Anal. Chem. 1998, 70, 1112-1118
Cathodic Electrogenerated Chemiluminescence of Luminol at Disposable Oxide-Covered Aluminum Electrodes S. Kulmala,*,† T. Ala-Kleme,† A. Kulmala,† D. Papkovsky,‡ and K. Loikas†
Department of Chemistry, University of Turku, FIN-20014 Finland, and Department of Biochemistry, University College, Cork, Ireland
Luminol exhibits strong electrogenerated chemiluminescence during cathodic pulse polarization of oxide-covered aluminum electrodes in aqueous solution. This electrogenerated chemiluminescence can be enhanced by the presence of dissolved oxygen or by the addition of other coreactants such as hydrogen peroxide, peroxydisulfate, or peroxydiphosphate ions. However, luminol detection is most sensitive in the presence of azide ions, which not only enhance the electrogenerated chemiluminescence intensity but also decrease the intrinsic electroluminescence of the thin aluminum oxide film on the electrodes mainly producing the blank emission. The present method is based on tunnel emission of hot electrons into an aqueous electrolyte solution and allows the detection of luminol, isoluminol, and its derivatives below nanomolar concentration levels. The linear logarithmic calibration range covers several orders of magnitude of concentration of luminol or N-(6-aminohexyl)-N-ethylisoluminol. Therefore, the above-mentioned labeling substances can be used as one of several available alternatives of simultaneous markers in multiparameter bioaffinity assays at disposable oxide-covered aluminum electrodes. The main advantage of the present electrochemiluminescence generation method is that luminescent compounds having very different photophysics and chemistry can be simultaneously excited, thus providing good possibilities for internal standardization and multiparameter bioaffinity assays. We have recently shown, in our lyoluminescence studies, that the generation of sulfate radical,1 phosphate radical,2 hydroxyl radical,1-3 or dichlorine radical ion3 in aqueous luminol solutions induces strong chemiluminescence (CL) of luminol. It has been also demonstrated that sulfate4,5 and hydroxyl radicals6 can be cathodically generated at pulse-polarized oxide-covered aluminum
electrodes in fully aqueous solution. The primary step of this cathodic generation of strongly oxidizing radicals has been suggested4,5 to be an injection of tunnel-emitted hot electrons into the conduction band of water followed either by a direct reaction of a dry hot quasi-free electron with compounds such as peroxydisulfate ion, hydrogen peroxide or molecular oxygen or by thermalization and solvation of the hot electrons and the successive reaction of the resulting hydrated electrons (e-aq) with the above-mentioned oxidizing radical precursors.5,7 If this is correct, it should then be possible to generate phosphate radicals from peroxydiphosphate ions via analogous pathways.8 It has been demonstrated elsewhere that disposable oxide-covered aluminum electrodes can be used as a solid phase of immunoassays utilizing aromatic Tb(III) chelates as electrochemiluminescent labels in both the heterogeneous and homogeneous cases and in either the absence or presence of peroxydisulfate ions.7,9 The present work was carried out to study the possible existence of electrogenerated chemiluminescence (ECL) of luminol induced by the cathodic generation of the above-mentioned oxidizing radicals as well as the analytical applicability of this type of luminol ECL. EXPERIMENTAL SECTION Reagents and Instrumentation. Luminol (5-amino-2,3-dihydrophthalazine-1,4-dione), 9-fluorenylmethyl chloroformate (FMOC), and ruthenium(II) tris(2,2’-bipyridine) chloride hexahydrate were purchased from Aldrich. N-(6-Aminohexyl)-N-ethylisoluminol (AHEI) and N-(6-aminobutyl)-N-ethylisoluminol (ABEI) are products of Wallac Oy. K2S2O8, H2O2, NaNO3, KBr, NaI, NaN3, NaSCN, and N2B4O7‚10H2O were pro analysi products of Merck. Potassium peroxodiphosphate was supplied by Polysciences, Inc. (Warrington, PA) and ethanol by Oy Alko Ab. Quartz-distilled water was used in all solutions. The purity of nitrogen and oxygen were 99.999 and 99.9%, respectively (Oy Aga Ab). The safety precautions required in handling of the reagents have been presented previously.1
†
University of Turku. University College. (1) Matachescu, C.; Kulmala, S.; Ala-Kleme, T.; Joela, H. Anal. Chem. 1997, 69, 3385-3390. (2) Kulmala, S.; Matachescu C.; Joela, H.; Lilius, E.-M.; Kupila, E.-L. J. Chem. Soc. Faraday Trans. 1997, 93, 3497-3503. (3) Matachescu, C.; Kulmala, S.; Laine, E.; Raerinne, P. Anal. Chim. Acta 1997, 349, 1-10. (4) Kulmala, S.; Ala-Kleme, T.; Hakanen, A.; Haapakka, K. J. Chem. Soc., Faraday Trans. 1997, 93, 165-168. ‡
1112 Analytical Chemistry, Vol. 70, No. 6, March 15, 1998
(5) Kulmala, S.; Ala-Kleme, T.; Heikkila¨, L.; Va¨re, L. J. Chem. Soc., Faraday Trans. 1997, 93, 3107-3113. (6) Kulmala, S.; Kankare, J.; Haapakka, K. Anal. Chim. Acta 1991, 252, 6576. (7) Kulmala, S., Academic dissertation, Finland, Turku, 1995. (8) Buxton, G.; Greenstock, C.; Helman, W.; Ross, A. J. Phys. Chem. Ref. Data 1988, 17, 513-886 and references therein. (9) Kankare, J.; Kulmala, S.; Na¨nto ¨, V., Eskola, J.; Takalo, H. Anal. Chim. Acta 1992, 266, 205-212. S0003-2700(97)00954-2 CCC: $15.00
© 1998 American Chemical Society Published on Web 02/07/1998
The methods of ECL excitation and the coulostatic pulse generator are described elsewhere.5,7,10,11 The ECL measurements were made in 0.2 M boric acid buffer at pH 9.2, normally using a coulostatic pulse generator adjusted to yield 120-µC cathodic pulses with -40-V applied pulse voltage using either Al cup10 electrodes with a Pt wire counter electrode or an aluminum plate5 electrode with a Pt wire counter electrode in a 1-cm quartz cuvette. The effective surface area of the oxide-covered working electrodes was 2.1 cm2 in the case of Al cup electrodes (350-µL samples) and 1.1 cm2 (1.0-mL samples) in that of Al plate electrodes. The disposable Al electrodes were cut from a nominally 99.9% pure aluminum band (Merck Art. 1057, batch 721 K4164557), and the electrodes were covered with the 2-3-nm-thick natural oxide film.12,13 With the coulostatic pulse generator, the ECL yield of various luminophores is directly proportional to the pulse charge when the aluminum base metal pulse potential is driven above the conduction edge of water.4,5,7,11 Boric acid buffer was used due to its unreactivity toward hydrated electrons and hydroxyl radicals, as well as toward sulfate radicals and dichlorine radical ions.7,8 Measurements were generally made through an interference filter having a transmission maximum of 450 nm and bandwidth ∼10 nm. The interference filter was originally selected by assuming that the luminol ECL emission maximum would be at 450 nm, the CL emission peak wavelength when luminol CL is induced by dichlorine radical ions in the presence of hydrated electrons in deoxygenated alkaline solution.3 Later, it was discovered that an interference filter with a transmission maximum of 420 nm would have yielded ∼2-fold ECL signal. However, the remaining measurements were also carried out using the original interference filter, because the sulfate radical-induced IEL4 of the oxide-covered aluminum electrode was known to have an emission maximum of 410-420 nm. The ECL spectra were recorded with a Perkin-Elmer LS-5 spectral luminometer excitation shutter closed. RESULTS AND DISCUSSION Basic Features of ECL. Yoshimi et al.14 have shown that luminol ECL can be cathodically generated at an indium-tin oxide (ITO) electrode by producing hydroxyl radicals cathodically from hydrogen peroxide and that luminol can be used as an electrochemiluminescent label of antibodies and antigens at an ITO cathode. Yoshimi et al. did not study the mechanism of this ECL but assumed that the reaction between hydroxyl radical and luminol produced the luminol radical, which finally reacted with hydrogen peroxide producing the luminol chemiluminescence.14 An ITO electrode cannot be driven to very cathodic potentials due to the reduction of ITO; hence luminol is not reduced in the usable potential window of ITO electrodes in aqueous solution.14,15 Therefore, an ECL pathway involving a reduction of luminol (10) Kankare, J.; Fa¨lden, K.; Kulmala, S.; Haapakka, K. Anal. Chim. Acta 1992, 256, 17-28. (11) Ala-Kleme, T.; Kulmala, S.; Latva, M. Acta Chem. Scand. 1997, 51, 541546. (12) Tajima, S. Electrochim. Acta 1977, 22, 995-1011. (13) Despic’A.; Parkhutik, V. In Modern Aspects of Electrochemistry; Bockris, J., White, R., Conway, B., Plenum Press: Eds.; New York, 1989; Vol. 20, Chapter 6, pp 400-503, and references therein. (14) Yoshimi, Y.; Haramoto, H.; Miyasaka, T.; Sakai, K. J. Chem. Eng. Jpn. 1996, 29, 851-857.
Figure 1. Effect of coreactants on luminol ECL intensity. Key: Hydrogen peroxide (2), peroxydisulfate (9), and peroxydiphosphate (b). Conditions: 0.2 M borate buffer, pH 9.2, 1 × 10-7 M luminol, pulse charge 120 µC, pulse voltage 40 V, and pulse frequency 20 Hz. ECL intensity is an integral of photon counts induced by 1000 excitation pulses.
radicals, resulting in excited luminol molecules, could be possible in addition to the well-known CL pathway of luminol induced by the parallel presence of hydrogen peroxide and hydroxyl radicals having 3-aminophthalate as the final excited fragmentation product of luminol.16 However, the required3 >3 eV of -∆G is not obtainable for the reduction step of luminol radical at the ITO electrode, and therefore, this excitation pathway in the system of Yoshimi et al.14 can be rejected. Air-saturated luminol solutions also exhibited a strong cathodic ECL at a pulse-polarized oxide-covered aluminum electrode in the absence of other added coreactants (Figure 1). However, the presence of peroxydisulfate ions, peroxydiphosphate ions, and hydrogen peroxide enhanced this ECL, the enhancing effect being weaker for hydrogen peroxide than for the other two coreactants (Figure 1). The ECL spectrum was always similar to the luminol CL spectrum produced by Fenton reagent, peaking at ∼425 nm (Figure S1). Figure 2 displays the ECL intensity produced by the first 1000 excitation pulses in a 1 µM luminol solution at pH 9.2, where the solution is either deoxygenated, oxygen-saturated, air-saturated, or air-saturated and containing 1 mmol/L N3- ions. According to Figure 2, it is clear that the first cathodic excitation pulse lasting ∼400 µs already produces luminol ECL, indicating that the ECL is observed prior to the diffusion of anodic products from the counter electrode to the working electrode. The deoxygenated and air-saturated solution gave an approximately equal signal at the first excitation pulse, and the presence of dissolved oxygen had an ECL-enhancing effect in general, though this effect was not very strong. The simultaneous presence of oxygen and azide ion had a quite strong enhancing effect on the ECL intensity (Figure 2). The ECL dependence on pH in airsaturated solutions was nontypical for luminol CL, exhibiting the maximum ECL intensity in neutral solutions and a strong decrease of ECL intensity at high pH where luminol CL is normally the strongest1-3,16 (Figure 3). (15) Armstrong, N.; Lin, A., Fujihira, M.; Kuwana, T. Anal. Chem. 1976, 48, 741-750. (16) Hodgson, E.; Fridovich, I. Photochem. Photobiol. 1973, 29, 451-455. Lind, J.; Merenyi, G.; Eriksen, T. E. J. Am. Chem. Soc. 1983, 105, 7655-7661. Merenyi, G.; Lind, J.; Eriksen, T. E. J. Am. Chem. Soc. 1986, 108, 77167726. Merenyi, G.; Lind, J.; Eriksen T. E. J. Biolumin. Chemilumin. 1990, 5, 53-56.
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Figure 2. Time profiles of luminol ECL in deoxygenated, airsaturated, and oxygen-saturated solutions and in an air-saturated solution containing 1 mmol/L N3- ions. Conditions: as in Figure 1, except luminol concentration was 1 × 10-6 mol/L. Each point of the curves represents ECL intensity induced by a single excitation pulse at a pulse frequency of 20 Hz.
Figure 4. Effects of free radical scavengers on luminol ECL. Hydrated electron scavengers: Co(NH3)63+ (b), NO3- (9), and H2O2 ([). Hydroxyl radical scavengers: Cl- (O), Br - (0), SCN- (4), N3(*), I - (3), oxalate (2), and ethanol (1). Conditions: as in Figure 1 except luminol concentration was 1 × 10-6 mol/L.
If a hydrated electron were involved in the present luminol ECL system, hydrated electron scavengers would have a strong effect on the observed ECL intensity. Co(NH3)63+ and nitrate ion are fast hydrated electron scavengers [k(e-aq + Co(NH3)63+) ) 8.7 × 1010 L mol-1 s-1, k(e-aq + NO3-) ) 9.7 × 109 L mol-1 s-1]8 which do not produce oxidizing secondary radicals by one-electron reduction as do peroxydisulfate, peroxydiphosphate ions, and hydrogen peroxide. Both these e-aq scavengers were found to strongly quench the luminol ECL in air-saturated solutions (Figure 4), and hydrogen peroxide [k(e-aq + H2O2) ) 1.2 × 1010 L mol-1 s-1]8 showed an ECL-enhancing effect analogous to the case of the 10-fold more diluted luminol solution shown above (Figures 1 and 4). When the hydrogen peroxide concentration became too high, the ECL intensity began to decrease, which also occurred in the case of other coreactants (Figures 1 and 4). We have observed, in the ECL of aromatic lanthanide(III) chelates at oxide-covered aluminum electrodes, that an oxidizing species is also formed in the cell.7,17 The properties of this oxidizing species closely resemble those of hydroxyl radical, and the hydroxyl radical scavengers and the hydrated electron
scavengers have strong effects on the ECL of aromatic lanthanide(III) chelates.7,17,18 Among the hydroxyl radical scavengers tested from the halide and pseudo-halide series, azide ion enhanced the luminol ECL (almost as much as hydrogen peroxide) while chloride and bromide had no significant effect on the ECL intensity (Figure 4). Iodide and thiocyanate ion had a minimal enhancing effect at low concentrations but a strongly quenching effect at high concentrations (Figure 4). Ethanol and oxalate, which produce strongly reducing secondary radicals in reaction with hydroxyl radical, quenched less than iodide and thiocyanate ions, the effect being stronger for ethanol, which has a higher reactivity against hydroxyl radical than oxalate [k(•OH + EtOH) ) 1.9 × 109 L mol-1 s-1, k(•OH + oxalate) ) 7.7 × 107 L mol-1 s-1, respectively].8 Mechanism of Luminol ECL. Cathodic pulse-polarization of oxide-covered aluminum, magnesium and n-silicon electrodes produce weak intrinsic electrogenerated luminescence (IEL) of the oxide films during tunnel emission of hot electrons into aqueous solutions. The IEL induced by the high-field conditions in thin insulating films is based on several parallel mechanisms, and the IEL may excite suitable luminophores adsorbed at the oxide/electrolyte interface by energy transfer.4,7 However, the oxide films cannot efficiently act as pulsed UV light sources; e.g., fluorescein-containing latex microparticles in contact with the electrode, and several strongly photoluminescent luminophore solutions in a quartz envelope above the ECL cell were found not to be excitable by cathodic pulse polarization of insulating filmcovered electrodes.7 The IEL can be only slightly affected in solution by electron or hole scavengers, whereas the ECL of luminophores in solution is strongly affected by the presence of electron and hole scavengers.4,5,7,17 If hot electron injection into aqueous electrolyte solution and subsequent generation of hydrated electrons at thin insulating filmcovered electrodes5,7,11 is accepted as the primary cathodic step at oxide-covered aluminum electrodes, the present ECL can be easily explained. The general scheme of tunnel emission and Fowler-Nordheim tunneling of hot electrons into aqueous electrolyte solutions has been described elsewhere.5 In principle,
(17) Kulmala, S.; Haapakka, K. J. Alloys Compd. 1995, 225, 502-506.
(18) Kulmala, S.; Ala-Kleme, T. Anal. Chim. Acta 1997, 355, 1-5.
Figure 3. Luminol ECL intensity as a function of solution initial pH. Conditions: as in Figure 1, except luminol concentration was 1 × 10-6 mol/L and supporting electrolyte was 0.1 M Na2SO4 solution adjusted to the desired pH with sulfuric acid or sodium hydroxide.
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the cathodic reductions at oxide-covered aluminum electrodes can be due to a direct action of hot dry electrons (e-quasifree), e-aq or heterogeneously transferred electrons from the bottom of the insulator conduction band or somewhere above it (e-CB of the insulator) and less energetic electrons via the surface states (e-SS of the 5 insulator).
e-hot(electrode) f e-quasifree(in the CB of water)
(1)
e-quasifree f e-aq
(2)
e-hot(electrode) f e-CB or SS of the insulator
(3)
However, coreactants such as peroxydisulfate ions do enhance the ECL of luminophores at oxide-covered aluminum electrodes only in the direct tunnel emission regime (oxide film thickness
