Anal. Chem. 2001, 73, 3282-3288
A Wireless Electrochemiluminescence Detector Applied to Direct and Indirect Detection for Electrophoresis on a Microfabricated Glass Device Arun Arora,† Jan C. T. Eijkel,† Werner E. Morf,‡ and Andreas Manz*,†
Astra Zeneca/SmithKline Beecham Centre for Analytical Sciences, Department of Chemistry, Imperial College of Science, Technology & Medicine, Exhibition Road, South Kensington, London, SW7 2AY, United Kingdom, and Sensors, Actuators and Microsystems Laboratory (SAMLAB), Institute of Microtechnology (IMT), University of Neuchaˆ tel, Rue Jaquet-Droz 1, CH-2007 Neuchaˆ tel, Switzerland
A novel electrochemiluminescence (ECL) detector is presented in this article. The detector is applied for micellar electrokinetic chromatographic separation of dichlorotris(2,2′-bipyridyl)ruthenium(II) hydrate [Ru(bpy)] and dichlorotris(1,10-phenanthroline)ruthenium(II) hydrate [Ru(phen)] on a microfabricated glass device. It consists of a microfabricated “U”-shape floating platinum electrode placed across the separation channel. The legs of the U function respectively as working and counter electrode. The required potential difference for the ECL reaction is generated at the Pt electrode by the electric field available in the separation channel during electrophoretic separation. Initial experiments demonstrate a micellar electrokinetic separation and direct ECL detection of 10-16 mol of Ru(phen) (10-6 M) and 4.5 × 10-16 mol of Ru(bpy) (5 × 10-6 M). Also, preliminary results show the indirect detection of three amino acids. The high voltage at the location of detection does not interfere with the electrochemistry. Electrochemiluminescence (ECL) is a highly specific and sensitive detection protocol used for a diversity of analytical applications. Duffard1 (1927) observed ECL first time in solutions of Grignard compounds in anhydrous ether. Harvey2 (1929) observed the ECL of luminol at 2.8 V in an aqueous alkaline solution. Paris and Brandt in 1959 discovered that dichlorotris(2,2′-bypyridyl)ruthenium(II) hydrate [Ru(bpy)] is photoluminescent.3 Chemiluminescence (CL) and ECL have advantages over fluorescence because they do not require a light source, which results in a simple instrumentation and low or zero background signal. The zero background signal allows optical detectors to be used at their maximum sensitivity. Various analytes have been successfully detected using ECL in a flow cell or a batch cell. They * Corresponding author: (e-mail)
[email protected]. † Imperial College of Science, Technology & Medicine. ‡ University of Neucha ˆ tel. (1) Duffard, R. T.; Nightingale, D.; Gaddum, L. W. J. Am. Chem. Soc. 1927, 49, 1848. (2) Harvey, N. J. Phys. Chem. 1929, 33, 1456. (3) Paris, J. P.; Brandt, W. W. J. Am. Chem. Soc. 1959, 81, 5001.
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include amines,4 amines surfactants,5 amino acids,6 glucose,7 ethanol,8 oxalate,9 naphthlene,10 erythomycin,11 codeine,12 pyruvate,13 polymerase chain reaction products,14 proviral DNA,15 and many others mentioned in the review papers.16-20 ECL was not investigated in detail until 1966. The ECL of Ru(bpy) has been investigated constantly since 1966 starting from the publication by Hercules.21 Bard and co-workers reported a series of investigations on the mechanism of Ru(bpy)33+ ECL reactions.22-26 Leland and Powell27 reported, for the first time, the use of tripropylamine (TPA) as a reducing agent for the ECL reaction. Brune and Bobbit28 studied the effect of pH on the ECL response of amino acids and Ru(bpy)2+in a continuous-flow electrochemical system. The ECL from Ru(bpy) in aqueous medium without addition of reducing agent was not reported until recently when Ficcabrino and co-workers29 observed ECL of only (4) Uchikura, K.; Kirisawa, M.; Sugii, A. Anal. Sci. 1993, 9, 121. (5) Alexander, C. J.; Ritcher, M. M. Anal. Chem. Acta 1999, 402, 105. (6) He, L.; Cox, K. A.; Danielson, N. D. Anal. Lett. 1990, 23, 195. (7) Martin, A. F.; Nieman, T. A. Anal. Chim. Acta 1993, 281, 475. (8) Lee, W. Y. Microchim. Acta 1997, 127, 19. (9) Egashira, N.; Kumasako, H.; Ohga, K. Anal. Sci. 1990, 6, 903. (10) Blatchford, C.; Humphreys, E.; Malcome-Lawes, D. J. J. Chromatogr. 1985, 329, 281. (11) Danielson, N. D.; He, L.; Noffsinger, J. B.; Trelli, L. J. Pharm. Biomed. Anal. 1989, 7, 1281. (12) Michel, P. E.; Fiaccabrino, G. C.; de Rooij, N. F. Anal. Chim. Acta 1999, 392, 95. (13) Knight, A. W.; Greenway, G. M. Analyst 1995, 120, 2543. (14) Kenten, J. H.; Casadei, J.; Link, J.; Lupold, S.; Willey, J.; Powell, M.; Rees, A.; Massey, R. Clin. Chem. 1991, 37/9, 1626. (15) Schutzbank, T. E.; Smith, J. J. Clin. Microbiolol. 1995, 33, 8, 2036. (16) Knight, A. W.; Greenway, G. M. Analyst 1994, 119, 879. (17) Knight, A. W. Trends Anal. Chem. 1999, 18, 47. (18) Bowie, A. R.; Sanders, P. J.; Worsfold, P. J. Biolumin. Chemilumin. 1996, 11, 61. (19) Rozhitskii, N. N.; Belash, E. M.; Bykh, A. I. J. Anal. Chem. USSR 1994, 49, 829. (20) Gerardi, R. D.; Barnett, N. W.; Lewis, S. W. Anal. Chim. Acta 1999, 378, 1. (21) Hercules, D. M.; Lytle, F. E. J. Am. Chem. Soc. 1966, 88, 4745. (22) Tokel, N. E.; Bard, A. J. J. Am. Chem. Soc. 1972, 92, 2862. (23) Chang, M. M.; Saji, T.; Bard, A. J. J. Am. Chem. Soc. 1977, 99, 5339. (24) Rubinstein, I.; Martin, C. R.; Bard, A. J. Anal. Chem. 1983, 55, 1580. (25) Fan, F. R. F.; Cliffel, D.; Bard, A. J. Anal. Chem. 1998, 70, 2941. (26) Ritcher, M. M.; Bard, A. J. Anal. Chem. 1998, 70, 310. (27) Leland, J. K.; Powell, M. J. J. Electrochem. Soc. 1990, 137, 10, 3127. (28) Brune, S. N.; Bobbit, D. R. Anal. Chem. 1992, 64, 166. (29) Fiaccabrino, G. C.; Koudelka-Hep, M.; Hsueh, Y. T.; Collins, S. D.; Smith, R. L. Anal. Chem. 1998, 70, 4157. 10.1021/ac0100300 CCC: $20.00
© 2001 American Chemical Society Published on Web 06/02/2001
Ru(bpy)2+ in water at a carbon interdigited microelectrode array of 2-µm width and spacing. Ru(bpy) has been the most studied and exploited inorganic ECL compound to date because of its capability of undergoing ECL at room temperature in aqueous buffered solutions, in the presence of dissolved oxygen and other impurities. It has been widely used in ECL flow cells and probe injection analysis systems to detect various chemicals and biochemical assays. The ECL generation from Ru(bpy) has been achieved in many ways. The majority of work reported uses a flow injection system or a batch method (electrochemical cell) using a three-electrode system to generate Ru(bpy)3+. Colins30 reported the use of Ru(bpy)3+ solution in a PTFE diffusion membrane and a flow cell mounted in front of a light-sensing device to detect the vapor of hydrazine and its derivatives. Egashira31 reported the use of Nafion film on a Pt gauze electrode to immobilize Ru(bpy)2+ and then detect oxalates in phosphate buffer. This approach increased the working pH range for ECL. It was, however, reported to be sensitive to temperature changes, and the system shows a reduced overall sensitivity. In CE, conventional three-electrode systems employed postcolumn32,33 show problems with the isolation from the high separation voltage. Some efforts have been reported to integrate an ECL detector with CE as a postcolumn detector.34-36 Forbes37 used a porous polymer junction near the end of the capillary to complete the CE circuit and inserted a working electrode into the capillary end to generate ECL. A parabolic reflector was used to direct emitted light to the PMT. He reported a detection limit of 0.6 µg mL-1 for β-blockers. In liquid chromatography (LC), Ru(bpy) ECL has been reported for precolumn,38 on-column,39,40 or postcolumn41,42 detection. Generally employing a large-area Pt electrode, the reported limit of detection in most cases was in the submicromolar range. The postcolumn generation of ECL requires the mixing of the separated analytes with Ru(bpy) and the transport of the mixture to the working electrode. In this process, analyte bands are broadened so that the sensitivity is reduced and separation severely compromised. Commercial ECL technology43 involves the use of Ru(bpy) assays on the surface of paramagnetic beads. Labeled analytes are bound to these beads and passed into a flow cell, where a magnet captures the beads on the surface of an electrode. A voltage is applied to the electrode to excite labeled analytes and (30) Colins, C. E. Sens. Actuators B Chem. 1996, 35, 202. (31) Egashira, H.; Kumasako, H.; Kurauchi, Y.; Ohga, K. Anal. Sci. 1994, 10, 405. (32) Gavin, P. F.; Ewing, A. G. J. Am. Chem. Soc. 1996, 118, 8932. (33) Wang, J.; Tian, B.; Sahlin, E. Anal. Chem. 1999, 71, 5436. (34) Dickson, J. A.; Ferris, M. M.; Milofsky, R. E. J. High Resolut. Chromatogr. 1997, 20, 643. (35) Bobbit, D. R.; Jackson, W. A. U.S. Patent 5614073, March 25, 1997. (36) Tsukagoshi, K.; Miyamoto, K.; Saiko, E.; Nakagima, R.; Hara, T.; Fujinaga, K. Anal. Sci. 1997, 13, 639. (37) Forbes, G. A.; Nieman, T. A.; Sweedler, J. V. Anal. Chim. Acta 1997, 347, 289. (38) Uchikura, K.; Kirisawa, M. Chromatography 1995, 16, 232. (39) Scotty, D. R.; Lee, W. Y.; Nieman, T. A. Anal. Chem. 1995, 68, 1530. (40) Ridlen, J. S.; Klopf, G. J.; Kissinger, P. T.; Nieman T. A. J. Chromatogr., B 1997, 694, 393. (41) Ridlen, J. S.; Klopf, G. J.; Nieman, T. A. Anal. Chim. Acta 1997, 341, 195. (42) Uchikura, K.; Kirisawa, M. Anal. Sci. 1991, 7, 971. (43) Igen Web Page, http://www.igen.com/technology.htm, 27th November 2000, 10:12 am.
the emitted light is recorded. Unbound labels, which are further from the electrode, are not excited. Hsueh44 reported the drawbacks to this technology. The nonconductive and paramagnetic beads limit the generation of ECL and their opacity partially blocks the signal from reaching the detector. Miniaturization of conventional analytical equipment45 offers many advantages such as short reaction time, safe handling, and safe storage of small quantities, low consumption of reactants, automation, and integration of separation and detection on the same chip. Microfabrication techniques of glass and silicon devices made it possible for capillary electrophoresis46 and other separation techniques to be carried out on a chip. High-throughput analysis,47 polymerase chain reaction,48 highspeed DNA sequencing,49 single DNA molecule detection,50 enzyme assays,51 indirect detection of phenols52 in water and photographic chemicals,53 and on-line synthesis of organic compounds and their detection by coupling the reactor with a mass spectrometer54 have been successfully demonstrated on microfabricated devices. To utilize the full potential of miniaturized analytical instruments, there is a need for integrated miniaturized detectors. A laser-induced fluorescence detector provides sensitivity even to the single-molecule limit,55,56 but having a detection system that is many times larger than the original device reduces one of the benefits of miniaturization. Electrochemical detectors can be smaller in size and offer a low detection limit. However, their use in CE on chip on column has not been yet reported because it is difficult to operate such a system in the presence of a high-voltage field in the separation channel. Electrochemical detectors at the end of column or off-column has been the subject of various publications.32,33,57-60 The Ewing32 group reported a postcolumn amperometric detector for on-chip CE, using an array of 100 Pt microelectrodes (each 95 µm wide and individually addressed). A Pt counter and a Ag/AgCl pellet reference electrode were placed in the same reservoir as the CE ground electrode and the current on each individual array electrode was monitored. This system detects picomole quantities of dopamine and catechol. Recently, Wang (44) Hsueh, T. T.; Collins, S. D.; Smith, R. L. Sens. Actuators B 1998, 49, 4. (45) Sanders, G. H. W.; Manz, A. J. Assoc. Lab Autom. JALA 2000, 5, 40. (46) Manz, A.; Harrison, D. J.; Verpoorte, E.; Widmer, H. M. Adv. Chromatogr. 1993, 33, 1-66. (47) Shi, Y.; Simpson, P. C.; Scherer, J. R.; Wexler, D.; Skibola, C.; Smith, M. T.; Mathies, R. Anal. Chem. 1999, 71, 5354. (48) Kopp, M. U.; De Mello, A. J.; Manz, A. Science 1998, 280, 1046. (49) Liu, S.; Shi, Y.; Ja, W. W.; Mathies, R. Anal. Chem. 1999, 71, 566. (50) Effenhauser, C. S.; Bruin, J. M.; Paulus, A.; Ehrat, M. Anal. Chem. 1997, 69, 3451. (51) Hadd, A. G.; Raymond, D. E.; Halliwell, J. W.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1997, 69, 3407. (52) Arundell, M.; Whalley, P. D.; Manz, A. Fresenius J. Anal. Chem. 2000, 367, 686. (53) Sirichai, S.; Demello, A. J. Analyst 2000, 125, 133. (54) Mitchell, M.C.; Spikmans, V.; Bessoth, F.; Manz, A. In Micro Total Analysis Systems 2000; van den Berg, A., Olthuis, W., Bergveld, P., Eds.; Kluwer Academic Publishers; Dordrecht 2000; pp 463-465. (55) Haab, B. B.; Mathies, R. A. Anal. Chem. 1995, 67, 3253. (56) Nie, S.; Chiu, D. T.; Zare, R. N. Science 1994, 226, 10181. (57) Woolley, A. T.; Lao, K.; Galzer, N.; Mathies, R. J. Anal. Chem. 1998, 70, 684. (58) Fermier, A. M.; Gostkowski, M. L.; Colon, L. A. Anal. Chem. 1996, 68, 1661. (59) Wanj, J.; Chatrathi, M. P.; Tian, B. Anal. Chem. 2000, 72, 5774. (60) Singhal, P.; Kuhr, W. G. Anal. Chem. 1997, 69, 3552.
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and co-workers33 reported the use of a thick, screen-printed, carbon electrode to detect electrophoretically separated dopamine and catechol in the submicromolar range. ECL detectors have been reported to be used successfully in small-volume detectors44,61 and microfabricated chips29,44,62,63 as integrated detectors and show high sensitivity. The light output of an ECL detector unlike that of an electrochemical detector is not affected by the presence of background electrical noise. Micellar electrokinetic capillary chromatography (MECC or micellar electrokinetic chromatography, MEKC, in general) was first reported by Terabe and co-workers64,65 to separate low molecular weight and nonionic analytes. The separation mechanism in MECC is based on the differential partitioning of solutes into a micellar pseudophase. The application of the technique has been proven very useful for a wide range of charged and uncharged compounds.66 This has been achieved by manipulation of the composition of micellar solutions and the incorporation of different types of chemical equilibria such as acid-base, complexation, ion exchange, or ion pairing. Floating electrodes are used for various applications. Under the name of bipolar electrodes,67 they are used in industrial reactors to reduce electrical connections and power dissipation in external circuits. Conducting electrode particles in a stream of electrolyte (referred to as fluidized bed electrodes) are used in electrochemical reactors.68,69 Fluidized bed electrodes offer large electrode area per volume and high mass and heat transfer. In high-voltage systems, floating electrodes are used to measure the electric field. They perform better than the conventional generating voltmeters (field mills) simply because floating electrodes do not have any mobile parts and offer contactless measurement of the electric field.70 The method presented also bears a slight resemblance to biamperometric71 equipment. In this study, Ru(bpy) and dichlorotris(1,10-phenanthroline)ruthenium(II) hydrate [Ru(phen)] were separated and then detected. A glass device with a microfabricated “U”-shaped Pt electrode across the separation channel was used, where its legs function as floating working electrode and counter electrode. Since the Pt electrode is not connected to any external power source, it is referred to as a floating electrode. The voltage required to carry out the ECL reaction on the floating electrode is the result of the electric field present in the separation channel during separation. (61) Arora, A.; de Mello, A. J.; Manz, A. Anal. Comm. 1997, 12, 393. (62) Fiaccabrino, G. C.; de Rooij, N. F.; Koudelka-Hep, M. Anal. Chim. Acta 1998, 359, 263. (63) Greenway, M.; Nelstrop, L. J.; Port, S. N. Anal. Chim. Acta 2000, 405, 43. (64) Terabe, S.; Otsukak, K.; Ichikawa, K.; Tsuchiya, A.; Ando, T. Anal. Chem. 1984, 56, 111. (65) Terabe, S.; Otsukak, K.; Ando T. Anal. Chem. 1985, 57, 834. (66) Landers, J. P. CRC Handbook of capillary Electrophoresis: A Practical Approach; CRC Press: London, 1994. (67) Picket, J. D. Electrochemical Reactor Design, 2nd ed.; Elsevier Scientific Publishing Co.: New York, 1979; p 34. (68) Kazdobin, K.; Shvab, N.; Tsapakh, S. Chem. Eng. J. 2000, 79, 203. (69) Matsuno, Y.; Tsutsumi, A.; Yoshida, K. Int. J. Hydrogen Energy 1997, 22, 6, 615. (70) Roman, F.; Cooray, V.; Scuka, V. J. Electrost. 1997, 40-41, 483. (71) Delahay, P. New instrumental methods in electrochemistry: theory instrumentation and applications to analytical and physical chemistry; Interscience: New York, 1954; p 258.
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Figure 1. Demonstration of the Pt floating electrode effect. (A) Weighing boat filled with universal indicator solution, with two Pt electrodes on each end (red, positive; blue, negative) connected to 30 V dc. (B) With a floating Pt foil electrode immersed. (C) With a U-shaped floating Pt wire electrode half immersed.
Since Ru(bpy) and Ru(phen) both have 2+ charge and their molecular weights are very close (748.63 and 712.61) their electrophoretic separation was effected using MEKC. EXPERIMENTAL SECTION Chemical Reagents. CE buffer containing 90 mM TPA was prepared by diluting a 180 mM stock solution (Procell, phosphate 300 mM, SDS 0.1%, pH 6.8 from Boehringer Mannheim) with high resistivity, deionized water (maxima Ultrapure Elga). Dichlorotris(2,2′-bypyridyl)ruthenium (II) hydrate (Fluka, Buchs, Switzerland) and dichlorotris(1,10-phenanthroline)ruthenium (II) hydrate (Aldrich) were used to prepare 10 mM stock solutions. Mixtures of these solutions in various proportions, as required, were used to prepare test samples. To demonstrate the floating electrode mechanism, a solution of universal pH indicator (Acros Organics, Geel, Belgium) was used. Indirect detection CE buffer containing 0.5 mM Ru(bpy) was prepared by mixing CE buffer with 10 mM Ru(bpy)‚H2O stock solution for the indirect detection of amino acids. L-Valine, L-aspartic acid, and L-alanine were obtained from Sigma Chemical Co. (St. Louis, MO). Their 1 mM stock solutions were prepared by dissolving the required quantities in deionized water. These stock solutions were mixed with the CE buffer solution for indirect detection to prepare sample solutions. Floating Electrode Mechanism Demonstration. A weighing boat (5 cm long, 3 cm wide, and 0.5 cm deep), as shown in Figure 1A, was prepared for demonstration by drilling two holes on two corners using a needle. Two 0.5-mm platinum wires were glued using Araldite rapid epoxy glue (Ciba-Geigy, Duxford, U.K.) in each hole. A drop of universal pH indicator was placed in the boat
Figure 2. (a) Schematic diagram of the bonded glass chip. The short separation channel structure (shown in dark lines) was used for all measurements. (b) Channel layout. (c) Photo of the device after drilling the holes and gluing the reservoirs. Key: 1, sample reservoir; 2, buffer reservoir; 3, sample waste, a vacuum tube was connected to this reservoir to fill sample in the double-T injector; 4, buffer waste; 5, double-T injector; 6, separation channel; 7, floating platinum electrode (leg width 50 µm, distance between legs 50 µm.); 8, sample filled into the double-T injector (sample volume 90 pL estimated from video images); 9, vacuum; 10, direction of plug during separation. Separation channel’s total length 6.1 cm from (2) to (4) and 2.5 cm from (5) to (7).
and diluated with ultrapure water to fill three-quarters of the boat. A dc power supply (Instek model 3030D, RS Components) at 30 V was used to provide the dc voltage. A Pt foil, 0.8 and 1.5 cm (Figure 1B), was used as a single floating electrode, and a 5-cmlong Pt wire of 0.5-mm diameter was used as a U-shaped floating electrode (Figure 1C). ECL Glass Chip. Corning glass devices measuring 5 cm × 2 cm were fabricated using a standard photolithography procedure followed by wet chemical etching and thermal bonding at 600 °C. The U-shaped Pt film electrodes (thickness 100 nm, legs 50 µm wide, spacing 50 µm) were fabricated on a thin underlayer of chromium using a liftoff method. The device structure is shown in Figure 2a. The structure shown with dark lines was used for all experiments. Figure 2b shows the schematic layout of the chip structure and a photo of the double-T injector filled with a sample. All channels in this structure were 60 µm wide and 20 µm deep. The channel length from buffer reservoir to buffer waste was 6.1 cm and the length between double-“T” injector and Pt electrode was 2.5 cm. The devices were made by the Alberta Microelectronic Centre (Alberta, Canada). Thermally bonded glass chip was prepared for experiment by drilling holes in the cover plate using the electrochemical discharge drilling method.72 In variance with the reported method the anode (a Pt wire of 0.5-mm diameter) was used as a drilling needle and 80 V was applied from a dc power supply (model (72) Shohi, S.; Esashi, M. Photoetching and Electrochemical Discharge Drilling of Pyrex Glass, Technical Digest of the 9th Sensor Symposium, Japan, 1990; p 27.
SAE2761, Shandon Scientific Co. Ltd., London, England) to produce a constant spark in 8 M sodium hydroxide solution. When a steel needle cathode was used as reported in ref 72, a large amount of brown precipitate was formed, which entered into the channel and created blocks. PVC pipets were glued around the access holes using rapid epoxy glue (Figure 2c). The chips were left overnight for proper curing. Experimental Setup. A Photomultiplier tube (PMT, model R3896, Hamamatsu, Hamamatsu City, Japan), operating at 1250 V, was placed inside the metal box for data collection. The emitted light from the reaction reaches the PMT window through a 2-mm aperture at the top of the metal box. A personal computer with a data acquisition card (model AT-MIO-16X, National Instruments) and Lab View (V 5.0) was used to control high-voltage power supplies (model 7E-12-500, FUG HCN). The output signal from the PMT was recorded using a Pico analog-digital convertor (ADC12, Pico Instrument). A microscope (model Leica DMIL, Leica Microsystems UK Ltd., London, U.K.) with mercury illumination source and a H3-513827 filter was used to optimize CE separation. A vacuum pump (Charles Austen Pumps Ltd., Surrey, England) was used to fill, empty, or clean the channels with buffer solution and to inject sample into the double-T region. Chip Cleaning Procedure. The glass chip was cleaned before each experiment by running chrome sulfuric acid (1 M), deionized water, hydrochloric acid (1 M), deionized water, sodium hydroxide solution (1 M), and deionized water, sequentially each for ∼5 mins. Finally, the chip was rinsed with TPA buffer. Vacuum-Driven Injection. As shown in Figure 2b, the sample was injected into the separation channel by applying vacuum to the sample waste reservoir. It was observed from the video images of the sample injection process that the actual injection volume of the sample filled in the double-T region was ∼90 pL. Reaction Mechanism. The reaction mechanism between TPA and Ru(bpy) used was described earlier.27 A graphical representation of the reaction mechanism on a floating electrode system is shown in Figure 3b. The reaction between Ru(phen) and TPA is equivalent to the reaction between Ru(bpy) and TPA. Ru(phen) [or Ru(bpy)] and TPA are oxidized in aqueous solution at the anode at ∼1.1 V versus Ag/AgCl. The TPA cation is unstable and becomes deprotonated almost immediately to form TPA′. Subsequent electron transfer from TPA′ to Ru(phen)3+ [or Ru(bpy)3+] causes the formation of an excited state Ru(phen) molecule [Ru(phen)*], which then relaxes radiatively back to the ground state (λemission ≈ 610 nm). It is noted that although TPA is consumed during the reaction, the Ru(phen) (or Ru(bpy)) is recycled. Theory. In the Supporting Information some theory is presented, describing the current through a floating electrode as a function of the potential difference between its ends. The theory considers the special case of an electrode of one piece, exposed to a solution containing a single irreversible couple. On the basis of the equality of anodic and cathodic current in a floating electrode (net current between the electrode and the solution is zero, and the electrode behaves as a complete electrochemical cell), a relation is derived between the current that flows through the electrode and the electrical potential difference V to which the electrode is exposed between its terminal ends. For the case considered, the current is shown to be proportional to V for V , Analytical Chemistry, Vol. 73, No. 14, July 15, 2001
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Figure 4. Effect of changes in separation voltage on light signal output in indirect detection buffer containing 1 mM Ru(bpy). Table 1 shows the steps used while switching voltage. Table 1. Separation Fields Strength and Floating Electrode Detection Voltage as a Function of Employed Separation Voltage
Figure 3. (a) Schematic drawing showing approximate potential window when the potential difference V applied between the ends of the floating electrode is 1.0 and 2.0 V. (b) Pictorial representation of the reaction of Ru(bpy) or Ru(phen) with TPA. Key: 1, separation (HV) anode; 2, separation (HV) ground; 3, U-shaped Pt film electrode over separation channel; 4, separation channel; 5, solution containing Ru(bpy) or Ru(phen) and TPA; 6, EOF direction.
0.1 V, changing to an exponential relationship for V . 0.1 V and increasing drastically. In the experiments described in this paper, several species will participate in the overall reactions at the electrode, and an increase of the applied separation potential will allow more couples to contribute to the overall current through it. As a result the average “absolute” potential of the floating electrode will not be defined by one redox couple alone but by several couples present, their concentrations, and reversibility. Such a situation is tentatively depicted in Figure 3a. It shows the approximate potentials for oxidation of water, TPA, and TBR at the anodic end of the electrode and the reduction of O2 and H2O at the cathodic end. Also indicated are the voltage area where the electrode is expected to be when it is exposed to voltage differences of respectively 1 and 2 V. At 1 V the current is expected to be limited by the limited availability of reducible substances (i.e., in this case, oxygen), making the average voltage more positive than at 2 V. RESULTS AND DISCUSSION Demonstration of Principle. The application of a dc voltage of 30 V between the two Pt electrodes suspended in the universal pH indicator solution (Figure 1A) shows the expected pH changes at cathode and anode due to electrolysis. When a Pt foil is placed 3286 Analytical Chemistry, Vol. 73, No. 14, July 15, 2001
voltage applied
separation field strength (V/cm)
detector voltage difference (V)
62.5 125 187.5 250 312.5 375 437.5 500 562.5 625 687.5 750 812.5 875
10.25 20.49 30.74 40.98 51.23 61.48 71.72 81.97 92.21 102.46 112.70 122.95 133.20 143.44
0.15 0.31 0.46 0.61 0.77 0.92 1.08 1.23 1.38 1.54 1.69 1.84 2.00 2.15
in the middle as shown in the Figure 1B, the side of Pt foil electrode facing the anode acts as a cathode and the side facing toward the cathode acts as an anode. The changes in the indicator color around the edges clearly show this phenomenon. A U-shaped Pt wire shows the same behavior (Figure 1C), the leg of the electrode facing the anode behaving as a cathode and the leg facing the cathode behaving as an anode. This phenomenon can be explained by considering that the Pt wire short-circuits the solution over its length, thus creating a potential difference between its ends proportional to the fraction of the total distance short-circuited. Application in on Chip CE. Figure 4 shows the light signal generated at a floating Pt electrode on application of a gradually increasing separation voltage between buffer reservoir and buffer waste when all channels were filled with 0.5 mM Ru(bpy) solution in TPA buffer. The voltage between the terminal points of the two electrode legs at the different applied voltages is shown in Table 1. A light signal was observed at separation voltages as low as 10.25 V/cm (0.15 V between the terminal points of the electrode). Since a cyclic voltammogram of Ru(bpy) suggested that the ECL reaction starts at 1.1 V versus Ag/AgCl or higher,61 it was interesting to observe a light signal at such a low voltage. A possible explanation is that species that were reduced at ∼0.9 V versus Ag/AgCl at the negative leg of the electrode were carried
by the electroosmotic flow to the surface of the positive leg to be oxidized there. This effect would be comparable to the ECL observed at an interdigitated electrode array.29 On application of a separation voltage higher than 133.2 V/cm, the current becomes unstable and the light signal drops sharply. This is due to the electrolysis of the aqueous buffer and the formation of gas bubbles displacing solution and decreasing the current density. The increase in the light signal with the increase of separation voltage will be due to the increase in the electrochemical reaction rate of Ru(bpy) as described in the theoretical section (Supporting Information). It is also possible that the increase in the electroosmotic flow (EOF) with increasing voltage increases the mass transfer of TPA to the electrode. The U-shaped platinum electrode has an underlayer of chromium to improve its adhesion to the glass surface. The chromium layer corroded by electrochemical reactions during the separation and was attacked by acid during the washing process, leaving the Pt film in the air, inside the channel. It was broken into pieces when vacuum was applied to fill the channel with buffer or by the EOF in the separation channel. According to Pourbaix,73 in the presence of chloride ions chromium metal corrodes heavily at -0.5 to -0.6 V vs NHE regardless of the pH of the solution. Pictures of devices with broken electrodes and with leakage are presented in the Supporting Information. The thickness of the Pt film was ∼100 nm, causing insufficient bonding around the electrode. In a few devices it led to visible leakage around the electrode which could be quite significant. In these devices, the Pt electrode life was reasonably longer. In the devices where this effect was absent, electrodes were eroded away after a few runs of CE. This is probably due to the fact that, in the devices with leakage, the thickness of the electrode film was much more than 100 nm. In the “leaky” devices, the direct detection of Ru(phen) and Ru(bpy) was not affected. However, a weak or no signal was detected during indirect detection because a large area of the electrode, not just the area above the channel, was responsible for ECL reaction, causing a overwhelming background signal. Separation of Ru(phen) and Ru(bpy). Various concentration ratios of Ru(bpy) and Ru(phen) were injected and separated by MEKC. Figure 5A shows the electropherogram obtained from the light emitted from the floating Pt electrode for a Ru(bpy)/Ru(phen) (3:4) mixture. A separation field strength of 133.2 V/cm was used. The Ru(bpy) peak appeared after 91.7 s (plate number N ) 5200, apparent migration velocity 273 µm/s) and the Ru(phen) peak after 97.25 s (N ) 4800, apparent migration velocity 257 µm/s). The electropherogram shown in Figure 5B was obtained on injection of a sample mixture of Ru(bpy)/Ru(phen) (3:1) under the same electrophoretic conditions. It is clear from the electropherograms that the ECL detector responds to the changes in the concentration of the analytes. Figure 5C was obtained on injection of a sample with only Ru(phen) under the same electrophoretic conditions as above (N ) 5800, apparent migration velocity 258 µm/s) (73) Pourbaix, M. Atlas of Electrochemical Equlibria; Pergamon Press: Oxford, U.K., 1966; p 265.
Figure 5. Electropherograms for Ru(bpy) and Ru(phen) separation and ECL detection. (A) Peaks for sample mixture of Ru(bpy) and Ru(phen) (3:4). (B) Peaks for sample mixture of Ru(bpy) and Ru(phen) (3:1). (C) Peak for sample of Ru(phen) only.
Figure 6 shows the log-log calibration plots obtained for Ru(bpy) and Ru(phen). Continuous lines represent the signal points observed when samples were injected into the separation channel and dashed lines represent the signal points observed when the channels were filled with sample. When samples were injected individually, the detection limits for Ru(bpy) and Ru(phen) were 5 × 10-6 and 6 × 10-6 M (S/N ) 3), respectively. On filling the channels with samples in sequence, concentrations as low as 8 × 10-7 M Ru(bpy) and 1.6 × 10-7 M Ru(phen) could be detected. The lower detection in the latter case can be simply explained by the fact that the sample concentration around the electrode is stable due to continuous supply of fresh sample. In contrast, after injection, the sample plug migrates toward the floating electrode and during migration band-broadening process dilutes the plug zone, which decreases the signal intensity. It is interesting to compare the detection limits obtained, which are in the low-micromolar range, with those of some other chipintegrated detection systems. The flow-through (non-separationbased) detection chips using CL63 and ECL62 obtain comparable Analytical Chemistry, Vol. 73, No. 14, July 15, 2001
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Figure 7. Background signal observed for the indirect detection buffer containing 0.5 mM Ru(bpy) at the application of 133.2 V/cm separation voltage. Indirect electropherogram for the separation of three amino acids (L-valine, L-aspartic acid, and L-alanine at 0.3 mM each in the sample) at a separation voltage 133.2 V/cm.
Figure 6. Calibration plots for Ru(bpy) (a) and Ru(phen) (b). Continuous lines represent points recorded when samples were injected and dashed lines represent points recorded when sample solutions were filled into the channel.
detection limits, 0.1 and 0.5 µM, respectively. Detection limits for CE chips with integrated electrochemical detection were reported to be 3-12 µM.57 Noting the inherent simplicity of the present detector, it therefore compares well with these devices. Indirect Detection of Amino Acids. To show that indirect detection in principle is possible using the floating electrode detector, a preliminary experiment was performed on indirect detection of amino acids. Figure 7a shows the background signal in the presence of the indirect detection buffer containing 0.5 mM Ru(bpy) at a separation field strength of 133.2 V/cm. The signal is built up of two responses. The first response is almost instantaneous and counts for about two-thirds of the total response. The slower second response represents one-third of the total light output. The slow response is possibly due to adhesion of ECL producing ions at or around the electrode. On switching off the separation voltage the signal drops down to zero. On switching the same voltage back on, the signal again shows the same behavior but now the fast response accounts for more than twothirds of the total, indicating some accumulation process. This slow response is also clearly visible in the indirect detection peaks of amino acids in Figure 7b. Figure 7b shows the negative peaks observed on the injection of a sample containing three amino acids (L-valine, L-aspartic acid, and L-alanine at 0.3 mM each) in the buffer solution. The identification of each amino acid was not performed as the objective of the experiment was to demonstrate the possibility of using ECL indirect detection. 3288 Analytical Chemistry, Vol. 73, No. 14, July 15, 2001
CONCLUSION The preliminary experiments presented in this paper demonstrate that the floating electrode ECL detector can be used successfully for the detection of Ru(bpy) and Ru(phen) following electrophoretic separation. In addition, it was shown that nonlabeled analytes following electrophoretic separation in Ru(bpy)TPA buffer can be detected indirectly. The problems related to the electrode deterioration and leakage are the major inhibiting factors in the present devices. Properly bonded devices with robust floating Pt electrodes are expected to show longer electrode lifetimes and increased reproducibility. Optimization of the distance between the floating electrode and the injection point will decrease the band broadening and will improve the detection limit for individually injected samples. ACKNOWLEDGMENT Authors thank Astra Zeneca and SmithKline Beecham for their financial support, the Worshipful Co. of Scientific Instrument Makers for its WCSIM Burser 1997/98 Award, Dr. John Crabtree of the Alberta Microelectronic Corp. for fabricating the microstructures, Dr. Darwin Reyes for help with photographs, Dr. Martin Arundell for constructing the CE setup, Dr. Chao-Xuan Zhang of the Imperial College, London, and Dr. Martin Boutelle of the King’s College, London, for the valuable personal discussions, and finally editor Dr. Royce W. Murray for his interest in this work and constructive comments. SUPPORTING INFORMATION AVAILABLE Theoretical Information. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review January 8, 2001. Accepted April 21, 2001. AC0100300
