8732
J. Phys. Chem. B 2001, 105, 8732-8738
High-Frequency Electrochemiluminescent Investigation of the Reaction Pathway between Tris(2,2′-bipyridyl)ruthenium(II) and Tripropylamine Using Carbon Fiber Microelectrodes† Erin M. Gross,‡ Paolo Pastore,§ and R. Mark Wightman*,‡ Department of Chemistry, UniVersity of North Carolina, Chapel Hill, North Carolina 27599-3290, and Dipartimento di Chimica Inorganica, Metallorganica e Analytica, UniVersita di PadoVa, Via Marzolo 1, 35131 PadoVa, Italy ReceiVed: April 17, 2001; In Final Form: June 27, 2001
The electrochemiluminescent (ECL) reaction mechanism between tris(2,2′-bipyridyl)ruthenium(II) (Ru(bpy)32+) and tripropylamine (TPrA) in aqueous solution at pH 7.4 was examined using fast potential pulses at carbon fiber microelectrodes. High-stability ECL emission was found with 0.5 ms pulses and a 25% duty cycle. In addition, stability was increased with negative rest potentials. Direct evidence for the strongly reducing free radical intermediate was obtained when the light was quenched upon addition of nitrobenzene derivatives. The formation of this free radical becomes rate-limiting at high concentrations of Ru(bpy)32+ and TPrA, as its production can be limited when there is an insufficient supply of one or both of its precursors (TPrA and TPrA•+) relative to Ru(bpy)32+. When TPrA is in sufficient excess of Ru(bpy)32+, the ECL efficiency (photons emitted/Ru(bpy)33+ generated) was determined to be very high (∼90%) by comparison to a Ru(bpy)33+/ Ru(bpy)3+ standard in acetonitrile. Rapid potential pulses also generated ECL from Ru(bpy)32+ when other tertiary amines, trimethylamine, diisopropylethylamine, and histamine, were used as co-reactants. The secondary amine epinephrine also produced light, but not norepinephrine, a primary amine.
Introduction Electrogenerated chemiluminescence (ECL) is the production of excited states via electron transfer between electrogenerated intermediates. These intermediates may be different redox states of the same species1 generated in aprotic solvents. For example, generation of the oxidized and reduced forms of tris(2,2′bipyridyl)ruthenium(II) (Ru(bpy)32+) is well-known to result in light emission by the following:
Ru(bpy)33+ + Ru(bpy)3+ f *Ru(bpy)32+ + Ru(bpy)32+ (1) However, it is possible to generate ECL in aqueous media when an oxidative-reductive type chemiluminescence is exploited.2 In such ECL mechanisms, upon oxidation of a lumophore and a co-reactant, the luminescent state is produced upon electron transfer between the oxidized form of the lumophore and a strong reductant formed upon irreversible oxidation of the coreactant. These types of reactions are best documented for Ru(bpy)32+ and a co-reactant such as oxalate (C2O42-) whose mechanism of light production has been determined.2,3 A similar reaction also occurs with tertiary and secondary amines.4 At an electrode Ru(bpy)32+ is reversibly oxidized to Ru(bpy)33+, which is able to oxidize the amine (R3N) to its radical cation.
†
Ru(bpy)32+ f Ru(bpy)33+ + e-
(2)
Ru(bpy)33+ + R3N f Ru(bpy)32+ + R3N•+
(3)
Part of the special issue “Royce M. Murray Festschrift”. * To whom correspondence should be addressed. Fax: (919) 962-2388. E-mail:
[email protected]. ‡ University of North Carolina. § Universita di Padova.
The radical cation is thought to form a free radical reducing agent (R•) via loss of a proton. This strong reducing agent then reacts to produce the luminescent state and other products.
R3N•+ f R• + H+
(4)
Ru(bpy)33+ + R• f *Ru(bpy)32+ + P
(5)
This aqueous ECL has created many possibilities for analytical applications.5 It has been used for the determination of oxalate6 and many other biologically important molecules.7-9 Using tripropylamine (TPrA) as the coreactant, Ru(bpy)32+ ECL has been used in immunoassays and DNA probe assays.10-17 Instruments that use these methods are now commercially available. The mechanism of chemiluminescence18 (CL) from metal complexes and alkylamines has been investigated in terms of structure-activity relationships.19,20 However, these investigations considered conditions where the catalytic oxidation of the amine by the metal complex was the rate-limiting step. In this paper we examine this reaction under conditions where the catalytic reaction competes with electrode processes. This is achieved by using a carbon electrode at neutral to slightly basic pH’s.21 An understanding of the mechanism and rate-determining step at this pH is important because these are the conditions for the most efficient light production.4,21,22 It has recently been reported that direct oxidation of the coreactant, specifically TPrA, leads to more intense light than if the co-reactant is homogeneously oxidized21 by Ru(bpy)33+ which has been shown to be the slow step in light production.20 The oxidation of TPrA was proceeded most efficiently at glassy carbon when compared to Au and Pt electrodes.
10.1021/jp011434z CCC: $20.00 © 2001 American Chemical Society Published on Web 08/17/2001
Reaction Pathway between Ru(bpy)32+ and TPrA In this work we have examined the oxidative-reductive ECL reaction using rapid potential pulses with carbon-fiber microelectrodes. Microelectrodes are useful tools for studying ECL reactions because they allow fast generation of reagents so that reactions may be studied in real time and kinetic information determined.23,24 For this specific reaction, short generation times ( 100 s stable > 100 s stable < 20 s
low amine, high Ru(bpy)32+ amine TPrA HIS EPI NE
rel intens
stability
1 stable > 100 s 0.5 stable > 100 s 0.4 stable > 100 s no light
a The following concentrations were used for these experiments: high amine, 50 mM; low Ru(bpy)32+, 50 µM; low amine, 20 µM; high Ru(bpy)32+, 1 mM. ECL was generated with the same waveform (+1.40 V Eapplied and -0.20 V rest potential) except with HIS, which required a +1.50 V Eapplied for maximum light production. Data were collected in the same manner as those in Figure 2B as each sample was run through the flow system.
ECL Reactions with Other Amines. Tertiary amines have been shown to produce luminescence more efficiently in both CL19 and ECL9 reactions than primary and secondary amines. The alkyl chains stabilize the radical intermediate, creating a more bright and stable signal. A linear correlation between the first ionization potential of the coreactant and the CL signal was observed.19 In an attempt to make a similar comparison for ECL and to investigate other co-reactants, we have compared the ECL of three different alkylamines, as shown in Table 1. For each reaction, the amine concentration was 50 mM and the Ru(bpy)32+ concentration was 50 µM. Tripropylamine was the most efficient and then diisopropylethylamine (DIEA) and trimethylamine (TMA). Trimethylamine was the only amine for which the ECL was not stable during the entire course of an injection into the flow system, which lasts ∼100 s. Aminecontaining molecules of biological interest also show ECL. Histamine (HIS), an imadazole, and epinephrine (EPI), a secondary amine, both produce ECL upon oxidation with Ru(bpy)32+. Norepinephrine (NE), a primary amine, did not produce any ECL. The relative ECL intensities for these amines (20 µM), using Ru(bpy)32+ at higher concentration (1 mM), are listed in Table 1. Conclusions At neutral pH’s, high-frequency generation of Ru(bpy)33+ and a strongly reducing coreactant using carbon fiber microelectrodes produces bright, stable light from Ru(bpy)32+ when various alkyl and biological amines serve as the co-reactant. Using potential pulses at carbon fiber microelectrodes, the amine can be directly oxidized, yet electrode fouling is not a factor. In addition to these experimental advantages, high-speed ECL also is advantageous for determining mechanistic information. Because of the short time scales of light generation, the temporal profiles of the light provide insight into the steps of the mechanism controlling light production. When the concentration of the amine is low and the concentration of Ru(bpy)32+ is high, the mechanism is rate-limited by the catalytic oxidation of TPrA (reaction 10) and is not temporally linked to the applied potential. When the concentration of the amine is high (mM) and Ru(bpy)32+ low (µM), the excited-state producing reaction (reaction 12) limits the rate of photon production. For this reason the temporal profiles are peak-shaped because they are limited by the rate of Ru(bpy)33+ production. However, when the concentration of Ru(bpy)32+ increases (mM), the light becomes dependent upon the rate of production of the free radical intermediate (reaction 8). This is demonstrated by the change in the temporal profile of the light at high concentrations of Ru(bpy)32+. With nitrobenzene derivatives that quenched the
8738 J. Phys. Chem. B, Vol. 105, No. 37, 2001 reaction, the central importance of the strongly reducing radical TPrA• in this ECL reaction was determined. Acknowledgment. This research was supported by a grant from the National Science Foundation. References and Notes (1) Bard, A. J.; Wallace, W. L. J. Phys. Chem. 1979, 83, 1350-1357. (2) Rubinstein, I.; Bard, A. J. J. Am. Chem. Soc. 1981, 103, 512-516. (3) Kanoufi, F.; Bard, A. J. J. Phys. Chem. B. 1999, 103, 1046910480. (4) Leland, J. K.; Powell, M. J. J. Electrochem. Soc. 1990, 137, 31273131. (5) Knight, A. W. Trends Anal. Chem. 1999, 18, 47-62. (6) Rubinstein, I.; Martin, C. R.; Bard, A. J. Anal. Chem. 1983, 55, 1580-1582. (7) Jameison, F.; Sanchez, R. I.; Dong, L.; Leland, J. K.; Yost, D.; Martin, M. T. Anal. Chem. 1996, 68, 1298-1302. (8) Brune, S. N.; Bobbitt, D. R. Talanta 1991, 38, 419-424. (9) Greenway, G. M.; Dolman, S. J. L. Analyst 1999, 124, 759-762. (10) Blackburn, G. F.; Shah, H. P.; Kenten, J. H.; Leland, J.; Kamin, R. A.; Link, J.; Peterman, J.; Powell, M. J.; Shah, A.; Talley, D. B.; Tyagi, S. K.; Wilkins, E.; Wu, T.; Massey, R. J. Clin. Chem. 1991, 37/9, 15341539. (11) Kenten, J. H.; Casadei, J.; Link, J.; Lupoid, S.; Willey, J.; Powell, M.; Rees, A.; Massey, R. Clin. Chem. 1991, 37/9, 1626-1632. (12) Gudibande, S. R.; Kenten, J. H.; Link, J.; Friedman, K.; Massey, R. J. Mol. Cell. Probes 1992, 6, 495-503. (13) Xu, X. H.; Yang, H. C.; Mallouk, T. E.; Bard, A. J. J. Am. Chem. Soc. 1994, 116, 8386-8387. (14) Xu, X.; Bard, A. J. J. Am. Chem. Soc. 1995, 117, 2627-2631. (15) Boom, R.; Sol, C.; Weel, J.; Gerrits, Y.; de Boer, M.; Wertheimvan Dillen, P. J. Clin. Microbiol. 1999, 37, 1489-1497. (16) Hsueh, Y. T.; Smith, R. L.; Northrup, M. A. Sensors and Actuators 1996, 33, 110-114. (17) Yu, H. J. Immun. Methods 1998, 218, 1-8. (18) Noffsinger, J. B.; Danielson, N. D. J. Chromatogr. 1987, 387, 520524.
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