Anal. Chem. 1996, 68, 1254-1257
Chemiluminescence of Luminol Catalyzed by Electrochemically Oxidized Ferrocenes R. Wilson* and D. J. Schiffrin
Department of Chemistry, University of Liverpool, P.O. Box 147, Liverpool L69 3BX, England
Ferrocenes oxidized at an indium tin oxide-coated glass electrode catalyze the chemiluminescent reaction of luminol with hydrogen peroxide. The catalytic reaction has been studied with ferrocene derivatives in solution and covalently attached to ovalbumin adsorbed on the electrode. It is shown that chemiluminescence is initiated by electrochemical oxidation of the ferrocene derivative. The chemiluminescent oxidation of luminol by hydrogen peroxide is catalyzed by Fe(III),1 ferricyanide,2 horseradish peroxidase,3 and other iron-containing compounds.4-6 These reactions have been used to detect trace amounts of material in a variety of analytical techniques, most notably immunoassays.7 Immunoassays can be divided into heterogenous, requiring a separation step, or homogenous, not requiring this step.8 The latter are less time consuming and avoid the errors that are associated with the separation step. For a homogenous immunoassay to be successful, there must be a way for distinguishing bound and unbound analyte. One way to do this is to modulate the signal from a label such as an enzyme,8 a fluorophore,9 or a redox compound such as ferrocene10 so that the signal is proportional to the amount of analyte in the sample. An alternative way is to activate the label when it binds to the solid phase with radioisotopes,11 fluorescently12 or electrochemically.13 In the present work it is shown that electrochemically oxidized ferrocenes catalyze the chemiluminescent oxidation of luminol by hydrogen peroxide. The reaction was studied under a variety of conditions in solution. The effect of oxidized ferrocenes is similar to that of horseradish peroxidase, which is used as a label in immunoassays, except that chemiluminescence can be initiated at the surface of an electrode where ferricinium is generated electrochemically. This difference means that ferrocene la(1) Obata, H.; Karatini, H.; Nakayama, E. Anal. Chem. 1993, 65, 1524-1528. (2) Seitz, W. R. In Methods in Enzymology; De Luca, M. A., Ed.; Academic Press: London, 1978; Vol. 57, pp 445-462. (3) Cormier, M. J.; Prichard, P. M. J. Biol. Chem. 1968, 243, 4706-4714. (4) Radi, R.; Thomson, L.; Rubbo, H.; Prodanov, E. Arch. Biochem. Biophys. 1991, 288, 112-117. (5) Adam, Y.; Bernadou, J.; Meunier, B. New J. Chem. 1992, 16, 525-528. (6) Ci, Y.-X.; Tie, J.-K.; Yao, F.-J.; Liu, Z.-L.; Lin, S.; Zheng, W.-Q. Anal. Chim. Acta 1993, 277, 67-72. (7) Coyle, P. M.; Thorpe, G. H. G.; Kricka, L. J.; Whitehead, T. P. Ann. Clin. Biochem. 1986, 23, 42-46. (8) Rubenstein, K. E.; Schneider, R. S.; Ullman, E. G. Biochem. Biophys. Res. Commun. 1972, 47, 846-851. (9) Dandliker, W. B.; Kelly, K. J.; Dandliker, J.; Levin, Immunochemistry 1973, 10, 219-227. (10) Di Gleria, K.; Hill, H. A. O.; McNeil, C. J.; Green, M. J. Anal. Chem. 1986, 58, 1203-1205. (11) Bosworth, N.; Towers, P. Nature 1989, 341, 167-168. (12) Kronick, M. N.; Little, W. A. Bull. Am. Phys. Soc. 1973, 18, 782. (13) Yamamoto, N. Y.; Nagasawa, Y.; Shuto, S.; Tsubomura, H.; Sawai, M.; Okumura, H. Clin. Chem. 1980, 26, 1569-1572.
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bels10,14,15 could be used to detect material bound to the surface of an electrode. This has been shown in the present work by detecting a layer of labeled ovalbumin adsorbed on an indium tin oxide-coated glass electrode as a model for a separation-free immunoassay. EXPERIMENTAL SECTION Materials. Sephadex-G15, horseradish peroxidase (type II), ovalbumin (grade V), ferrocenemonocarboxylic acid (Sigma), ferroceneacetic acid (Aldrich), and luminol (3-aminophthalhydrazide) (Fluka) were used as received. All other reagents were of AnalaR grade. Indium tin oxide (ITO)-coated glass was from Balzer Ltd., Buckinghamshire, England. It was cut into 24 × 9 mm wafers. Equipment. Electrochemiluminescence measurements were carried out in a three-electrode cell made of a cuvette which was placed in a Perkin-Elmer MPF-43 spectrofluorometer. The reference electrode, unless otherwise indicated, was a silver chloridecoated silver wire immersed in the solution under study; the counter electrode was a platinum wire, and these were located in the cell behind an ITO-coated glass working electrode. The ITO surface faced the detector, which was set at 425 nm with a slit width of 20 nm. Potentials were controlled with an in-house-built potentiostat and a waveform generator (PPR1, Hi-Tek Instruments, Buckinghamshire, England). Cyclic voltammetry experiments were carried out in a two-compartment cell using the same potentiostat and waveform generator. One compartment contained a saturated calomel reference electrode (SCE) and the other the ITO-coated glass working electrode and a platinum mesh counter electrode. Spectrophotometric studies were carried out in a Hewlett-Packard Model 8452A diode array spectrophotometer. Linear Sweep Voltammetry. Reactions were carried out in 0.1 M TRIS buffer, pH 8.0, that contained 0.1 M NaCl. Solutions of ferrocenemonocarboxylic acid, luminol, and hydrogen peroxide were mixed before the experiment so that the final concentrations were 100 µM ferrocene, 100 µM luminol, and 1 mM hydrogen peroxide. Light intensity was recorded as the potential was swept from 0 to 350 mV at 1 mV s-1. In the control experiment, ferrocenemonocarboxylic acid was omitted from the solution. Electrochemiluminescence Reaction at a Fixed Potential. Reactions were carried out in the same solution described previously except that electrochemiluminescence was initiated by stepping the potential from 0 to 350 mV. Light and current were measured simultaneously. The effect of pH on the reaction was investigated by adjusting the pH of the solutions with hydrochloric (14) Schumann, W.; Ohara, T. J.; Schmidt, H.-L.; Heller, A. J. Am. Chem. Soc. 1991, 113, 1394-1397. (15) Suzawa, T.; Ikariyama, Y.; Aizawa, M. Anal. Chem. 1994, 66, 3889-3894. 0003-2700/96/0368-1254$12.00/0
© 1996 American Chemical Society
acid. The influence of EDTA (10 mM) and p-iodophenol (0.5 mM) on light intensity was also investigated. Spectrophotometric Investigation of the Reaction Mechanism. Hydrogen peroxide and horseradish peroxidase were added to a 10 mM solution of ferrocenemonocarboxylic acid in 0.1 M TRIS buffer, pH 8.0, that contained 0.1 M NaCl, to final concentrations of 0.03% and 0.01 mg mL-1, respectively. The solution was allowed to stand for 10 min, and during this time it changed color from yellow to green owing to the oxidation of ferrocene to ferricinium. Horseradish peroxidase was separated from the ferricinium-monocarboxylic acid by loading 10 mL of solution onto a column packed with Sephadex G-15 (void volume 11 mL) and collecting the first 10 mL of green eluate, which was then scanned from 400 to 700 nm before and after dissolving luminol in it. Ferricinium-monocarboxylic acid has an absorbance maxima at 630 nm, but ferrocenemonocarboxylic acid does not absorb at this wavelength. The purpose of these experiments was to see if the absorbance at 630 nm decreased when luminol was added. Because ferricinium is unstable, experiments were carried out as soon as possible after eluting it from the column. Cyclic Voltammetry and Reaction Mechanism. ITO electrodes in a solution of 100 µM ferrocenemonocarboxylic acid in 0.1 M TRIS buffer, pH 8.0, that contained 0.1 M NaCl, were cycled between -200 and +600 mV vs SCE at 10 mV s-1. This was repeated for 1 mM luminol in place of ferrocenemonocarboxylic acid, and finally for luminol and ferrocenemonocarboxylic acid together. The purpose of these experiments was to see whether a catalytic wave could be observed for ferrocene oxidation in the presence of luminol. Preparation of Ferrocene-Labeled Ovalbumin. Ovalbumin was dissolved in 0.1 M phosphate buffer, pH 7.5, that contained 0.1 M NaCl. The N-hydroxysuccinimide ester of ferroceneacetic acid (SFA) was prepared as described previously.16 It was dissolved in ethanol and added to the ovalbumin solution so that the final concentrations were 10%(v/v) ethanol, 10 mg mL-1 ovalbumin, and 1 mM SFA. The solution was allowed to stand for 1 h at room temperature and then loaded onto a column packed with Sephadex G-15 (void volume 11 mL) and eluted with distilled water. The first 3 mL of eluate after the void volume was retained. The concentration of protein in the retained eluate was determined colorimetrically with coomassie blue.17 The concentration of iron was determined by flame atomic absorption spectroscopy after digesting the ovalbumin with nitric acid at 100 °C. All experiments were repeated with an ovalbumin solution that had not been treated with SFA. Cyclic Voltammetry of Adsorbed Ovalbumin. ITO-coated glass electrodes were immersed for 10 min in the retained eluate diluted 1:1 with 0.2 M citric acid and then thoroughly washed with 0.1 M citric acid. The electrodes were cycled between 0 and 700 mV vs SCE at 10 and 100 mV s-1 in 0.1 M citric acid. The area of the working electrode was 1 cm2. Electrodes were also cycled in 0.1 M TRIS buffer, pH 9.0, that contained 0.1 M NaCl. Electrochemiluminescence of Adsorbed Ovalbumin. The potential of electrodes with a layer of adsorbed ovalbumin was stepped from 0 to 350 mV in 0.1 M TRIS buffer, pH 9.0, that contained 0.1 M NaCl, and the light intensity was recorded. A similar experiment was carried out with unlabeled ovalbumin.
RESULTS Electrochemiluminescence in Solution. The chemiluminescent reaction of electrochemically oxidized luminol with hydrogen peroxide is well-known.18 Recent work has shown that the potential at which this occurs when the electrode is made from ITO is more positive than on metallic electrodes.19 Therefore ferricinium, which is generated at less positive potentials, catalyzes chemiluminescence because the electrochemical oxidation of luminol is not significant in this potential range. The potential dependence of chemiluminescence is shown in Figure 1. When ferrocenemonocarboxylic acid was present, light emission was observed at potentials of about 200 mV more negative than when it was absent. Figure 2 shows the effect on current and light intensity of stepping the potential from 0 to 350 mV. Current decayed as expected for a diffusionally controlled process, but
(16) Wilson, R.; Schiffrin, D. J. Analyst 1995, 120, 175-178. (17) Spector, T. Anal. Biochem. 1978, 86, 142-146.
(18) Haapakka, K. E.; Kankare, J. J. Anal. Chim. Acta 1982, 138, 263-275. (19) Kremesko ¨tter, J. Ph.D. Thesis, University of Liverpool, Liverpool, UK, 1995.
Figure 1. Dependence of chemiluminescence on potential at pH 8.0, in 0.1 M TRIS buffer with 0.1 M NaCl, 1 mM hydrogen peroxide, and 100 µM luminol: (A) Catalyzed by 100 µM ferrocenemonocarboxylic acid; (B) uncatalyzed. Sweep rate 1 mV s-1.
Figure 2. (A) Current and (B) light intensity transients for a potential step from 0 to 350 mV for 100 µM ferrocenemonocarboxylic acid, at pH 8.0 in 0.1 M TRIS buffer with 0.1 M NaCl, 1 mM hydrogen peroxide, and 100 µM luminol.
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Figure 3. Dependence of chemiluminescence on pH at 350 mV in 0.1 M TRIS buffer, with 0.1 M NaCl, 1 mM hydrogen peroxide, and 100 µM luminol: (A) Catalyzed by 100 µM ferrocenemonocarboxylic acid; (B) uncatalyzed.
light intensity continued to increase for a long period of time. The effect of pH on light emission is shown in Figure 3. The increase in light intensity with pH is typical of luminol reactions.2,6,18,19 When horseradish peroxidase is used to catalyze the chemiluminescent reaction between luminol and hydrogen peroxide, compounds such as p-iodophenol greatly enhance the light intensity.7 However, this compound has no effect on the reaction catalyzed by ferrocenes. EDTA, which is capable of chelating the iron center in ferricinium,20 reduced the light intensity by 25%. Reaction Mechanism. Oxidation of ferrocenes to ferricinium ions catalyzed by horseradish peroixidase, followed by gel filtration to remove the enzyme, is a convenient way of preparing ferriciniums ions. An absorbance spectrum of the product has a peak at 630 nm that corresponds to ferricinium-monocarboxylic acid, which has an extinction coefficient of 420 cm-1 M-1 at this wavelength.21 Absorbance spectra of ferrocenemonocarboxylic acid and luminol show no absorbance at this wavelength. When luminol was added to a solution of ferricinium-monocarboxylic acid, it had no effect on absorbance at 630 nm, indicating that ferriciniums are not reduced by luminol. No evidence for reduction of ferricinium to ferrocene was observed by cyclic voltammetry either, and a voltammogram of luminol and ferrocenemonocarboxylic acid solution corresponded to the sum of the responses of the individual components. Ferrocene-Labeled Ovalbumin. The concentration of ovalbumin in the retained eluate from the Sephadex G-15 column was 8.6 mg mL-1 for the ferrocene-labeled ovalbumin and 8.7 mg mL-1 for the unlabeled ovalbumin solution. This suggests that the incorporation of ferroceneacetic acid into the protein did not interfere with the method used for protein determination. Ovalbumin has a molecular mass of 44 000 Da,22 and therefore, the concentration in the eluate was 0.19 mM. Atomic absorbance spectroscopy indicated that the concentration of iron in the labeled ovalbumin solution was 26 ppm. Assuming all the additional iron was present as ferrocene, these results imply that there were two to three ferrocene molecules attached per molecule of ovalbumin. Ferricinium ions are unstable in aqueous solution.21 This is particularly noticeable in cyclic voltammetric experiments when (20) Smith, T. D. J. Inorg. Nucl. Chem. 1960, 14, 290-291. (21) Szentrimay, R.; Yeh, P.; Kuwana, T. In Electrochemical Studies of Biological Systems; ACS Symposium Series 38; Sawyer, D. T.; American Chemical Society: Washington, DC, 1977; p 152. (22) Florkin, M.; Stotz, E. H. Comprehensive Biochemistry; Elsevier: London, 1963; Vol. 7, p 50.
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Figure 4. Cyclic voltammogram of ferrocene-labeled ovalbumin adsorbed on an ITO electrode in 0.1 M citric acid: (A) 100 mV s-1; (B) 10 mV s-1. Potentials vs SCE.
they are present as a monolayer on the surface of an electrode.16 When ITO-coated electrodes were immersed in ferrocene-labeled ovalbumin in citric acid, a layer of labeled protein adsorbed onto the electrode as shown by cyclic voltammetry (Figure 4). Strong surface attachment was evident because these peaks did not change when the electrode was washed with 0.1 M citric acid or distilled water; no peaks were observed for electrodes immersed in unlabeled ovalbumin. The average charge under the peaks (0.6 µC cm-2) corresponded to about four ferrocene molecules/ nm2. Ovalbumin is a disk-shaped protein with a major and minor axis of 23 and 6.3 nm, respectively.22 If each molecule contained 2 equiv of ferrocene and the electrode surface was perfectly smooth, an adsorbed monolayer should contain a maximum of 0.5 ferrocenes/nm2. A value of 4 ferrocene molecules/nm2 suggests that there was more than one layer of protein adsorbed on the surface and/or that the electrode surface was not perfectly smooth. The surface of ITO is indeed very rough on an atomic scale as shown by scanning tunneling and atomic force microscopies.19 Repeated cycling of the electrodes did not reduce the area under the ferrocene peaks, but when they were cycled in pH 9.0 TRIS buffer, the peaks rapidly decayed. This may be due to the decrease in stability of ferricinium ions in the absence of citric acid, which is known to stabilize it.20 Electrochemiluminescence of Adsorbed Ferrocene-Labeled Ovalbumin. ITO-coated electrodes with a layer of adsorbed ferrocene-labeled ovalbumin were washed with 0.1 M TRIS buffer, pH 9.0, that contained 0.1 M NaCl. These were then immersed in the same buffer that also contained 10 mM hydrogen peroxide and 100 µM luminol in the cell fluorometer electrochemical cell. The potential was stepped from 0 to 350 mV and the light intensity recorded. This procedure was repeated with electrodes treated with unlabeled ovalbumin, and Figure 5 compares light emission for labeled and unlabeled ovalbumin. DISCUSSION Electrochemiluminescence detection of ferrocene labeled ovalbumin adsorbed on an electrode suggests that it may be possible to detect ferrocene-labeled antibodies in the same way. This could be used to determine the amount of analyte in solution in competitive immunoassays in which labeled antibodies partition between analyte in solution and analyte attached to an electrode. Ferrocenes have already been used in a number of immunoassays, and some of these could be carried out electrochemiluminescently.
Figure 6. Proposed mechanism for the catalytic effect of ferricinium on the chemiluminescent reaction of luminol with hydrogen peroxide.
Figure 5. Electrochemiluminescence of ITO electrodes with adsorbed ovalbumin when the potential was stepped from 0 to 350 mV vs AgCl at pH 9.0 in 0.1 M TRIS buffer, with 0.1 M NaCl, 1 mM hydrogen peroxide, and 100 µM luminol: (A) Ferrocene-labeled ovalbumin; (B) unlabeled ovalbumin.
For example, ferrocene attached to bovine serum albumin labeled with digoxin becomes electroinactive in the presence of antibodies to digoxin, and electrochemiluminescence detection may increase the sensitivity.23 Ferrocenes could also be used instead of ruthenium tris(bipyridyl) labels in electrochemiluminescence immunoassays24 and DNA assays.25 An attractive, but more speculative, possibility is to use ferrocene as an antigen label in homogenous immunoassays for small molecules like drugs and pesticides. The ferrocene molecule is small and therefore it may become electroinactive when enveloped by the antibody or because the diffusion coefficient decreases when it is bound by the antibody, in the same way that daunomycin-labeled biotin becomes electroinactive in the presence of avidin.26 If the effect of ferrocenes on chemiluminescence involved reduction of ferricinium to ferrocene by luminol, this should be apparent in spectrophotometric and cyclic voltammetric studies, but no evidence for reduction was obtained by either technique. An alternative mechanism, which is in agreement with the results presented here, is that ferricinium ions catalyze chemiluminescence as shown in Figure 6. This mechanism resembles the way that heme proteins such as horseradish peroxidase, catalase, and microperoxidase catalyze the chemiluminescent reaction between luminol and hydrogen peroxide.27 These proteins contain Fe(III), which is thought to react with hydrogen peroxide and form a (23) Toshiyuki, S.; Ikariyama, Y.; Aizawa, M. Bull. Chem. Soc. Jpn. 1995, 68, 165-171. (24) Gattomenking, D. L.; Yu, H.; Bruno, J. G.; Goode, M. T.; Miller, M.; Zulich, A. W. Biosens. Bioelectron. 1995, 10, 501-507. (25) Yu, H.; Bruno, J. G.; Cheng, T. C.; Calomiris, J. J.; Goode, M. T. J. Biolumin. Chemilumin. 1995, 10, 239-245. (26) Sugawara, K.; Tanaka, S.; Nakamura, H. Anal. Chem. 1995, 67, 299-302. (27) Cambell, A. K. Chemiluminescence: Principles and Applications in Biology and Medicine; Ellis Horwood: Chichester, UK, 1988; pp 414-423.
complex that oxidizes luminol chemiluminescently. If ferricinium forms a similar complex, the effect of EDTA on the chemiluminescent reaction would be explained by its ability to compete with hydrogen peroxide for the iron center in ferricinium. Similar to the heme proteins mentioned before, ferricinium is a very efficient catalyst of luminol chemiluminescence and subnanomolar amounts were readily detected even though the fluorometer used in this work was not designed for luminometry. To account for the delay between the start of the electrochemical reaction and maximum chemiluminescence, it is proposed that ferricinium decays and light reaches a maximum when the rate at which ferricinium is generated electrochemically is equal to the rate at which it is destroyed. Ferricinium is unstable and decays to Fe(III),20 which also catalyzes the chemiluminescent reaction between luminol and hydrogen peroxide.27 Close inspection of Figure 4 shows that after the electrochemical reaction light does not return to the original baseline, as would be expected if the decay product was a less efficient catalyst of chemiluminescence such as Fe(III). CONCLUSIONS The results presented here show that oxidized ferrocenes catalyze the chemiluminescent reaction between luminol and hydrogen peroxide. As a result, ferrocene-labeled proteins can be detected by electrochemiluminescence methods. This suggests the possibility of using ferrocenes as a label in electrochemiluminescence immunoassays. ACKNOWLEDGMENT The authors thank Mr. S. Apter for his help with the atomic absorbance measurements. The support of the European Union Environment Programme, Contract EV5V-CT92-0067, Biopticas, for R.W., is gratefully acknowledged.
Received for review December 6, 1995.X
October
10,
1995.
Accepted
AC951023C X
Abstract published in Advance ACS Abstracts, February 1, 1996.
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