. Electrochemiluminescence Detection in Bioanalysis. In New Frontiers in Ultrasensitive Bioanalysis: Advanced Analytical Chemistry Applications in Nanobiotechnology. Single Molecule Detection, and Single Cell Analysis; Xiao-Hong, N. X., Ed.; John Wiley & Sons, Inc.: New York, 2007; Vol. 172, p 258. (34) Zuo, X.; Xiao, Y.; Plaxco, K. W. J. Am. Chem. Soc. 2009, 131 (17), 6088– 6089. (35) Leland, J. K.; Powell, M. J. J. Electrochem. Soc. 1990, 137 (10), 3127–3131.

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Figure 1. ECL optical imaging cell (75 × 25 × 1 mm). Left panel: side view. Right panel: top view (Ag quasi-reference electrode, 8 mm2; FTO working electrode, 16 mm2; FTO counter electrode, 20 mm2). Scheme 1. Method I: Direct Binding of Ru(bpy)32+-NH2 to Carboxylate Beads

ethyl-carbodiimide hydrochloride (EDC), N,N′-dicyclohexylcarbodiimide (DCC), and ferrocenemethanol (Fc-MeOH) were purchased from Sigma-Aldrich Co. (St. Louis, MO). Water was freshly poured from a Milli-Q ddH2O system. The polystyrene carboxylated microbeads (diameter 8 µm) were purchased from Spherotech (Libertyville, Illinois). Fluorine-doped tin oxidecoated glasses (20 Ω) were purchased from Flexitec Electronica Organica (Curitiba Parana`, Brazil). Imaging Instrumentation. For microscopy imaging, a BX60 epifluorescence microscope (Olympus Optical, Tokyo, Japan) equipped with a liquid nitrogen-cooled ultrasensitive CCD camera (LN/CCD Princeton Instruments, Roper Scientific, Trenton, NJ) was used. The microscope was enclosed in a dark box to avoid interference from ambient light and was equipped with an OptiScan ES103 motorized microscope stage (Prior Scientific Instruments Ltd., Fulbourn, England) for sample positioning. The integrated system also includes a potentiostat/galvanostat (model 2059, AMEL Instruments, Milan, Italy) suitable to provide the needed potential for the ECL triggered reaction. Glass Electrochemical Cell Fabrication. The prototype cell was prepared with conventional technology. A microscope FTOglass (2.5 × 7.5 × 0.1 cm) was employed for the development of a transparent standard three electrode cell (see Figure 1). Sticky tape was used to create a mask on the conductive face of the FTO microscope glass. The electrode area (i.e., the surface not covered with the sticky tape) was then painted with a thin layer of nail polish to protect the FTO. After tape removal, the glass was treated with a Zn/H2O paste and was dipped in a 37% v/v HCl solution to remove the exposed FTO layer. The nail polish was then removed with acetone, and the cell was rinsed with distilled water and sonicated for 5 min in H2O/ethanol (7:3 v/v). In order to create a working volume on the cell and to better delimitate electrode areas, the cell was covered with a sticky tape limiter (inner area 1.5 × 1.0 cm, 110 µm thick). A quasireference electrode was prepared by flash galvanic deposition (from a 3 M AgNO3 solution applying a constant negative 6236

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current of 3 mA) directly on one of the FTO electrodes. The shapes and distances of the three electrodes were optimized to obtain the configuration shown in Figure 1 (Ag quasireference electrode (8 mm2); FTO working electrode (16 mm2); FTO counter electrode (20 mm2); volume of cell 20 µL). Electrochemical Cell Characterization. Cyclic voltammetry and ECL measurements were carried out with an AUTOLAB electrochemical station (Ecochemie, Holland) at a 0.2 V/s scan rate. The electrochemical behavior of the cell was monitored by cyclic voltammetry using a 10-4 mol/L Fc-MeOH solution in 0.1 mol/L PBS (pH 7.5). The ECL signal was collected by a photomultiplier tube (Hamamatsu R255, biased at 750 V) placed a few millimeters in front of the working electrode. To register the ECL signal, the photomultiplier output signal was sent to an ultralow noise current preamplifier (Acton Research model 181). Reproducibility and stability were evaluated by means of light/current/potential and light/current/time plots using a 10-4 M Ru(bpy)32+ solution in 0.1 M PBS (pH 7.5). The potential program was E1 ) +1.5 V; E2 ) 0.0 V; t1 ) t2 ) 1 s for a chrono-pulsed reproducibility test. pH optimization was carried out using a 10-4 M Ru(bpy)32+ solution and a 3 × 10-2 M TPA solution in 0.1 M phosphate buffer (pH range from 6.0 to 9.0). Preparation of Ru(bpy)32+ Labeled Microsphere. Method I: Direct Binding of Ru(bpy)32+-NH2 to Carboxylate Beads (Scheme 1). Ru(bpy)32+-NH2 was conjugated to carboxylated beads (1 in Scheme 1) according to the following protocol. The suspension of beads (200 µL of 8 µm) was washed three times in 0.1 mol/L borate buffer (pH 9.6) and two times in 0.1 mol/L MES buffer (pH 5.5). The beads were finally resuspended in 250 µL of MES buffer. Carboxylic groups were activated by adding EDC and s-NHS to a final concentration of 50 mM and 2 mM, respectively. The reaction mixture was gently mixed for 1 h at RT. After a washing cycle (see above), 500 µL of a 0.75 mM Ru(bpy)32+-NH2 solution in 0.1 mol/L borate buffer (pH 8.6) was added, and the suspension was incubated overnight at 4 °C. The obtained

Scheme 2. Method II: Indirect Binding of Ru(bpy)32+ Using a Biotin-Streptavidin Interaction

labeled beads (2 in Scheme 1) were finally washed three times in PBS. Method II: Indirect Binding of Ru(bpy)32+ Using a BiotinStreptavidin Interaction (Scheme 2). To obtain labeled streptavidin, Ru(bpy)32+-COOH was dissolved in DMF to obtain 70 µL of a solution with a concentration of 7.1 × 10-3 M. Equivalents (1.5) of DCC and NHS were added, and the reaction solution was gently mixed for 4 h at RT. A streptavidin solution (630 µL of a 2.0 × 10-5 M) in 0.1 M borate buffer (pH 9.4) was then added (activated Ru(bpy)32+-COOH/streptavidin 40:1 mol/mol). The solution was incubated overnight, and the labeled protein was subsequently purified with dialysis against 5 L of PBS. Beads (1 in Scheme 1) were subsequently conjugated with an aminoderivative of biotin. The previously described procedure was used to wash and activate the beads. After 1 h of RT incubation, 500 µL of 9 mM biotin cadaverine in 0.1 mol/L borate buffer (pH 8.6) was added. The mixture was incubated overnight at 4 °C, and the solution was finally washed three times in PBS to obtain the beads (3 in Scheme 2). Beads (3 in Scheme 2) were successively coupled with the streptavidin-Ru(bpy)32+ complex to form beads (4 in Scheme 2) using the following procedure. A suspension (50 µL) of beads (3 in Scheme 2) was centrifuged, and after buffer removal, 50 µL of labeled streptavidin was added. The mixture was gently mixed for 2 h at RT, and then three washing steps were performed by centrifugation and resuspension with PBS. ECL Microscope Imaging. The suspension of microbeads labeled with Ru(bpy)32+ and a 3 × 10-2 M TPA solution in 0.1 M phosphate buffer at pH 7.5 was added to the transparent electrochemical cell. The live image (Figure 6A) was recorded with the transmitted light using a conventional microscope lamp, and this was used to locate the position of the beads on the electrode surface. A potential of +1.2 V was then applied to the electrode for 30 s, and the emitted light was recorded with the CCD. The ECL image was elaborated in a pseudocolor scale, and an overlay of this image with the live image was created, which allowed the probe to be properly localized on the bead surface. According to the lense magnification, we can calculate the pixel size and the resolution of the obtained image. RESULTS AND DISCUSSION Cell Design and Geometry. The cell has been designed to be used as a microscope slide (7.5 × 2.5 cm) with a conventional optical microscope. The geometry has been designed to cover the field of view of the objective with magnification ranging from 10 to 100×. The electrode size and dimension have been optimized

as a function of the thickness and cell volume. The best performance has been obtained with a three electrode cell (Figure 1): Ag quasi-reference electrode (8 mm2); FTO working electrode (16 mm2); FTO counter electrode (20 mm2); and a cell volume of 20 µL. The working electrode surface shape and size were tailored to be operated with real tissues or cells on a standard optical microscope system. The selected geometry (Figure 1) allows one to obtain a uniform luminescent signal on the surface of the working electrode and, therefore, to potentially analyze any micrometersized biological sample such as bacteria, mammalian cells, or tissue sections labeled with the Ru(bpy)32+ complex. ECL Probe Preparation. Two different ruthenium trisbipyridine derivatives with a spacer arm of four carbon atoms with an amine or a carboxy reactive group were used. The spacer length was chosen to allow an efficient conjugation of the probe with the activated solid surface, facilitating the contact of the labeled microbeads with the electrode surface, thus, increasing the accuracy of the ECL measurements. The amine group present in ruthenium trisbipyridine (Ru(bpy)32+-NH2) was used for labeling carboxylate microbeads through the formation of a stable amide bond as shown in Scheme 1. The fluorescence from the functionalized beads (2 in Scheme 1) confirmed their efficient derivatization. As a negative control, beads (1 in Scheme 1) without active carboxyl groups were incubated with Ru(bpy)32+-NH2, and as expected, no fluorescence was detected. Alternatively, a ruthenium trisbipyridine derivative with the carboxyl reactive group (Ru(bpy)32+COOH) was used for labeling microspheres by indirect binding using a biotin-streptavidin interaction (Scheme 2). First, the ruthenium complex was bioconjugated to streptavidin (Method II), and such a reaction yielded a ruthenium/strepavidin ratio of about 5:1 mol/mol. Subsequently, the functionalized complex was reacted with biotinylated conjugated beads (3 in Scheme 2). Fluorescence was detected also in this case after functionalization. As a negative control, beads (1 in Scheme 1) without activation of a carboxyl group were incubated with a streptavidin-Ru(bpy)32+ complex; also, in this case, no fluorescence was detected. Basic Electrochemical and ECL Characterization. The conditions for the electrochemically generated luminescence reaction were optimized in order to obtain linearity of light emission in an as wide as possible concentration range with the highest detectability and sensitivity. The necessity to use a conducting material which combines transparency with an adequate resistivity to realize the needed Analytical Chemistry, Vol. 81, No. 15, August 1, 2009

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Figure 2. Cyclic voltammetry of ferrocene registered with an FTO cell (10-4 M Fc-MeOH in PBS, pH 7.5, scan rate of 0.5 V/s).

current densities restricts the selection among the transparent materials practically employable. FTO glass was selected since it fulfilled the electrochemical conditions needed to trigger the ECL reaction better than other conducting transparent materials such as the more common indium tin oxide (ITO) glass. The homemade glass cells were developed to include a standard three electrode electrochemical configuration on a microscope slide and, therefore, to combine optical and ECL imaging. The transparency allows one to visualize the microscope image of a given cell or structure and the electrochemical cell to trigger the ECL reaction in the same chamber. The overlay of the optical image with the ECL image allows one to localize the analyte. Cell characterization was carried out first by monitoring the basic electrochemical and ECL behavior. The electrochemical response was evaluated using a Fc-MeOH solution. Cyclic voltammetry showed the expected ferrocene reversible oxidation process falling in the correct range of E1/2 vs Ag (see Figure 2).36 The second basic experiment consisted of a voltammetric scan on a Ru(bpy)32+ solution with 0.1 M PBS in the presence of tripropylamine (TPA) and simultaneous measurements of the light emission. As expected, the light emission peak was observed at +1.15 V vs Ag corresponding to the electrochemical oxidation of Ru(II) to Ru(III)4 (see Figure 3). ECL Signal Optimization. The time stability of ECL intensity was monitored by applying repeatedly a constant potential of +1.2 V for 1 s. The light/current/time plot (Figure 4) evidences a fairly constant ECL intensity. The slight decay of ECL signal during repeated measurements can be associated with the progressive formation of filming products at the electrode. This phenomenon can also explain the progressive increase of the oxidation current and the decrease of the reduction current in the reverse voltammetric scan where the cyclic voltammetry curve tends to flatten and broaden. The best ECL signal imaging conditions compatible with the electrochemical performance of the electrode was the application (36) Abruna, H. D. J. Electroanal. Chem. 1984, 175, 321–326.

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Figure 3. Light/current/potential curve registered with an FTO cell from a 10-4 M Ru(bpy)32+ solution with 0.1 M PBS (pH 7.5) and 3 × 10-2 M TPA; PMT bias, 750 V; amplification factor, 10-5 A/V. Switching method: CV 0.2 V/s. Potential is vs onboard Ag.

Figure 4. Chrono-pulsed measurement of light/current vs the potential for a Ru(bpy)32+ solution with 3 × 10-2 M TPA and 0.1 M PBS, pH 7.5. Potential program: E1 ) +1.2 V, E2 ) +0.0 V, t1 ) t2 ) 1 s; sample time, 0.05 s; PMT, 750 V; amplification, 10-5 A/V. Potentials are vs quasi-reference Ag.

of a constant electrical potential of 1.2 V for 30 s, which also ensures a reasonable detectability with the nitrogen cooled CCD camera used. A progressive increase of cell resistance is mild and slow enough to allow 5-8 reuses of the cell within an error below 15%. A cleaning step consisting of water rinsing and sonication between successive measurements was found ineffective in preventing the decrease of the signal. Moreover, a disposable single use of the cell will further improve the analytical performance by increasing the acquisition time. The precision of the ECL signal was evaluated by nine replicates in repeated light/current/potential measurements by changing the solution and washing the cell before each run. The

Figure 5. Dependence of the log of ECL intensity (10-8 A/V, amplification range) on the molar concentration of Ru(bpy)32+. (Note: the sample was measured starting from the lower concentration of the ruthenium complex. PMT, 750 V; photocurrent amplification, 10-8 A/V; 0.1 M PBS solution (pH 7.5) in the presence of 3 × 10-2 M TPA. Background has been subtracted.)

mean peak intensity and standard deviation of eight ECL signal replicates was 1.33 ± 0.17 au suggesting an acceptable precision of the measurements. Analytical Dose-Response Curve. Bulk Measurement. The mean IECL experimental values of five replicates (±SD) as a function of the Ru(bpy)32+ complex concentration in solution are reported in Figure 5. A good correlation between ECL intensity and the Ru(bpy) 32+ complex concentration was observed in the 10-8-10-6 M range where the SD values showed good reproducibility of the data. At a higher concentration of 10-5 M, the measurements present a high variability due to the electrode working performance deterioration with a rapid formation of filming products at its surface. The detectability is mainly affected by the background emission, and the specific electrode response is efficient at a concentration higher than 10-8 M. The relatively high background emission could be associated with the thin layer cell geometry characterized by extended interphases and with the intrinsic nature of the electrode material that may cause partial adsorption of the Ru(bpy)32+ complex on its surface. According to these data, the dynamic range of the calibration curve was limited to 10-8-10-6 M and IECL data were fitted with the following regression equation: Y ) (0.4050 ± 0.03175)X +

(-0.2133 ± 0.06860); R2 ) 0.994 (n ) 6) (Figure 5). The limit of detection (LOD) defined as the concentration giving a signal corresponding to the mean signal of the blank plus three times its standard deviation was 2.0 × 10-8 M. The optimal working pH ranged from 7.5 to 8.1 similar to that of conventional electrochemical cells,35 and these values are also compatible with common immunological and hybridization reactions. The IECL values obtained with the Ru(bpy) 32+ complex in solution and with a PMT detector can be applied to ECL microscope imaging of microbeads with a diameter of 8-10 µm using a nitrogen cooled CCD camera with a high sensitivity and low dark noise. Imaging Measurements. Considering the electrochemical cell volume of 20 µL, the transparent electrode surface of 16 mm2, and a concentration of the Ru(bpy)32+ complex of 10-7 M, we can calculate that the IECL derived from a 1 µm2 sample surface corresponds to a concentration of 6.25 × 10-20 mol/µm2. From the imaging acquisition using the CCD camera, we were able to obtain an image on a single bead as low as ∼104 Ru(bpy)32+ complex molecules. These data show that ECL performance is similar to those obtained with conventional CL using HRP/luminol as a labeling system. The device needs the use of an ultrasensitive CCD where the instrumental dark noise is highly minimized by cooling the camera with liquid nitrogen at -100 °C; in addition, the red emitted light (620 nm) corresponds to the higher wavelength region of the CCD sensitivity. The resolution of the acquired images varied from 1 (20× objective) to 0.4 (60× objective) µm/image pixel according to the different numerical aperture (NA) of the used lenses. The 60× magnification objective presents a NA of 0.95, and therefore, with a light emission at 620 nm, a resolution of 0.4 can be achieved, which allows one to accurately localize the Ru(bpy)32+ complex on the microbead surface. ECL Microscope Imaging of Labeled Microbeads. The polystyrene carboxylated microbeads (8 µm diameter) covalently coupled to an ECL-active ruthenium reproduce a real situation on which an analyte, localized on cells, needs to be imaged on a microscope glass slide. Carboxylated beads (1 in Scheme 1) were conjugated with the Ru(bpy)32+-NH2 derivative to form a stable amide bond. The suspension containing beads (2 in Scheme 1) was deposited on the cell glass surface in the presence of 10-2 M TPA, and

Figure 6. Microscope imaging 20× objective/30 ms. (A) Transmitted light image and (B) ECL image of Ru(bpy)32+-conjugated polystyrene microbeads. (C) Overlay of the reflected light image and the false color elaborated CL image. Analytical Chemistry, Vol. 81, No. 15, August 1, 2009

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Figure 7. Scheme of the ECL optical imaging cell (75 × 25 × 1 mm) and microscope imaging 20× objective/30 ms. (A) Transmitted light image and (B) ECL image of Ru(bpy)32+-conjugated polystyrene microbeads using the biotin-streptavidin interaction.

images were acquired. The live image (Figure 6A) was obtained with the transmitted light from the microscope lamp and was used to locate the bead positions. The ECL image (Figure 6B) was obtained by applying the potential to the cell and acquiring the ECL light for 30 s from the sample (with the source lamp from the microscope turned off). The ECL image showed a good overlap with the live image (Figure 6C), which allowed both the detection and the spatial localization of labeled beads in the microbead sample. In order to better simulate the biological environment, the conventional biotin-streptavidin reaction (Method II) was used. ECL images were acquired showing a good match (Figure 7). The ECL signal is produced only by the microbeads in correspondence to the FTO electrode and not by the microbeads localized on the nonconducting glass support. This clearly demonstrated that the emitted luminescence is triggered only on the labeled beads present in the proximity of the electrode surface. Signal-to-noise ratio for Method I and Method II ECL images were, respectively, 3.2 ± 0.2 and 2.3 ± 0.2. The lower value obtained for Method II is likely to be caused by the lower surface density of the ECL label for beads (4 in Scheme 2) with respect to beads (2 in Scheme 1) due to steric hindrance. CONCLUSIONS In this work, a new transparent electrochemical cell using FTO glass was developed and optimized for ECL microscope imaging. The developed cell presents optimal electrochemical properties allowing cyclic voltammetric and ECL light/current/voltage measurements in a standard PBS/Ru(bpy)23+ complex solution. The cell geometry and dimensions were selected to fit with the configuration of a conventional optical microscope, and the working electrode area of 16 mm2 was compatible with the field of view obtained by the magnification of the conventional 6240

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microscope lenses. The cell volume of 20 µL allows the addition of coreactants such as TPA for the ECL or allows the use of a conventional cell for CL or other luminescent-based measurements such as TRF on the same sample. The microscope imaging of the ECL probe on the microbeads can be obtained with high resolution and detectability. The intrinsic properties of the ECL-based detection method allowed one to minimize the background noise since no spontaneous CL reaction can be obtained. In addition, the ECL signal can be switched on or off anytime, offering the opportunity to perform other measurements in an acceptable time scale. As electrode material, FTO proved to be a good choice in terms of stability and reproducibility of the cell. However, FTO like ITO is a relatively delicate material that can be easily scratched and electrically passivated. Furthermore, it is important to consider that these are not the “ideal” electrode materials in terms of electrical resistance and maximum current density. For these reasons, gold or platinum surfaces would improve system performances significantly but cannot be employed because of their opacity even if used as a very thin coating. The electrochemical cell allows one to achieve sensitive imaging of the ECL labeled beads with the nitrogen cooled CCD camera with an acquisition time of 30 s, which is the optimized condition compatible with the light signal intensity and with the quality of the electrochemical measurements and the lifetime of the electrode. Under these conditions, the electrode can be reused several times. Moreover, the prototype of the cell reported here has been obtained with a simple manual procedure to demonstrate the feasibility of this technique; the use of microfabrication technology for cell production will improve the ECL emission intensity and reproducibility and, thanks to the low cost of the materials

used, the cell can be proposed as a disposable single-use test. The present method demonstrated the possibility to detect and precisely localize by ECL the binding of labeled antibodies or DNA on a biological sample; thus, it was applied to the most common diagnostic and research techniques based on microscopy imaging. The method could also be used in multiplex analysis employing different ECL labels such as the osmium system (Os(bpy)32+ E1/2(ox) ) 0.85 V vs Ag/AgCl)36 or luminol (E1/2(ox) ) 0.500 V in the presence of H2O2 vs Pt).11 This device can also be used for metal ion metabolism inside cells studied by the formation of ECL-active metal ion complexes.37,38 In addition, different luminescence-based methods such as bioluminescence (BL) and CL can be employed to

simultaneously localize different analytes and achieve an ultrasensitive multiplexed imaging technique. ACKNOWLEDGMENT We are grateful to Dr. M. Marcaccio and Professor M. Guardigli for useful discussions and helpful support. We are also grateful to Dr. E. Marzocchi for help in bioconjugation. Received for review April 8, 2009. Accepted June 5, 2009. AC900756A (37) Bruno, J. G.; Collard, S. B.; Kuch, D. J.; Cornette, J. C. J. Biolumin. Chemilumin. 1996, 11, 193–206. (38) Bruno, J. G.; Collard, S. B.; Andrews, A. A. J. Biolumin. Chemilumin. 1997, 12, 155–164.

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