Hot Electron-Induced Electrogenerated Chemiluminescence of Ru

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Anal. Chem. 1999, 71, 5538-5543

Hot Electron-Induced Electrogenerated Chemiluminescence of Ru(bpy)32+ Chelate at Oxide-Covered Aluminum Electrodes T. Ala-Kleme,*,† S. Kulmala,‡ L. Va 1 re,† P. Juhala,§ and M. Helin‡

Department of Chemistry, University of Turku, FIN-20014 Turku, Finland, Helsinki University of Technology, P.O. Box 6100, FIN-02015 HUT, Finland, and Orion Diagnostica, P.O. Box 425, FIN-20101 Turku, Finland

Ruthenium(II) tris-(2,2′-bipyridine) chelate (Ru(bpy)32+) is a well-known label molecule in bioaffinity assays such as immuno and DNA-probing assays.1 Salts of Ru(bpy)32+ are very stable, water-soluble compounds that can be chemically modified with reactive groups on one of the bipyridyl ligands to form activated species with which, for example, proteins, haptens, and nucleic acids are readily labeled.2 Therefore, Ru(bpy)32+ has been used as label on the basis of different techniques among others in the electrogenerated chemiluminescence (ECL) based analysis.3 Traditionally, the ECL of Ru(bpy)32+ has been measured in the conventional three-electrode cell using gold or platinum electrodes as both counter and working electrodes and Ag/AgCl as a reference electrode.4 The ECL mechanism of Ru(bpy)32+ normally utilized is based on the use of coreactants which produce reducing

radicals upon one-electron oxidization. In commercial kits the coreactant is typically a tertiary amine, tripropylamine (TPA). Both Ru(bpy)32+ label and TPA are oxidized at the surface of the working electrode (Au or Pt), forming Ru(bpy)33+ derivative and TPA+•, respectively. In the appropriate pH range, the TPA+• loses a proton, forming strongly reducing TPA•, which successively reacts with strongly oxidizing Ru(bpy)33+ resulting in the excited label molecule Ru(bpy)32+*. The resulting 3MLCT excited state decays to the ground state by emitting light at 620 nm. In principle, the excitation cycle may occur repeatedly several times for each label molecule.4 Another alternative, and constructionally a more simple system, is based on the use of cathodically pulse-polarized disposable oxide-covered aluminum electrodes with a quite freely selectable counter electrode. According to previous studies, strongly oxidizing species such as sulfate and hydroxyl radicals can be cathodically generated at pulse-polarized oxide-covered aluminum electrodes in fully aqueous solutions.5 The cathodic pulse polarization of thin oxide film-covered aluminum electrodes induces, as the primary step, a tunnel emission of hot electrons (ehot) into aqueous electrolyte solutions, probably resulting in a subsequent generation of hydrated electrons (eaq-) and oxidizing radicals such as sulfate radicals (SO4•-) from added coreactants.6 Hot, or hydrated, electrons can react with compounds that are hard to reduce, and therefore, cathodic reductions usually not possible to carry out in aqueous solutions can be made.7 Analogous phenomena have also been observed at oxide-covered silicon electrodes.8 It is probable that not all of the emitted hot electrons are reacting at the aluminum oxide/solution interface with solute species during the high-amplitude cathodic pulse polarization. If tunnel-emitted electrons have enough energy, they can enter into the conduction band of water and become hydrated electrons after

* Corresponding author: (fax) 358-2-3336700; (e-mail) [email protected]. † University of Turku. ‡ Helsinki University of Technology. § Orion Diagnostica. (1) Sammes, P. G.; Yahioglu, G. Nat. Prod. Rep. 1996, 1-28. (2) Juris, A.; Balzani, V.; Barigelletti, F.; Campagna, S.; Belser, P.; Zelewsky, A. Coord. Chem. Rev. 1988, 84, 85-277. (3) Collinson, M.; Pastore, P.; Maness, K.; Wightman, R. J. Am. Chem. Soc. 1994, 93, 165-168. (4) Blackburn, G.; Shah, H.; Kenten, J.; Leland, J.; Kamin, R.; Link, J.; Peterman, J.; Powell, M.; Shah, A.; Talley, D.; Tyagi, S.; Wilkins, E.; Wu, T.-G.; Massey, J. Clin. Chem. 1991, 37, 1534-1539.

(5) Kulmala, S.; Kankare, J.; Haapakka, K. Anal. Chim. Acta 1991, 252, 6576; Kulmala, S and Haapakka, K, J. Alloys Compd. 1995, 225, 502-506; Kulmala, S.; Kulmala, A.; Ala-Kleme, T.; Pihlaja, J. Anal. Chim. Acta 1998, 367, 17-31; Kulmala, S.; Ala-Kleme, T.; Latva, M.; Loikas, K.; Takalo, H. J. Fluoresc. 1998, 8, 59-65. (6) Kulmala, S.; Ala-Kleme, T.; Hakanen, A.; Haapakka, K. J. Chem. Soc., Faraday Trans. 1997, 93, 165-168.; Kulmala, S.; Ala-Kleme, T.; Heikkila¨, L.; Va¨re, L. J. Chem. Soc., Faraday Trans. 1997, 93, 3107-3113. (7) Kulmala, S.; Ala-Kleme, T.; Joela, H.; Kulmala, A. J. Radioanal. Nucl. Chem. 1998, 232, 91-95. (8) Ala-Kleme, T.; Kulmala, S.; Latva, M. Acta Chem. Scand. 1997, 51, 541546.

Ruthenium(II) tris-(2,2′-bipyridine) chelate exhibits strong electrogenerated chemiluminescence during cathodic pulse polarization of oxide-covered aluminum electrodes in aqueous solutions. The present method is based on a tunnel emission of hot electrons into an aqueous electrolyte solution. The method allows the detection of ruthenium(II) tris-(2,2′-bipyridine) and its derivatives below nanomolar concentration levels and yields linear log-log calibration plots spanning several orders of magnitude of concentration. This method allows simultaneous excitation of derivatives of ruthenium(II) tris-(2,2′-bipyridine) and Tb(III)-chelates. The former label compounds have a luminescence lifetime of the order of microseconds, while the latter compounds generally have a luminescence lifetime of around 2 ms. Thus, the combined use of these labels easily provides the basis for two-parameter bioaffinity assays by either using wavelength or time discrimination or their combination.

5538 Analytical Chemistry, Vol. 71, No. 24, December 15, 1999

10.1021/ac981336i CCC: $18.00

© 1999 American Chemical Society Published on Web 11/09/1999

thermalization and solvation processes.9 This precludes that the concentration of the coreactant added to produce oxidizing radicals is not too high and electron species are not too efficiently scavenged by oxidizing radical precursors, such as peroxodisulfate ion, hydrogen peroxide, or molecular oxygen. Peroxodisulfate ions, hydrogen peroxide, and molecular oxygen react near a diffusion-controlled rate with hydrated electrons and produce highly oxidizing sulfate or hydroxyl radicals upon one-electron reduction.10 Hence, highly reducing and oxidizing conditions are simultaneously achieved in the vicinity of the electrode surface by appropriate selection of the concentration of the cathodic coreactant.6 ECL of aromatic compounds at active metal electrodes in aqueous solutions is generally impossible for energy reasons. However, the simultaneous presence of hydrated electrons (E° ) -2.9 V vs SHE)10 and sulfate radicals (E° ) 3.4 V vs SHE)11 is capable of generating chemiluminescence (CL) from a wide variety of compounds that otherwise are hard to reduce or hard to oxidize.5-7 It has previously been shown that cathodic hotelectron injection into aqueous electrolyte solutions can induce ECL from aromatic Tb(III) chelates and some organic luminophores, which can also be used as labels in immunoassays.5-7 Lyoluminescence studies have shown that simultaneous genera•- by dissolution of alkali halides containing tion of eaq and SO4 trapped electrons into peroxodisulfate solutions induces a strong CL of Ru(bpy)32+ chelate in fully aqueous solutions.12 Furthermore, we have recently demonstrated that luminol also shows strong ECL during cathodic pulse polarization of oxide-covered aluminum electrodes in aqueous solution when highly oxidizing radicals are cathodically generated.13 However, luminol shows strong CL in the presence of sulfate radicals generated by other methods, as well, and does not require necessarily the reduction of luminol or its follow-up products.14 When sulfate radicals are generated in aqueous Ru(bpy)32+ solution in the absence of strong reductants no CL is generated.15 On this basis, Ru(bpy)32+/S2O82- aqueous system should produce strong ECL at oxide-covered aluminum electrodes, if the above concept of forming primary radicals is correct. Ru(bpy)32+ is known to produce cathodic ECL in the presence of peroxodisulfate ions at active metal electrodes in acetonitrile and acetonitrile/water mixtures, but not in fully aqueous solutions.16 Bard et al. have suggested that either Ru(bpy)3+ is not produced, or is a highly unstable species, or peroxodisulfate is always reduced with concerted two-electron transfer at an active metal electrode (9) Pleskov, Y.; Rotenberg, Z. Photoemission of Electrons from Metals into Electrolyte Solutions as a Method for Investigation of Double-Layer and Electrode Kinetics. In Advances in Electrochemistry and Electrochemical Engineering; Gerischer, H.; Tobias, C., Eds.; Wiley: New York, 1978; pp 3-115. Hart, E.; Anbar, M. The Hydrated Electron; Wiley: New York, 1970, and references therein. (10) Buxton, G.; Greenstock, C.; Helman, W.; Ross, A. J. Phys. Chem. Ref. Data 1988, 17, 513-886, and references therein. (11) Memming, R. J. Electrochem. Soc. 1969, 116, 785-790. (12) Kulmala, S.; Hakanen, A.; Raerinne, P.; Kulmala, A.; Haapakka, K. Anal. Chim. Acta 1995, 309, 197-210. (13) Kulmala, S.; Ala-Kleme, T.; Kulmala, A.; Papkovsky, D.; Loikas, K. Anal. Chem. 1998, 70, 1112-1118. (14) Matachescu, C.; Kulmala, S.; Ala-Kleme, T.; Joela, H. Anal. Chem. 1997, 69, 3385-3390. (15) Kulmala, S. Ph.D academic dissertation, University of Turku, Finland, Turku, 1995. (16) Ege, D.; Becker, W. ja Bard, A. Anal. Chem. 1984, 56, 6: 2413-2417.

in fully aqueous solutions.16 The present work was carried out to study the mechanism of cathodic ECL of Ru(bpy)32+ chelate in fully aqueous solution and its analytical applicability. EXPERIMENTAL SECTION Reagents and Instrumentation. Ruthenium(II) tris-(2,2′bipyridine) chloride hexahydrate, benzophenon-4-carboxylate, and Tween 20 were purchased from Aldrich. K2S2O8, Na2B4O7‚10H2O, H2O2, NaNO3, NaNO2, NaBr, NaI, NaN3, NaSCN, and HCOONa were pro analysi or suprapur products of Merck. Ethanol was supplied by Oy Alko Ab and suprapur Na2SO4 by Merck. Hexammine cobalt(III) chloride was a product of Ventron. Quartzdistilled water was used in all solutions. The safety precautions required in handling of the reagents have been presented previously.13,14 The ECL measurements were made in 0.2 M boric acid buffer at pH 9.2. Boric acid buffer was used because of its unreactivity toward sulfate radicals and hydrated electrons. The methods of ECL excitation and both the coulostatic and potentiostatic pulse generator are described elsewhere.5,6,15 Aluminum electrodes were made from a nominally 99.9% pure aluminum band (Merck Art. 1057) and were covered with natural about 2-3 nm thick or thicker anodically oxidized oxide film.6 All measurements were made through an interference filter having a transmission maximum of 620 nm and bandwidth of about 10 nm. The apparatus has been described earlier elsewhere.17 Immunoassay of hTSH. Oxide-covered aluminum disks with diameters of 20.0 mm were cut from aluminum band and degreased with hexane. The electrodes were coated with antihTSH antibodies in a Teflon container that restricted the active area to a circle with a diameter of 15.0 mm in the middle of the disks. The coating was carried out in a buffer that contained 2-(NMorpholino)ethanesulfonic acid, 0.1 mol/L, and boric acid, 0.3 mol/L, in pH 6.5 bovine gammaglobulin 0.005%, 1% glutaraldehyde. The coating antibody was treated prior to coating at pH 2.5 with sodium citrate for 15 min. The final concentration of anti-hTSH (MOAB anti-TSH MIT 0406 Lot M-21310) was 1.5 µg/mL, and incubation time was 3 h. After coating, the disks were saturated with bovine serum albumin by incubating them for 1 h in a solution containing 0.05 M Tris pH 7.5 (pH adjusted with H2SO4), 0.1% bovine serum albumin, 0.1% Tween 20, 0.1% NaN3. After saturation the disks were dried in a vacuum (1 Torr) at a temperature of 37 °C and additionally incubated at 80 °C for 30 min. Another monoclonal anti-TSH antibody (Medix Biochemica clone 5404 anti-hTSH lot sp077) was labeled with Ru(bpy)32+ derivative (Igen, Inc., Gaithersburg, MD) according to the manufacturers instructions. One hundred nanograms of the labeled antibody, 300 µL of hTSH standards (Orion Oyj, Finland), and 700 µL of incubation buffer were added in each Teflon container with the disposable oxide-covered aluminum electrode. The incubation buffer was 0.05 M Tris buffer at pH 7.8 (pH adjusted with H2SO4) and contained 0.9% NaCl, 0.5% BSA, 0.05% NaN3, 0.05% bovine gammaglobulin, 0.01% Tween 20, 20 µmol/L DTPA. Incubation was carried out for 1 h in a shaker at room temperature. (17) Kankare, J.; Fa¨lde´n, K.; Kulmala, S.; Haapakka, K. Anal. Chim. Acta 1992, 256, 17-28.

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Figure 1. Effect of excitation mode on the ECL of Ru(bpy)32+. DC polarization -9 V and pulse polarization amplitude -9 V, pulse time 200 µs, frequency 100 Hz. Conditions: 1.0 × 10-5 M Ru(bpy)32+ in 1.0 M Na2SO4/0.2 M boric acid-borate buffer at pH 9.2, Pine Instruments RD4 potentiostat, homemade pulse generator.

After incubation the disks were washed twice with a solution that contained 0.05 mol/L Tris pH 7.4, 0.1% Tween 20, 0.2% BSA, 0.05% NaN3. The ECL was measured with the slightly modified apparatus of ref 17 using a 0.2 M borate buffer at pH 7.8 containing 1.0 mmol/L K2S2O8 as a measuring buffer. The measuring cell was modified to contain a stainless steel cylinder with an inner diameter of 10.0 mm serving as an anode through which the light was detected from above with a photon counter. The working electrode was sealed and insulated from the counter electrode cylinder with a Teflon gasket. The electrolyte volume was 350 µL. RESULTS AND DISCUSSION Basic Features of Ru(bpy)32+ ECL. An ECL emission spectrum of Ru(bpy)32+ measured at the cathodically polarized oxide-covered aluminum electrode having the maximum at the wavelength of 620 nm has been previously measured.6 The ECL spectrum6 demonstrates the occurrence of the well-known emission from the 3MLCT excited state of Ru(bpy)32+. The ECL intensity depends strongly on the electrogenerated excitation mode, i.e., the use of cathodic direct current (DC) or pulse polarization. The big difference between the ECL profiles of these two excitation modes can be seen in Figure 1 where the ECL intensity of Ru(bpy)32+ has been presented as a function of time in both pulse and DC polarization methods at the oxide-covered aluminum cathodes. After a cathodic flash, the direct-currentinduced ECL is totally faded away within 20 ms in buffered solutions. If the cathodic polarization is continued for a longer time, the ECL yield gradually starts to increase as the alkalinization of the electrode surface and the dissolution of the oxide film advances. However, in unbuffered solutions at extremely high current densities (e.g., 250 mA/cm2) DC polarization-induced alkalinization of the electrode surface dissolves the oxide film within a couple of seconds, yielding CL of these chelates in the presence of peroxodisulfate ions. This CL can be simulated by an addition of alkali hydroxide in the sample solution (Figure S1, Supporting Information) and, on the other hand, after disconnection of strong cathodic DC current CL fades away only after diffusion has removed the pH gradient in the cell.15 Pulsed excitation produces naturally a similar ECL yield during the first excitation pulse to the flash occurring at the moment of cathodic 5540 Analytical Chemistry, Vol. 71, No. 24, December 15, 1999

Figure 2. Ru(bpy)32+ ECL intensity as a function of solution initial pH. Conditions: 1.0 × 10-6 M Ru(bpy)32+ in 0.1 M Na2SO4 supporting electrolyte; solution was adjusted to the desired pH with sulfuric acid or sodium hydroxide. Coulostatic pulse generator, pulse charge 300 µC, voltage -40 V, and frequency 40 Hz.

DC polarization, but the successive excitation pulses produce an increasing ECL intensity of around as much as 4 s (pulse time 200 µs and frequency 100 Hz) after which the ECL intensity remains unchanged during several tens of seconds (Figure 1). After the cathodic pulse, a strong anodic current transient must be allowed to pass through the cell as has been observed earlier in the case of other luminophores.5,7 As shown earlier,5 many aromatic Ln(III) chelates efficiently produce cathodic ECL at thin oxide film-covered aluminum electrodes, impossible to produce at active metal electrodes in fully aqueous solutions. These ECL processes become possible only if the base metal of the metal/insulator/electrolyte junction is cathodically pulse-polarized to a more cathodic potential than ∼-3 V vs SHE.6,7 After that potential value, the ECL intensity rapidly starts to increase. Although the redox and luminescence properties of Tb(III) and Ru(bpy)32+ chelates differ strongly, their cathodic ECL onset pulse potentials coincide. This indicates that the mechanism of the ECL of all these luminophores is based on the same primary process. The ECL onset pulse potential of oxidecovered aluminum metal is close to the conduction band edge of water, which suggests that the electrochemical generation of hydrated electrons at oxide-covered aluminum by electron injection into the conduction band of water can be the common cathodic process in both of these electrogenerated chemiluminescences. In addition, it has been recently shown that the effects of oxide film thicknesses on the ECL intensity of Tb(III) and Ru(bpy)32+ chelates and some other luminophores are analogous.7 In all these cases, the ECL, or time-resolved ECL, intensity started to decrease exponentially when the aluminum oxide film thickness exceeded 4-5 nm. Thus, also Ru(bpy)32+ chelate seems to behave in the same way as all the other tested luminophores6,7 and show a strong ECL only in the direct field assisted tunneling regime.6,7,18 The ECL dependence of Ru(bpy)32+ chelate on pH in airsaturated solutions is presented in Figure 2. The ECL intensity seems to be practically constant in the pH range 3-11. In the alkaline region the chemiluminescence effect has to be taken into consideration. The effect of hydroxide ion concentration on the CL intensity of Ru(bpy)32+ in the presence of peroxodisulfate ions is presented in Figure S1. The CL intensity of Ru(bpy)32+ starts to rapidly and linearly increase from the hydroxide ion concentra(18) DiMaria, D.; Cartier, E. J. Appl. Phys. 1995, 78, 3883-3894.

the secondary radicals produced by the free-radical scavenger used above is displayed in Figure 4 at flat-band potential. A schematic energy diagram of the actual tunnel emission and Fowler-Nordheim tunneling at a thin insulating film-covered electrode in contact with an aqueous solution has been presented earlier.6 When eaq are available, sulfate radicals can be conveniently generated from peroxodisulfate ions according to reaction 1. 2ef SO4•- + SO42aq + S2O8

Figure 3. Dependence of the ECL intensity on the Ru(bpy)32+ and peroxodisulfate ion concentrations. Ru(bpy)32+ concentration varied, 1.0 × 10-3 M K2S2O8 solution (9), and peroxodisulfate concentration varied, 1.0 × 10-5 M Ru(bpy)32+ solution (b). Conditions: coulostatic pulse generator, pulse charge 300 µC, voltage -40 V and frequency 40 Hz, 0.2 M boric acid-borate buffer at pH 9.2.

tion of 10-4 to 10-1 M (S1). At higher hydroxide ion concentration metallic ruthenium is clearly observed in the aluminum cup, and the chemiluminescence is naturally decreased.12 However, it is clear that the present strong ECL occurs only in the pH range where the aluminum oxide film is sufficiently stable (Figure 2), and therefore, the chemiluminescence induced by aluminum metal in contact with the aqueous solution is not the basis of the ECL during cathodic pulse polarization. An analogous situation was previously observed in the case of the ECL of aromatic lanthanide chelates.15 Figure 3 shows that the ECL intensity at the wavelength of 620 nm depends linearly on the concentration of Ru(bpy)32+ at oxide-covered disposable aluminum electrodes during high current density pulsed excitation. However, the calibration plot of the peroxodisulfate ion is more mildly sloping. According to the calibration curve (Figure 3) the Ru(bpy)32+ specific ECL shows a linear response for Ru(bpy)32+ concentration over 6 orders of magnitude from 10-5 to 10-11 mol/L concentration level. One order of magnitude lower detectability of Ru(bpy)32+ is achieved by adding Tween 20 as a detergent into the sample (Figure S2). The optimum peroxodisulfate ion concentration was observed to be 10-3 M (Figure 3). Mechanism of Ru(bpy)32+ ECL at Cathodically Pulse Polarized Oxide-Covered Aluminum Electrodes. If hydrated electrons and sulfate radicals are involved, as assumed in the present Ru(bpy)32+ ECL system, hydrated electron and sulfate radical scavengers should have a strong effect on the observed ECL intensity. The effects of the different free-radical scavengers are presented and discussed in the Supporting Information section. In short, all the tested hydrated electron scavengers were observed to quench the ECL signal in the order of their reactivities with the eaq (Figure S4). In addition, sulfate radical scavengers quenched the ECL more strongly the higher the rate of their scavenging reaction rate constant for sulfate radical was, and the weaker the resulting secondary oxidizing radical was as an oxidant (Figure S4). Cathodic pulse polarization of oxide-covered aluminum electrodes induces, as the primary step, tunnel emission of hot electrons into aqueous electrolyte solutions, which can become hydrated electrons after thermalization and solvation processes as has been discussed in detail elsewhere.6,7 The energy diagram of the present system containing also the energetics of some of

(1)

After these initial processes, there are two obvious pathways to raise the Ru(bpy)32+ to its excited 1MLCT or 3MLCT states at energies of 2.7 and 2.1 eV, respectively.2 The ECL excitation route of Ru(bpy)32+ can occur by the reduction-initiated oxidative excitation (red-ox) pathway (reactions 2a and 2b) or the oxidation-initiated reductive excitation (ox-red) pathway (reactions 3a and 3b). + Ru(bpy)32+ + eaq f Ru(bpy)3

(2a)

Ru(bpy)3+ + SO4•- f 1Ru(bpy)32+* + SO42-

(2b)

Ru(bpy)32+ + SO4•- f Ru(bpy)33+ + SO42-

(3a)

2+ 1 Ru(bpy)33+ + eaq f Ru(bpy)3 *

(3b)

In these excitation processes, Ru(bpy)32+ is rapidly reduced 2+ 10 -1 by hydrated electron [k(eaq + Ru(bpy)3 ) ) 8.2 × 10 l mol -1 10 s ] or oxidized by a sulfate radical. The next step is the formation of 1Ru(bpy)32+* or 3Ru(bpy)32+*, i.e., the Ru(bpy)32+ in its excited 1MLCT or 3MLCT state, by the oxidation of Ru(bpy)3+ (reaction 2b) or the reduction of Ru(bpy)33+ (reaction 3b) with the second-order rate constant of k3b ) 5.2 × 1010 l mol-1 s-1 but the reaction rate constant k2b is unknown.10 According to basic thermodynamics, the calculated enthalpies for reactions 2b and 3b are 4.5 and 4.0 eV, respectively, when the standard reduction potentials of SO4•-/SO42-, Ru(bpy)32+/Ru(bpy)3+, Ru(bpy)33+/Ru(bpy)32+, and hydrated electron are 3.4 V, -1.28 V, 1.26 V, and -2.9 V vs SHE.2,10,11 These results clearly demonstrate that the excitation steps (2b and 3b) are sufficiently energetic to leave the resulting Ru(bpy)32+ even in its 1MLCT state at 2.7 eV above the ground state, but due to a fast intersystem crossing (kISC > 1010 s-1)2, the 1MLCT state is essentially exclusively relaxed (reaction 4) to the 3MLCT state at 2.1 eV, which is further radiatively relaxed to the ground state (reaction 5) inducing the 620-nm emission peak.

Ru(bpy)32+* f 3Ru(bpy)32+*

(4)

Ru(bpy)32+* f Ru(bpy)32+ + hν (620 nm)

(5)

1

3

As far as we know, the rate constant k2b has not been determined. If the sulfate radical is assumed to show a RehmWeller type of behavior, the reaction 2b can be considered to occur at nearly a diffusion-controlled rate. However, in the increasing order of the oxidizing power of the following oxidants, their Analytical Chemistry, Vol. 71, No. 24, December 15, 1999

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Figure 4. Schematic energy diagram of an Al/Al2O3/aqueous solution interface at flat-band potential.6 The redox potentials of inorganic couples and Ru(bpy)32+ are taken from refs 2 and 10.

second-order reduction rate constants k(CO3•- + Ru(bpy)32+), k(Br2•- + Ru(bpy)32+), and k(Cl2•- + Ru(bpy)32+) have been reported to be 4 × 108, 3.1 × 109, and 1.6 × 109 L mol-1 s-1, respectively (standard reduction potentials 1.5, 1.9, and 2.1 V yielding ∆G values -0.24 eV, -0.64 eV, and -0.84 eV for chelate oxidation).19 If the decrease of rate constant in the above series between dibromine and dichlorine radical anion is taken as a sign of oxidation of Ru(bpy)32+ and reduction of the sulfate radical following a Marcus type of behavior, the reaction 2b can be considered to be quite slow in comparison with reaction 2a due to the very high Gibbs energy change, -2.14 eV, of step (2b). In this case, step (2a) is very rapid, and the oxidation of Ru(bpy)3+ by sulfate radical would be slow if resulting in the ground state of Ru(bpy)32+ (∆G ) -4.68 eV) but more rapid if resulting in 1MLCT (∆G ) - 1.98 eV) or 3MLCT (∆G ) -2.58 eV) excited states. Also, the oxidation rate constant should then be higher in cases of excited-state end products. Thus, both red-ox and oxred excitation pathways are energetically possible, but the predominant excitation pathway is red-ox pathway, if the sulfate radical is assumed to show a Marcus inverted region in its oxidizing reactions. On the other hand, it is well-known that the hydrated electron shows Rehm-Weller type of behavior10 in its reactions which allows the second step of the ox-red excitation pathway to be extremely rapid. However, results supporting the red-ox excitation pathway as the predominant excitation mechanism have been presented by Mulazzani et al.20 The third theoretical pathway to raise the Ru(bpy)32+ in its excited 3MLCT state is a comproportionation reaction in which (19) Neta, P.; Huie, R.; Ross, A. J. Phys. Chem. Ref. Data 1988, 17, 1027-1284, and references therein. (20) Mulazzani, Q.; Emmi, S.; Fuochi, P.; Venturi, M.; Hoffman, M.; Simic, M. J. Phys. Chem. 1979, 83, 1582-1590; Mulazzani, Q.; Emmi, S.; Fuochi, P.; Hoffman, M.; Venturi, M. J. Am. Chem. Soc. 1978, 100, 981-983; Casalboni, F.; Mulazzani, Q.; Clark, C.; Hoffman, M.; Orizondo, P.; Perkovic, M.; Rillema, D. Inorg. Chem. 1997, 36, 2252-2257.

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Ru(bpy)3+ and Ru(bpy)33+ formed by reactions 2a and 3a are reacting together, forming 3Ru(bpy)32+* and Ru(bpy)32+ with enthalpy of 2.3 eV. Unfortunately, our experimental data do not allow definite conclusions to be drawn about predominance of any of these three different excitation pathways. It is possible that all the above-mentioned excitation pathways proceed in parallel in the case of solvated Ru(bpy)32+ chelate. However, we propose that in the case of actual immunoassay only red-ox and ox-red excitation pathways are important, especially taking into account that Ru(bpy)32+ labels are not free in the solution but bound onto the surface of the working electrode, located relatively far away from each other and not diffusing freely. Analytical Applicability of Ru(bpy)32+ ECL at Cathodically Pulse Polarized Oxide-Covered Aluminum Electrodes. The present method provides a linear calibration curve for Ru(bpy)32+ spanning over several orders of magnitude of Ru(bpy)32+ concentration (Figure 3). The addition of Tween 20 to the measurement solution as a detergent was observed to improve sensitivity (Figure S2). The special advantage of the present excitation method is that various label molecules having different redox and optical properties can be simultaneously excited. Another benefit is that both wavelength and time discrimination can be utilized in the separation of the signals emerging from different labels. Thus, the basis is easily created for two-parameter assays, internal standardization, or even multicomponent analysis. For example, Ru(bpy)32+ can be used as a pair with aromatic Tb(III) chelates (Figure 5), where one parameter is labeled with a Ru(bpy)32+ label exhibiting a short-lived luminescence and the other parameter with a Tb(III) chelate with a long luminescence lifetime. To demonstrate the usability of the cathodic ECL of the Ru(bpy)32+ label in bioaffinity assays, an immunoassay of human thyroid stimulating hormone (hTSH) was carried out using disposable aluminum electrodes. The calibration curve of hTSH (Figure S5) measured exploiting a Ru(bpy)32+ label prepared using a commercial labeling

On these grounds, Ru(bpy)32+ based labels must be used in the case of the present excitation method only in the assays not requiring ultimate sensitivity, and in two-parameter assays, the parameter requiring highest sensitivity must be labeled with a Tb(III) chelate21 and the less demanding parameter with a Ru(bpy)32+ derivative. The most important advantage of the present excitation method is the possibility to simultaneously excite molecules emitting light in a very wide spectral range, from the UV to the NIR, and molecules having very different luminescence lifetimes. In comparison, the multiparameter assay possibilities of the traditional anodic ECL generation method of Ru(bpy)32+ labels are very modest in this respect. ACKNOWLEDGMENT Financial support by the Technology Development Center, Finland, and Orion Diagnostica is gratefully acknowledged. Figure 5. Simultaneous ECL excitation of Ru(bpy)32+ and a Tb(III) chelate.

kit gives a linear response over 3 orders of magnitude of concentration to as low as 1 mIU/L level (y ) 1.054x + 3.083; R ) 0.9989, SD ) 5.6%, N ) 5).

NOTE ADDED IN PROOF We have noticed that the following important papers on hotelectron electrochemistry have appeared since the submission of the present paper: Gaillard, F.; Sung, Y.-E.; Bard, A. J. J. Phys. Chem. B 1999, 103, 667-674; Sung, Y.-E.; Bard, A. J. J. Phys. Chem. B 1998, 102, 9806-9811; Sung, Y.-E.; Gaillard, F.; Bard, A. J. J. Phys. Chem. B 1998, 102, 9797-9805.

CONCLUSIONS Cathodic pulse polarization of disposable oxide-covered aluminum electrodes provides a basis for relatively sensitive detection of Ru(bpy)32+ based labels. However, the traditionally used anodic ECL of these labels at nondisposable noble metal electrodes gives better detection limits. This is mainly based on the very low blank emission of the anodic ECL chemistry at suitably preconditioned gold electrodes. The drawback of the present method is the solidstate luminescence induced by the aluminum oxide film under high-field conditions necessary for the hot-electron emission into the aqueous solution and variations in the blank emission on the basis of purity and fabrication method of the aluminum oxide film.

SUPPORTING INFORMATION AVAILABLE The effect of hydroxide ion concentration on dissolution of aluminum-induced chemiluminescence of Ru(bpy)32+ (Figure S1), ECL calibration plot of Ru(bpy)32+ in the presence of Tween 20 (Figure S2). Results obtained with hydrated electron and sulfate radical scavengers are discussed in the SI section (Figures S3 and S4). Calibration plot of immunometric hTSH immunoassay (Figure S5). This material is available free of charge via the Internet at http://pubs.acs.org.

(21) Kankare, J.; Karppi, A.; Takalo, H. Anal. Chim. Acta 1994, 295, 27-35.

AC981336I

Received for review September 23, 1999.

December

1,

1998.

Accepted

Analytical Chemistry, Vol. 71, No. 24, December 15, 1999

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