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Neutral and Dianionic Ru(II) Bathophenanthrolinedisulfonate Complexes: A Route To Enhance Electrochemiluminescence Performance in Aqueous Media Leopoldo Della Ciana,*,† Simone Zanarini,*,‡ Rossana Perciaccante,† Ettore Marzocchi,† and Giovanni Valenti‡ Cyanagen srl, Via Stradelli Guelfi 40/C, 40138 Bologna, Italy, and Dipartimento di Chimica “G. Ciamician”, UniVersita’ di Bologna, Via Selmi 2, 40126 Bologna, Italy ReceiVed: NoVember 6, 2009; ReVised Manuscript ReceiVed: January 14, 2010
We report the strong enhancement of ECL intensity and duration in neutral and dianionic Ru(II) complexes bearing mixed 2,2′-bipyridine (bpy) and bathophenanthrolinedisulfonate (BPS) ligands. In aqueous conditions, using the tripropylamine-assisted method and applying a constant potential, we observed for Ru(BPS)(bpy)2 a remarkable 26-fold ECL integrated intensity increase with respect to Ru(bpy)32+. The results herein obtained reveal that the cause of reduced ECL intensity of [Ru(BPS)3]4- and [Ru(BPS)2(bpy)]2- can be ascribed to surface effects related to the interaction of the oxidized complex with the electrode. The expected reduction or absence of electrostatic interactions with biomolecules, together with the strongly enhanced performance, makes the zwitterionic Ru(BPS)(bpy)2 complex a highly promising candidate for the development of very efficient ECL labels for ultrasensitive bioassays and functional imaging applications. Introduction Electrochemiluminescence (ECL), i.e., luminescence generated by an electrochemical reaction, is far superior to photoluminescence spectroscopy in terms of intrinsically low noise and sensitivity.1 Through ECL, the concentration or the presence of specific proteins and DNA sequences can be accurately detected.1,2 The benefits of the electrochemical generation of the excited state are the absence of scattering from the excitation source, an ultralow background noise, and the possibility to control light generation both spatially and temporally. Last, but not least, ECL requires relatively low cost of instrumentation. It is therefore surprising that only one type of ECL label, based on the well-known complex of ruthenium, Ru(bpy)32+, is currently used in bioanalysis (bpy ) 2,2′-bipyridine, see Chart 1 for acronyms and structures). On the other hand, many compounds are known to be ECL active in a nonaqueous environment, often with a degree of efficiency much higher than that of Ru(bpy)32+.3 However, bioanalytical applications require at least some degree of water solubility. Our initial efforts aimed at finding more efficient labels were focused on the Ru(bathophen)32+ complex (bathophen ) 4,7-diphenyl-2,2′-bipyridine). This species is known to have a considerably higher fluorescent quantum yield compared with Ru(bpy)32+, while retaining very similar oxidation potential.4 Since Ru(bathophen)32+ salts are very hydrophobic and not soluble at all in aqueous buffers, we studied a hexasulfonated derivative, Na4Ru(BPS).5 The ECL efficiency of this compound did indeed prove to be higher than that of Ru(bpy)32+ by a factor of 5. These somewhat surprising results have led us to investigate the behavior of the two heteroleptic Ru(II) complexes with bpy and BPS ligands, and then to compare ECL performances in aqueous buffers/TPA in the series of [Ru(BPS)n(bpy)3-n](2n-2)- complexes, where as n decreases from 3 to 1, the net charge changes from -4 to 0. * To whom correspondence should be addressed. Phone: +39 051534063. E-mail:
[email protected]. † Cyanagen srl. ‡ Universita’ di Bologna.
CHART 1: Structures and Acronyms of the Complexes and Ligands of the Present Work
Experimental Section Materials. RuCl3 · 3H2O, bathophenanthrolinedisulfonic acid disodium salt trihydrate (Na2BPS · 3H2O), and 2,2′-bipyridine (bpy) were purchased from Fluka. Bpy was purified by sublimation. According to a recent study,6 the sulfonate substituents in the phenyl rings in BPS occur predominantly in the meta position, and are accordingly represented in Chart 1. [Ru(BPS)3]Na4 was prepared according to a previously reported procedure.5 Synthesis of Ru(BPS)2Cl2 · 9H2O.7 Na2BPS · 3H2O (500 mg, 0.85 mmol), RuCl3 · 3H2O (110.12 mg, 0.42 mmol), and LiCl
10.1021/jp910596z 2010 American Chemical Society Published on Web 02/09/2010
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(107.08 mg, 2.53 mmol) were added to 2.5 mL of argon degassed DMF. The mixture was refluxed at 160 °C for 2 h under argon. The dark solution was then added dropwise to 300 mL of rapidly stirred diethyl ether. The precipitate was filtered and washed with diethyl ether (100 mL) and acetone (200 mL) to afford 580 mg of the final product, C48H28Cl2N4Na4O12RuS4 · 9H2O, MW 1407.09, yield 98%. The amount of water of crystallization was determined by differential thermogravimetry (loss in weight 11.8%). 1H NMR: δ (ppm) 8.38-8.05 (m, 7H), 7.91-7.36 (m, 21H). Synthesis of [Ru(BPS)2bpy]Na2 · 9H2O and [Ru(BPS)2bpy](TBA)2. Ru(BPS)2Cl2 · 9H2O (100 mg, 0.071 mmol) and bpy (15 mg, 0.096 mmol) were added to 8 mL of argon-degassed 1:1 ethanol/water. The mixture was refluxed at 90 °C for 3 h under argon. The solvents were removed by rotavapor and the residue was dissolved in 2 mL of methanol and added dropwise to 300 mL of rapidly stirred diethyl ether. The precipitate was filtered, washed with diethyl ether, and dried under reduced pressure over P2O5 to afford 96 mg of the desired product, C58H36N6Na2O12RuS4 · 9H2O, MW 1446.39, yield 93%. The amount of crystallization water was determined by differential thermogravimetry (loss in weight 11.1%). 1H NMR: δ (ppm) 8.93 (apparent d, 2H, J ) 9.0 Hz), 8.38-8.13 (m, 11H), 7.89-7.79 (m, 12H), 7.74-7.49 (m, 11H). The tetrabutylammonium salt was prepared by ion exchange. A Dowex 50WX8-200, 1 × 7 cm column, sodium form, was exchanged with tetrabutylammonium bromide (1 M) and subsequently washed with water. A 25 mg sample of the ruthenium complex dissolved in 300 µL of water was loaded onto the column. The orange product was eluted with water, collected, and evaporated under reduced pressure. Synthesis of Ru(BPS)(bpy)2 · 4H2O. Ru(bpy)2Cl2 · 2H2O8 (100 mg, 0.19 mmol) and Na2BPS · 3H2O were added to 10 mL of argon-degassed 1:1 ethanol/water. The mixture was refluxed at 90 °C for 3 h under argon. The solution was then cooled to room temperature. An orange precipitate formed and was collected by filtration, washed with cold H2O, ethanol, and diethyl ether, and dried under reduced pressure to afford 157 mg of the product, C44H30N6O6RuS2 · 4H2O, MW 976.01, yield 85%. The amount of crystallization water was determined by differential thermogravimetry (loss in weight 8.7%). 1H NMR: δ (ppm) 8.89 (apparent d, 2H, J ) 8.8 Hz), 8.86 8.38 (apparent d, 2H, J ) 8.8 Hz), 8.24-8.12 (m, 8H), 7.86-7.74 (m, 10H), 7.75-7.73 (m, 2H), 7.65-7.59 (m, 6H), 7.46-7.43 (apparent t, 2H, J ) 8.8 Hz). Differential thermogravimetry assays were carried out with a Nietzsch STA 409 PC/PG instrument. NMR spectra were collected with a Varian Mercury-Plus 400 MHz instrument (ASWPFG probe), using DMSO-d6 as solvent and 1 s delay at 25 °C. Photophysical Measurements. Absorbance spectra were collected with a varian Cary 5 UV-vis-NIR spectrophotometer. Photoluminescence was investigated with a Varian (model Cary Eclipse) spectrofluorimeter. Relative quantum yields of photoluminescence (PL) were obtained with Ru(bpy)32+ in aerated solution as reference.9 The results of the basic photophysical characterizations are summarized in Table 1 and Figure S2 in the Supporting Information. Electrochemistry. Electrochemical one-compartment airtight cells were used for electrochemical and ECL measurements in ultra-anhydrous conditions. The working electrode consisted of either a side oriented Pt disk electrode sealed in glass (φ ) 3 mm) or an analogous side oriented Au electrode (φ ) 1.5 mm). The counter electrode consisted of a Pt spiral while the
Ciana et al. TABLE 1: Summary of Photophysical Properties of the Ruthenium Complexes Measured in H2Oa compd
MLCT abs λmax, nm
ε, M-1 cm-1
PL λmax, nm
ΦPL,b %
452 452 432 464
14 000 18 000 18 000 25 000
610 620 618 615
2.8 3.6 4.0 4.5
2+
Ru(bpy)3 Ru(BPS)(bpy)2 [Ru(BPS)2(bpy)]Na2 [Ru(BPS3)]Na4 a
Aerated solutions. b [Ru(bpy)3]2+ was taken as standard reference according to literature data, ref 9.
quasireference electrode was a silver spiral. Further details about the electrochemical cell were described elsewhere.10 Acetonitrile (MeCN, spectroscopy grade from Merck) was refluxed over CaH2, then distilled under vacuum at room temperature with a high refluxing ratio. Tetrabutylammonium hexafluorophosphate (TBAH, electrochemical grade from Sigma-Aldrich) was used without further purification as supporting electrolyte in organic media. ECL and electrochemical measurements were carried out with an AUTOLAB electrochemical station (Ecochemie). The E1/2 values for reductive and oxidative processes were calculated by adding ferrocene as an internal standard. In aqueous media, phosphate buffer solution (PB) was used as the supporting electrolyte. Electrochemiluminescence. Annihilation ECL measurements were carried out in ACN solution with TBAH as supporting electrolyte, under strictly aprotic conditions. The three-electrode cell described above was fitted with a Pt side oriented 2 mm diameter disk working electrode, a Pt spiral counter electrode, and a quasireference Ag wire. The annihilation reaction was obtained by alternating the working electrode between the first oxidation and the first reduction peak potential of the complex with a pulse width of 0.1 s. For ECL generation in 0.1 M, pH 7.5 phosphate buffer, TPA was added as oxidative coreactant at the concentration of 3 × 10-2 M. ECL was obtained in a single oxidative step by generating, at the same time, the TPA and the Ru complex in their oxidized forms according to well-established methods.1 The ECL signal during cyclic voltammetry was measured with a photomultiplier tube (PMT, Hamamatsu model R928P) placed at a few millimeters distance in front of the working electrode, inside a darkbox. A voltage in the range 400-1000 V was supplied to the PMT. The light/current/voltages curves were recorded by collecting the preamplified PMT output signal (by an ultralow noise Acton research mod. 181) with the second input channel of the ADC module of the AUTOLAB instrument. ECL spectra were recorded by inserting the same PMT in a dual exit monochromator (ACTON RESEARCH Spectra Pro2300i) and collecting the signal as described above. Results and Discussion The primary scope of the present investigation is to clarify the role of the net charge on the electrochemical and ECL properties of a series of Ru(II) complexes, in order to optimize ECL tracers. It is important to consider that charge also affects solubility in aqueous and organic solvents in opposite ways. In fact, [Ru(BPS)3]4- is poorly soluble in MeCN, but highly soluble in water; on the other hand, [Ru(BPS)2(bpy)]2- and Ru(BPS)(bpy)2 are relatively soluble in MeCN, while retaining at least some water solubility. Electrochemical Investigations in Anhydrous Acetonitrile. The electrochemical behavior of [Ru(BPS)2(bpy)]2- and Ru(BPS)(bpy)2 was first investigated in ultradry MeCN. Typical
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Figure 1. Cyclic voltammetric curves of 0.5 mM Ru(BPS)(bpy)2 in 0.1 M TBAH/MeCN solution. Working electrode: Pt disk (diameter 3 mm). T ) 25 °C. Scan rate: (a) 0.5 and (b) 0.2 V/s. Cyclic voltammogram under the same experimental conditions including the third (black line), fourth (red line), and fifth (blue line) reduction process occurring at the edge of the solvent discharge.
TABLE 2: Summary of the Electrochemical Properties of the Analyzed Compounds process E1/2/V vs Fc/Fc+
Ru(BPS)(bpy)2
[Ru(BPS)2(bpy)]TBA2
[Ru(BPS)3]TBA4a
I 1 2 3 4
+0.86 -1.74 -1.91 -2.14 -2.57
+0.75 -1.71 -1.85 -2.08 (-2.52)
+0.77 -1.71 -1.86 -2.07 (-2.65)
a
Values from ref 5.
voltammetric curves of Ru(BPS)(bpy)2 are reported in Figure 1. Unlike [Ru(BPS)3]4- and [Ru(BPS)2(bpy)]2- (see Table 2), Ru(BPS)(bpy)2 showed a completely reversible first oxidation process (labeled as I in Figure 1), with a potential E1/2 slightly shifted to more positive potentials (∆V ) +50-90 mV). This behavior can be related to the lack of net charge, making the Ru complex less electron rich, while at the same time limiting the formation of adsorption films at the electrode when positive potential is applied. The oxidation can be confidently assigned to the Ru(II) metallic center.12 In the case of [Ru(BPS)2(bpy)]2-, as already discussed for [Ru(BPS)3]4-,5 the oxidation process is overlaid with an adsorption process. In the negative potential region, four fully reversible reduction peaks (labeled respectively as 1, 2, 3, and 4, respectively Figure 1b) were registered for Ru(BPS)(bpy)2 and [Ru(BPS)2(bpy)]2-, while a fifth peak is visible only in the neutral complex at the limit of the solvent discharge. As evidenced in Figure 1b, when the voltammetric forward scan includes the partially irreversible peak 5, a product of follow-up reaction is detected on the reverse scan (peak 5′). In Table 2 the potentials E1/2 of oxidation and reduction processes for the [Ru(BPS)n(bpy)3-n](2n-2)- complexes series are reported. The comparison highlights clearly that the E1/2 of the reductive processes 1, 2, and 3 of [Ru(BPS)2(bpy)]2- and Ru(BPS)(bpy)2 are, as expected, very similar to those of the homoleptic complexes with small negative shifts for the neutral compound (∆V ≈ -30 to -60 mV). Furthermore, by observing the first three reductive processes, one can note that the first and the second reductions are less spaced than the second and the third ones, as typically happens for Ru(bpy)32+.12 This confirms that the first three reductions are essentially monoelectronic and localized separately into the three distinct ligands.
ECL Generation by Cation-Anion Annihilation. The presence of a reversible or partially reversible oxidative process and of at least two fully reversible reductions in [Ru(BPS)2(bpy)]2- and Ru(BPS)(bpy)2 made the generation of ECL possible with the well-known mechanism of cation-anion direct annihilation.1 The typical ECL profile registered during a cyclic voltammetry of Ru(BPS)(bpy)2 is shown in Figure 2a, where, in contrast to [Ru(BPS)3]4- and [Ru(BPS)2(bpy)]2-, a high-intensity emission occurs during oxidation while during reduction light generation has a rather low efficiency. This behavior did not change by raising the positive limiting potential or alternating oxidative and reductive 100 ms impulses (Figure 2c). However, in the chronoamperometric experiments an increase in reductive ECL intensity was observed. From the shape of the peaks reported in Figure 2c, it is evident that ECL emission decay is longer during the oxidation than the reduction process. Considering that the species present in the diffusion layer are the Ru(II) complex, its radical cation, and its radical anion, and that the emission originates only from the excited state decay of the complex, the intensity and lifetime of ECL emission during reduction can thus be attributed to a shorter lifetime of the radical cation with respect to the radical anion. This explanation is confirmed by the difference of relative intensity in CV and fast potential impulses. In the case of [Ru(BPS)2(bpy)]2- the ECL behavior is intermediate, in the sense that ECL intensity is higher during reduction as for [Ru(BPS)3]4- in CV (0.5-5 V/s scan rate). If, however, fast alternate positive and negative potential steps are applied, ECL emission increases enormously in intensity and becomes prevalent during oxidation as with Ru(BPS)(bpy)2. This allows an intense ECL spectrum to be obtained also for [Ru(BPS)2(bpy)]2- in the appropriate chronoamperometric conditions (see Table 3). The fact that, unlike [Ru(BPS)3]4-, the two heteroleptic complexes become stable and produce highly intense ECL signal during oxidation is an experimental evidence that the partial irreversibility of the oxidation peak in the homoleptic complex and its reduced ECL intensity are a consequence of the highly negative charge, which promotes the formation of filming products at the electrode during oxidation. This hypothesis is in agreement with the reported adsorption phenomena of Ru(bpy)32+-like complexes.13 In the case of Ru(BPS)(bpy)2 it is also interesting to note that if the potential is swept between the first oxidation
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Ciana et al. TABLE 3: ECL Quantum Yield by Cation-Anion Direct Annihilation for the Investigated Complexesa complex
ΦECL,rel,b %
Ru(BPS)(bpy)2 [Ru(BPS)2(bpy)]2[Ru(BPS3)]4-
24 13 0.08
a These values have been measured in ultra dry MeCN. quantum efficiency relative to Ru(bpy)32+.
b
ECL
Figure 3. Comparison of ECL profile of [Ru(BPS)n(bpy)3-n](2n-2)series during ciclic voltammetry. Experimental details: W ) Au disk 1.5 mm; reference ) Ag/AgCl; 10-5 M Ru/3 × 10-2 TPA/PB 0.1 M (pH 7.5); scan rate and potential range ) 0.5 V/s; E1 ) 0 V, E2 ) -1.0 V, E3 ) +1.3 V. Detection: PMT bias 750 V. Current range: 10-6 A/V.
Figure 2. Typical light/current/voltage profiles of 0.5 mM Ru(BPS)(bpy)2 in 0.1 M TBAH/MeCN solution in ultra dry conditions. PMT bias ) 750 V; ECL current range ) 10-6 A/V; scan rate ) 1 V/s, E1 ) +1.5 V; E2 ) -1.3 (first reduction) (a) and -1.6 (first and second reduction) (b). ECL emission is generated here by direct cation-anion annihilation. (c) Light/current/time profiles for the cation-anion direct annihilation of 0.5 mM Ru(BPS)(bpy)2 in 0.1 M TBAH/MeCN solution. Double potential step program: E1 ) +1.5 V (first oxidation), E2 ) -1.45 V (first reduction) with t1 ) t2 ) 0.1 s. All of the potentials are vs Ag wire. Sample time: 1 ms. PMT bias: 750 V. ECL current range: 10-4 A/V.
and the second reduction, ECL intensity is approximately twice the value obtained when only the first reduction is included. This effect can be clearly seen by comparing panels a and b of Figure 2 and is related to the relative amounts of electrogenerated radical anion. This behavior is an experimental proof of the relatively long lifetime of [Ru(BPS)(bpy)2]- and [Ru(BPS)(bpy)2]2- radical anions and of their equivalent activity in the generation of the excited state by combination with [Ru(BPS)(bpy)2]+ radical cation.
Since ECL quantum efficiency is the product of luminescence quantum efficiency and the probability of combination between radical cation and radical anion1,11 its value was estimated for the three complexes under investigation as reported in the Experimental Section. The results, summarized in Table 3, show a progressive increase of ΦECL with the decrease of n in the formula [Ru(BPS)n(bpy)3-n](2n-2)-. The real nature of the filming product is hard to elucidate experimentally because of the reversibility of his formation; however, what we demonstrated here is that the formation of the filming product is strictly correlated to the complex overall charge and with ECL performance. The comparison with Ru(bpy)32+ ECL intensity by annihilation evidences that the standard oxidized and reduced form has a higher stability even if his luminescence quantum yield is lower than that of the RuBPS series. Electrochemical and ECL in Aqueous Buffers. The investigations in aqueous buffers have been performed in the conditions of a typical bioanalytical test, i.e., in phosphate buffer solution (PB) at a physiological pH (7.5) in the presence of TPA as an oxidative ECL coreactant. In Figure 3 are reported the light/current/potential curves registered with homoleptic and heteroleptic complexes. Note that the ECL intensity was monitored after a preliminary scan in the negative potentials region generating electrochemically H2 at the electrode for surface preconditioning. This well-known pretreatment method14 was effective in increasing ECL signal reproducibility.
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Figure 4. Comparison of ECL profile of Ru(BPS)(bpy)2 (a) and [Ru(BPS)3]4- (b) during the first three voltammetric cycles. Experimental details: W ) Au disk 15 mm; reference ) Ag/AgCl; 10-5 M Ru/3 × 10-2 TPA/PB 0.1 M (pH 7.5); scan rate and potential range ) 0.5 V/s; E0 ) 0 V, E1 ) -1.0 V, E2 ) +1.3 V. Detection: PMT bias 750 V. Current range: 10-6 A/V.
These experiments revealed that, despite a luminescence quantum yield decrease (see Table 1), a reduction in the number of bathophenanthrolinedisulfonated ligands causes an increase in ECL maximum intensity. This behavior can be explained by considering the previously discussed filming product formation observed in MeCN. In Figure 4, it is shown how the progressive decay of ECL intensity for repeated CV cycles is strictly connected to the charge of the complex. In particular, the first, second, and third cycles are shown in black, blue, and red, respectively. Ru(BPS)(bpy)2 ECL emission appears much more stable than that of [Ru(BPS)3]4- during repeated voltammetric scans. It is also noteworthy that under analogous conditions Ru(bpy)32+ shows a signal decrease similar to that observed for [Ru(BPS)3]4-; this implies that filming products are generated by all charged Ru(II) species and can contribute to shorten ECL lifetime by progressively limiting the active surface of the electrode. Additional evidence of the strong influence of the charge on ECL time transients were obtained by applying a constant positive potential (Figure 5). By comparing the light/current/ time curve for the different complexes it is clear that by decreasing the number of BPS ligands maximum ECL intensity increases by about 16 times and remains significantly higher after 2 s of potential biasing. On the other hand, the time necessary to decay to 50% of the maximum remains substantially unaltered. This aspect reveals that the increased intensity is caused by the higher concentration of the electrogenerated Ru(II) radical cation in the diffusion layer, strictly dependent on the condition of the electrode surface. The rate of time decay was unchanged because it is controlled by the diffusion rate (IECL with good approximation is proportional to the square root of t) and not by the concentration of the radical cation. Finally, a comparison of ECL integrated intensity for Ru(bpy)32+ and the [Ru(BPS)n(bpy)3-n](2n-2)- complexes series during cyclic voltammetric scans is reported in Figure 6. By normalizing the emission data with respect to Ru(bpy)32+, the sulfonated complexes show an intensity enhancement of approximately 5 to 26 times with a standard deviation around (20%. If filming products are formed during oxidation even in the absence of TPA as demonstrated by direct annihilation experiments, in the presence of coreactant the possibility of side products is much greater due to the great reactivity of TPA radical species. The reactivity and on the other side the chemical
Figure 5. Comparison of ECL/time profile of [Ru(BPS)n(bpy)3-n](2n-2)complexes during a single chronoamperometric impulse. Experimental details: W ) Au disk 1.5 mm; reference ) Ag/AgCl; 10-5 M Ru/3 × 10-2 TPA/PB 0.1 M (pH 7.5). Potential program: conditioning at -1 V for 30 s then E ) +1.3 V vs Ag/AgCl; t ) 2 S. Sample time: 1 mS. Detection: PMT bias 750 V. Current range: 10-6 A/V.
stability of the RuBPS series oxidized form was thus experimentally correlated with the complex neat charge. Concluding Remarks. In heteroleptic complexes of the [Ru(BPS)n(bpy)3-n](2n-2)- series ECL emission was efficiently generated by direct cation-anion annihilation. In the case of Ru(BPS)(bpy)2, an ECL quantum efficiency was found to be very similar to that of Ru(bpy)32+. The voltammetric behavior of Ru(BPS)(bpy)2 was comparable to that of Ru(bpy)32+ and the first oxidative process was completely reversible and stable unlike [Ru(BPS)3]4- and [Ru(BPS)2(bpy)]2-, where an adsorption process was overlaid to the faradic one. This interesting result has been confirmed in phosphate buffer solutions by using TPA as the oxidative coreactant. In aqueous conditions we observed a 4-fold increase of ECL maximum intensity for Ru(BPS)(bpy)2 with respect to [Ru(BPS)3]4- during cyclic voltammetry. In the same way, by applying a constant positive potential, a maximum intensity increase of around 16 times was
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Ciana et al. Supporting Information Available: Method for estimating ECL quantum efficiency for cation-anion direct annihilation, typical time profile of current and photocurrent during a single potential impulse, and absorption and photoluminescence spectrum of heteroleptic complexes. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes
Figure 6. Comparison of ECL integrated intensity for Ru(bpy)32+ (assumed as unity) and the three complexes bearing BPS ligands. Experimental details: W ) Au disk 15 mm; reference ) Ag/AgCl; 10-5 M Ru/3 × 10-2 TPA/PB 0.1 M (pH 7.5); scan rate and potential range ) 0.5 V/s; E0 ) 0 V, E1 ) -1.0 V, E2 ) +1.4 V. Detection: PMT bias 750 V. Current range: 10-6 A/V.
recorded by comparing the complexes with three and one BPS ligands. The voltammetric experiments also showed a progressive slower decay rate of ECL intensity in repeated scans for the heteroleptic complexes upon reduction of BPS ligands number from three to one, thus confirming that the high negative charge was the reason for the irreversibility of electrochemical oxidation. In view of these results, the cause of the reduced ECL quantum yield of [Ru(BPS)3]4- in both annihilation and coreactant-assisted methods appears to be the formation of a filming product at the electrode during oxidation; this does not occur when the negative charge is reduced or eliminated. The highly enhanced stable ECL emission intensity together with the expected absence of electrostatic interactions with proteins and nucleic acids thus makes Ru(BPS)(bpy)2 a highly promising candidate for the development of tracers for ultrasensitive bioassays and imaging applications, adding new potentialities to the already powerful electrochemiluminescence technique. Acknowledgment. The authors are grateful to Massimo Marcaccio, Francesco Paolucci, Luca Prodi, and Danilo De Marchi for the useful discussions. This work was accomplished in the framework of LATEMAR (www.latemar.polito.it), Centre of Excellence funded by MIUR (Italian Ministry for Education, University and Research) Grants FIRB 2003-2004 for public/ private structures involved in research fields characterized by strategic value. Additional financial support from Fondazione Cassa di Risparmio in Bologna is also acknowledged. Thanks are due to Enrico Modena from Polycrystalline srl (Bologna, Italy) for TGA assays.
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