Anal. Chem. 2007, 79, 4521-4528
Inhibited Ru(bpy)32+ Electrochemiluminescence Related to Electrochemical Oxidation Activity of Inhibitors Yuwu Chi, Yongqiang Dong, and Guonan Chen*
Ministry of Education Key Laboratory of Analysis and Detection Technology for Food Safety (Fuzhou University), and Department of Chemistry, Fuzhou University, Fuzhou, Fujian, 350002, China
Electrochemiluminescence (ECL) has been accepted by the analytical chemist as a powerful tool for detection of many inorganic and organic compounds. Ru(bpy)32+ has been the most popular ECL system, and many investigations have been focused on the application based on the enhancement or inhibition of Ru(bpy)32+ ECL system. However, not much attention has been paid to the theoretical investigation of this ECL system, especially to the inhibiting mechanism for the Ru(bpy)32+ ECL system. In the present study, many of the inorganic and organic compounds with electrochemical oxidation activity were found to strongly inhibit Ru(bpy)32+ ECL. To explain these inihibited ECL phenomena, a new “electrochemical oxidation inhibiting” mechanism has been proposed via the establishment of a corresponding model. The effects of applied potential, uncompensated resistance, and concentration of inhibitor on the inhibited ECL derived from the model have been verified by experiments. The new ECL inhibition mechanism can be commonly used to explain many kinds of inhibited ECL presently observed, and it is envisioned to result in finding of more inhibitors of this type and establishment of new sensitive ECL detection methods for them. Enhanced tris(2,2-bipyridine)ruthenium(II) (Ru(bpy)32+) electrochemiluminescence (ECL) has been studied quite extensively in terms of mechanism and applications.1-10 In contrast, inhibited Ru(bpy)32+ ECL has been relatively seldom studied. Up to now, only a few inhibited Ru(bpy)32+ ECL systems have been reported.5,9,11-14 Richter’s group reported the inhibition of Ru(bpy)32+/tripropylamine (TPA) ECL by phenol, substituted phe* Corresponding author. Fax: 86-591-83713866. E-mail:
[email protected]. (1) Knight, A. W.; Greenway, G. M. Analyst 1996, 121, 101R. (2) Gerardi, R. D.; Barnett, N. W.; Lewis, S. W. Anal. Chim. Acta 1999, 378, 1-41. (3) Fahnrich, K. A.; Pravda, M.; Guilbault, G. G. Talanta 2001, 54, 531-539. (4) Kulmala, S.; Suomi, J. Anal. Chim. Acta 2003, 500, 21-69. (5) Chi, Y.; Duan, J.; Zhao, Z. F.; Chen, H.; Chen, G. Electroanalysis 2003, 15, 208-218. (6) Chen, G.; Chi, Y.; Wu, X.; Duan, J.; Li, N. Anal. Chem. 2003, 75, 66026607. (7) Yin, X. B.; Dong, S.; Wang, E. TrAC-Trends Anal. Chem. 2004, 23, 432441. (8) Richter, M. M. Chem. Rev. 2004, 104, 3003-3036. (9) Chi, Y.; Xie, J.; Chen, G. Talanta 2006, 68, 1544-1549. (10) Chi, Y.; Duan, J.; Lin, S.; Chen, G. Anal. Chem. 2006, 78, 1568-1573. 10.1021/ac0702443 CCC: $37.00 Published on Web 05/10/2007
© 2007 American Chemical Society
nols, hyroquinones, catechols, and benzoquinones.11,12 Li and coworkers observed ECL of Ru(bpy)32+/TPA inhibited by adrenaline.13 Lin et al and Cui et al. reported inhibited Ru(bpy)32+/TPA ECL by gallic acid, phenols, and anilines,14,15 In our previous studies, the Ru(bpy)32+ ECL was found to be inhibited by homogentisic acid at pH e10,5 and by allopurinol at pH e11.9 The study of inhibited ECL is very important to further uncover the ECL mechanism and to enlarge the analytical application of the ECL method. It is known from the literature that most analytes involved in the inhibited ECL are limited to some special compounds, i.e., phenols and catechols and their derivatives,5,11-15 which means that vast other types of compounds need to be studied before extensive application of the inhibited ECL method. The reaction mechanism of inhibited Ru(bpy)32+ ECL by these phenolic moietycontaining compounds has been proposed to involve energy transfer from the excited luminophore, Ru(bpy)32+*, to benzoquinones by McCall and Richter,11,12 as well as other researchers.13-15 However, the mechanism of inhibited ECL remains unclear for several questions still need to be answered. One question is about the inhibition efficiency of ECL. McCall et al.11,12 reported that the inhibition of ECL happened only when ratios of inhibitor (quencher) to luminophore (Ru(bpy)32+) were higher than 100 whereas the other researchers13-15 showed that inhibited ECL began to appear when the ratios were ∼1 × 10-4-1 × 10-3 (estimated from detection limit of inhibitors) and half-inhibition was reached at ∼1:1 of inhibitor/Ru(bpy)32+.13 This means that the ECL inhibition efficiencies reported by McCall et al. are much smaller than those reported by the other researchers. Apparently, the great difference in ECL inhibition efficiencies cannot be explained merely by the benoquinone inhibiting mechanism; consequently, some other inhibition mechanism must be sought. It can be found from the comparison of the work reported by McCall et al. and the other researchers that there was an significant difference in experimental conditions between them. The weak ECL inhibitions were observed by McCall et al. in bulk (11) McCall, J.; Alexander, C.; Richter, M. M. Anal. Chem. 1999, 71, 25232527. (12) McCall, J.; Richter, M. M. Analyst 2000, 125, 545-548. (13) Li, F.; Cui, H.; Lin, X. Q. Anal. Chim. Acta 2002, 471, 187-194. (14) Lin, X. Q.; Li, F.; Pang, Y. Q.; Cui, H. Anal. Bioanal. Chem. 2004, 378, 2028-2033. (15) Cui, H.; Li, F.; Shi, M. J.; Pang, Y. Q.; Lin, X. Q. Electroanalysis 2004, 17, 589-598.
Analytical Chemistry, Vol. 79, No. 12, June 15, 2007 4521
Figure 1. Schematic diagram of thin-layer flow-through electrochemiluminescent cell. Table 1. Inhibitors Found for Ru(bpy)32+/TEA ECL System types inorganic compounds organic compounds
inhibitors NO2-, NO, H2O2, [Fe(CN)6]4-, NH2OH.HCl, Iphenol, tyrosine, catechol, hydroquinone, homogentisic acid, imidazole, methimazole, indole, indole-3-aceticacid, aniline, allopurinol, oxypurinol, xanthine,hypoxanthine, uric acid, 2-thiouracils, 4-thiouracil, dithiouracil, adenine, guanine, thymine, cytosine, uracil
solution whereas the strong ECL inhibitions were reported by the other researchers in flow injection analysis (FIA). It is well-known that solution resistances in FIA are much higher than those in bulk solutions; thus, a question would be arised about whether the high solution resistances resulted in the strong ECL inhibition. Another question is about the inhibitors. Unlike phenols and catechols, which inhibited ECL probably by their oxidation into benzoquiones of quenching activity, some other type of electroactive compounds, for example, HNO2 also exhibited strong inhibition to Ru(bpy)32+ ECL, although its electrooxidative product, HNO3, is unlikely a quencher of the excited-state luminopore. This implies that it is the electroactivity of the inhibitor rather than its oxidative product that plays an important role in the ECL inhibition of Ru(bpy)32+. In this paper, we report that the inhibited Ru(bpy)32+ ECL in the thin-layer flow cell is related to the electrochemical activity of the inhibitor. In a given ECL system, for example, Ru(bpy)32+/ triethylamine (TEA), ECL inhibition is observed when an electroactive species is injected into the ECL system, no matter what the electrooxidative product is. The inhibited ECL effect is explained by the fact that the electroactive inhibitor causes a significant change of IR drop in the FIA-ECL cell and thus decreases the electrochemical generation of Ru(bpy)33+ for ECL. The new ECL inhibition mechanism and its potential analytical application are demonstrated in detail by taking the inhibition of Ru(bpy)32+/TEA ECL by hypoxanthine as an example. EXPERIMENTAL SECTION Materials. Tris(2,2-bipyridine)ruthenium(II) chloride hexahydrate (Ru(bpy)3Cl2‚6H2O) and TEA, and some ECL inhibitors, imidazole, methimazole, indole-3-acetic acid, allopurinol, oxypurinol, hypoxanthine, hypoxanthine, uric acid, 2-thiouracil, 4-thiou4522 Analytical Chemistry, Vol. 79, No. 12, June 15, 2007
Figure 2. Inhibition of Ru(byp)32+/TEA ECL observed in flow injection analysis by (A) nitrite and (B) [Fe(CN)6]4-. Carrier solution: 5.0 × 10-5 mol L-1 Ru(byp)32+ + 5.0 × 10-4 mol L-1 TEA + 0.1 mol L-1 PBS (pH 10.0). Sample solution: the carrier solution + 5.0 × 10-5 mol L-1 nitrite or [Fe(CN)6]4-. Flow rate was 1.0 mL min-1. Applied potential was +1.3 V (vs Ag/AgCl).
racil, dithiouracil, adenine, guanine, thymine, cytosine, and uracil, were purchased from Sigma-Aldrich and used without further purification. All other chemicals were all of analytical purity. The 1 × 10-2 mol L-1 various stock solutions were prepared with double-distilled water and stored in a refrigerator. Phosphate buffer solutions (PBS) were prepared by neutralizing a certain concentrations of phosphoric acid solutions with a concentrated sodium hydroxide solution to the required pH value. Carrier solutions were prepared by diluting Ru(bpy)32+ and TEA cosolution with PBS. Sample solutions were prepared by adding the required volume of the inhibitor stock solutions into the carrier solutions. Apparatus and ECL Measurement. The ECL measurements were performed on a homemade FIA-ECL system, which was similar to that previously descriped.9 The thin-layer flow-through electrochemiluminescent cell equipped in the FIA-ECL system is shown in Figure 1. The cell had a Pt working electrode (22.1 mm2), a Ag/AgCl (3 M KCl) reference electrode, and a stainless steel counter electrode. The working electrode was positioned at the center of a solution thin layer (1.6 cm × 0.45 cm × 50 µm), the thickness of which was determined by a Teflon spacer (50 µm). Two solution channels (i.d. 0.10 cm × 0.5 cm) were drilled at the two ends of the solution thin layer and connected with an inlet and an outlet of the flow cell, respectively. The counter electrode made of a piece of stainless steel pipe was placed at the outlet, and the reference electrode was mounted at the end of the solution channel near the outlet. Apparently, this cell configuration had a large uncompensated resistance, i.e., the resistance between the surface of Pt working electrode and the tip of reference electrode. It can be known from the sizes of the solution thin layer and the channels that most of the uncompensated resistance resulted from the solution resistance in the thin layer rather than the solution in the channels. The uncompensated resistance of this flow-through thin-layer cell was estimated to be
Figure 3. Electrochemical oxidation-related ECL inhibition model for Ru(bpy)32+-TEA/ inhibitor ECL system in flow injection analysis at a constant and high enough applied potential. The inhibited ECL peaks (the solid line curves drawn by modified Gaussian-shape model16a) are generated upon injecting inhibitors of electroactivity into ECL thin-layer flow-through cell. The ECL background (the dotted line) was drawn by use of the Nernst equation.
more than 10 kΩ when using 0.1 mol L-1 KCl as an electrolyte.16b The uncompensated resistance was dependent on both the size (length, width, and thickness) of the solution thin layer and the concentration of salts in the test solutions.16b However, after fabrication of the flow cell, it was more convenient to change the value of uncompensated resistance by altering the concentration of salts (ionic strength). All experiments were carried out at 25 °C room temperature. RESULTS AND DISCUSSION Inhibition of Ru(bpy)32+/TEA ECL by Inhibitors with Electrochemical Oxidation Activity. In recent experiments, we found that many electroactive compounds besides phenols and catechols had an inhibitiory effect on Ru(bpy)32+/TEA ECL (see Table 1). Apparently the ECL inhibition by these compounds cannot be explained by the electrochemical generation of benzoquiones of quenching activity or other possible quenchers. The typical compounds are NO2- and [Fe(CN)6]4-, which strongly inhibit the Ru(bpy)32+/TEA ECL (see Figure 2A and B, in which the inverse peaks demonstrate the inhibition of ECL). It is wellknown that the electrooxidative products of NO2- and [Fe(CN)6]4are NO3- and [Fe(CN)6]3-, respectively:
NO2- - 2e- + H2O ) NO3- + 2H+ 4-
[Fe(CN)6]
-
3-
- e ) [Fe(CN)6]
Model for Inhibited Ru(bpy)32+/TEA ECL. Since the previously proposed product-quenching mechanism cannot account for the inhibition of Ru(bpy)32+/TEA ECL by inhibitors capable of electrochemical oxidation, a new inhibited ECL mechanism is thus being sought. Based on the facts that the inhibited ECL is highly dependent on the electrochemical activity of the inhibitors, and strong inhibited ECLs were often observed in flow injection analysis, where thin-layer flow-through cells were used, we propose here an “electrochemical oxidation inhibiting” mechanism to explain the inhibition of Ru(bpy)32+/ TEA ECL in the presence of electroactive inhibitors. In the model shown in Figure 3, the ECL intensity of Ru(bpy)32+/TEA (background ECL, the dotted line curve) is the function of the potential of working electrode, Ew; i.e. the higher Ew is, the stronger the ECL intensity observed. Apparently, in the thin-layer flow cell, the value of the IR drop between the working and the reference electrodea is significant, and makes Ew less than the applied potential, V:
Ew ) V - IR ) V - (II + IRu)R
(3)
Ew ) V - (I0Ru + ∆I)R
(4)
or,
(1) (2)
In terms of molecular structure, it is impossible that the two electrooxidative products (NO3- and [Fe(CN)6]3-) can be served as inhibitors of the Ru(bpy)32+/TEA ECL. Actually, NO3- and [Fe(CN)6]3- were experimentally proved that they indeed did not inhibit the Ru(bpy)32+/TEA ECL. (16) (a) Novic, M.; Novic, M.; Zupana, J.; Zafranb, N.; Pihlarb, B. Anal. Chim. Acta 1997, 348, 101-111. (b) Chen, T.; Dong, S.; Xie, Y. J. Electroanal. Chem. 1994, 379, 239-245.
where I0Ru is the limiting oxidation current of Ru(bpy)32+, R is the resistance of the thin-layer solution between the working and reference electrodes, II and IRu are the oxidation currents of the inhibitor and Ru(bpy)32+, respectively, and ∆I is the difference between I and I0Ru. The oxidation of an inhibitor results in an increase of ∆I. Generally, the concentration of the inhibitor is much lower than that of buffer; the value of R is hence kept constant. Therefore, according to eqs 3 and 4, the potential of the working electrode, Ew, will be decreased when the inhibitor Analytical Chemistry, Vol. 79, No. 12, June 15, 2007
4523
Figure 4. ECL inhibition model for Ru(bpy)32+-TEA/inhibitor ECL system in flow injection analysis at a random applied potential. H0 and H are the background ECL intensity of Ru(bpy)32+-TEA at a high enough applied potential and an any given applied potential, respectively. h is the inhibited ECL intensity.
is oxidized at the electrode, and then the ECL intensity of Ru(bpy)32+ is decreased; i.e., the inhibition happens. Obviously, the inhibited peak height of ECL (h in Figure 3) is the function of the concentration of the inhibitor. It has been demonstrated by Zu and Bard that the Ru(bpy)32+/TPA ECL mainly follows an electrocatalytical route at Pt and Au working electrodes for the direct oxidation of TPA is inhibited significantly by the surface oxides formed at anodic potential,17 and the Ru(bpy)32+/TEA ECL might involve the same mechanism: 2+
Ru(bpy)3
-
3+
- e f Ru(bpy)3
(6)
TEA•+ f TEA• + H+
(7)
Ru(bpy)33+ + TEA• f Ru(bpy)32+* + TEA+
(8)
Ru(bpy)32+* f Ru(bpy)32+ + hν
(9)
When the concentration of TEA is much higher (e.g., 10 times) than that of Ru(bpy)32+, the oxidation current of Ru(bpy)32+ is kinetically controlled:18a
IRu RT ln nF I0 - I Ru Ru
I0Ru ) nRuFAC*Ru2+(Dk′C*TEA)1/2
(10)
(17) Zu, Y.; Bard, A. J. Anal. Chem. 2000, 72, 3223-3232. (18) (a) Bard, A. J.; Faulkner, L. R. Electrochemical Methods-Fundamentals and Applications, 2nd ed.; John Wiley & Sons Inc.: New York, 2001. (b) Kanoufi, F.; Zu, Y.; Bard, A. J. J. Phys. Chem. B 2001, 105, 210. (c) Klatt, L. N.; Blaedel, W. J. Anal. Chem. 1968, 40, 512.
Analytical Chemistry, Vol. 79, No. 12, June 15, 2007
II ) nIFAmI(C*I - Cx)0 I )
(12)
In most cases, formal standard potentials of inhibitors, such as catechols, phenols, and other inhibitors listed in Table 1 are much lower than that of Ru(bpy)32+; thus in the oxidation potential range of Ru(bpy)32+, the inhibitors will be oxidized completely at the electrode, and we have
II ) I0I ) nIFAmIC*I
(13)
From (3), (10) and (13), we obtain
(I0I + IRu)R ) V - ERu 1/2 -
IRu RT ln 0 nF I - I Ru Ru
(14)
In Figure 3, it is assumed that the applied potential, V, is high enough; thus, a maximum Ru(bpy)32+/TEA ECL intensity, H0, is obtained. However, as is well-known, V is often an important parameter for ECL measurements. The effect of V on ECL should be considered. Therefore, we proposed an ECL inhibition model at a random V in Figure 4. At the applied potential of V, the ECL intensity of Ru(bpy)32+/TEA is H, which forms the baseline of a FIA-ECL curves. At the baseline, the inhibitor is absent, thus we have
(11)
Generally, eqs 10 and 11 are used for quitescent solutions; however, the rate constant of chemical reaction between Ru(bpy)32+ and TEA (eq 6) is very large,18b, which results in that the catalytic current being independent of flow rate of the solutions.18c Therefore, these two equations still hold for flow
4524
solutions involved in the present study. The oxidation current of the inhibitor, II, is determined by
(5)
Ru(bpy)33+ + TEA f Ru(bpy)32+ + TEA•+
Ew ) ERu 1/2 +
Figure 5. Variation of Ru(bpy)32+-TEA ECL intensity with oxidation current of Ru(bpy)32+. Testing solution: 5.0 × 10-5 mol L-1 Ru(byp)32+ + 5.0 × 10-4 mol L-1 TEA + 0.1 mol L-1 PBS (pH10.0). Flow rate was 1.0 mL min-1.
Ru V ) I0′ RuR + E1/2 +
I0′ Ru RT ln 0 nF I - I0′ Ru Ru
(15)
2+ where I0′ Ru is the catalytic oxidation current of Ru(bpy)3 /TEA at the baseline H. Combination of eq 14 and 15 results in
K)
I0I I0Ru
)
I0′ Ru - IRu I0Ru
(
+
)
I0Ru - IRu I0′ Ru 0.059 • log IRu I0RuR I0Ru - I0′ Ru
(T ) 25 °C) (16)
Figure 6. Variation of inhibited ECL peak height, h, with background ECL intensity, H, at different K and I0RuR values: I0RuR ) (A) 0.1, (B) 0.2, and (C) 0.5 V. H0 is used as the unit for both h and H.
The catalytic oxidation current of Ru(bpy)32+, IRu, is related to the ECL intensity IECL. The experimental result shown in Figure 5 indicates that there is a good linear relationship between IRu and IECL, then
H0 H h ) ) 0′ ) I0Ru I0′ I IRu Ru Ru H0 - H H0 - H + h H-h ) 0 ) 0 (17) 0′ IRu IRu - IRu IRu - IRu
(
and eq 16 is changed to
)
h H H 1- 0+ 0 0 h H H H 0.059 K) 0+ 0 lg • H H h H IRuR 1- 0 H H0 H0
(18)
According to eqs 11, 13, 16, and 18, we have
C*I C*Ru
1 ) K) k
{ (
)}
H h H 1- 0+ 0 0.059 H0 H H 1 h + 0 lg H h k H0 H IRuR 1- 0 - 0 0 H H H
(19)
where
k)
nIFAmI nRuFA(Dk′C*TEA) K)
1/2
)
( )
nImI nRu(Dk′C*TEA)1/2
k C*I C*Ru
(20a)
(20b)
Apparently, k is the ratio of the mass-transfer coefficient of an inhibitor to Ru(bpy)32+. Equation 20b indicates that K is linear proportional to the concentration of the inhibitor when the concentration of Ru(bpy)32+ and other experimental parameters
are kept unchanged; herein, we call K the concentration factor of the inhibitor. Equation 19 suggests that at a given Ru(bpy)32+/ TEA system where C*Ru, k, I0Ru, and H0 are kept constant, h (the inhibited ECL intensity) is a function of C*I (inhibitor concentration), R (uncompensated resistance), and H (ECL background). From eqs 15 and 17, we obtain
V)
H RT H 0 ln 0 I R + ERu 1/2 + 0 Ru nF H H -H
(21)
Equation 21 shows that H is determined by V; thus, it follows that h is also a function of V. Both eq 18 and eq 19 can be used to analyze effects of experimental conditions on the inhibited ECL intensity, h; however, it is more convenient to use eq 18 than eq 19 for the former includes the unknown constants of k and I0Ru in the variables of K and I0RuR, respectively. Therefore, some typical plots of h/H0 versus H/H0 at different K, I0RuR were drawn and shown in Figure 6A-C. Effect of Applied Potential. The plots in Figure 6 clearly indicate that only an appropriate H/H0 (or potential, V) value is good for obtaining sensitive ECL inhibition. This can be explained when applying over high V, the excess potential will compensate the decrease of the effective potential of the working electrode caused by the oxidation of the inhibitor. Thus, h is decreased with further increase in V (or H), while when applying over low V, the ECL background (H) itself becomes very low, which determines that h is also very small, because h is always less than H. To verify this theoretical result, inhibition of Ru(bpy)32+/TEA ECL by hypoxanthine at various applied potentials was investigated. The experimental result shown in Figure 7 indicates that h first increases with potential V (or H) in the low potential range (Figure 7a-d), then reaches a maximum value at a medial potential (Figure 7d), and finally, h decreases with further increasing potential (Figure 7d-h). Apparently, this experimental result fits the model (see Figure 6) very well. The optimum H to obtain the most sensitive ECL inhibition is more or less affected by K. In the low K value range (K e 0.1), the maximum h is obtained where the H is 0.5H0, the position is Analytical Chemistry, Vol. 79, No. 12, June 15, 2007
4525
Figure 7. Ru(bpy)32+-TEA ECL inhibited by hypoxanthine of a low concentration (5.0 × 10-6 mol L-1) at different applied potentials: (a) 1000, (b) 1050, (c) 1100, (d) 1150, (e) 1200, (f) 1250, (g) 1300, and (h) 1400 mV. Carrier solution: 0.1 mol L-1 pH 10 PBS + 5.0 × 10-5 mol L-1 Ru(bpy)32+ + 5.0 × 10-4 mol L-1 TEA. Sample solution: carrier solution + 5.0 × 10-6 mol L-1 hypoxanthine.Flow rate was 1.0 mL min-1.
Figure 9. Effect of solution electroconductivity on the inhibited ECL intensity. FIA-ECL responses were recorded for PBS solution (pH 10) of different concentrations: (A) 0.05, (B) 0.1, and (C) 0.25 mol L-1. Carrier solution: PBS + 5.0 × 10-5 mol L-1 Ru(bpy)32+ + 5.0 × 10-4 mol L-1 TEA. Sample solution: carrier solution + 1.0 × 10-4 mol L-1 hypoxanthine. Applied potential was +1350 mV; flow rate was 1.0 mL min-1.
Figure 8. Ru(bpy)32+-TEA ECL inhibited by hypoxanthine of a high concentration (1.0 × 10-4 mol L-1) at different applied potentials: (a) 1000, (b) 1050, (c) 1100, (d) 1150, (e) 1200, (f) 1250, (g) 1300, (h) 1350, (i) 1400, (j) 1450,; (k) 1500, and (l) 1550 mV. Carrier solution: 0.1 mol L-1 pH 10 PBS + 5.0 × 10-5 mol L-1 Ru(bpy)32+ + 5.0 × 10-4 mol L-1 TEA. Sample solution: carrier solution + 1.0 × 10-4 mol L-1 hypoxanthine. Flow rate was 1.0 mL min-1.
seldom dependent on K. From eq 21, the optimum applied potential, Vopt, can be obtained:
Vopt )
1 0 I R + ERu 1/2 2 Ru
(22)
After measuring Vopt, I0Ru, and ERu 1/2 by experiment, eq 22 can be used to estimate the uncompensated resistance, R, of the ECL cell. However, in the high K value range (i.e., K > 0.1), the optimum H, noted as Hopt, is dependent on K; i.e., Hopt is no longer 0.5H0, but shifts to the range of 0.5H0-H0. Moreover, the higher K that is used, the larger Hopt that is found. This was confirmed by our investigation on the inhibition of Ru(bpy)32+/TEA ECL by high concentrations of hypoxanthine. Figure 8 shows that, at high concentration of the inhibitor (i.e., high K), the maximum inhibition indeed occurred at the ECL baseline near H0 (see peak h in Figure 8). Effect of Uncompensated Resistance. By comparison of panels A-C in Figure 6, it can be seen that the uncompensated resistance (R), or IR drop, affects inhibition intensity (h/H0) 4526 Analytical Chemistry, Vol. 79, No. 12, June 15, 2007
significantly. Under certain experimental conditions, the maximum inhibition intensity, (h/H0)max, is increased with increasing I0RuR value. Corresponding experiments were designed to test this effect as follows: FIA-ECL responses were recorded for Ru(bpy)32+/ TEA/hypoxanthine in PBS of different concentrations. The experimental results shown in Figure 9 verify the theoretical prediction that the inhibition ECL intensity is increased with increasing uncompensated resistance (or with decreasing salt concentration). The effect of uncompensated resistance on the inhibition intensity gives us valuable information that the detection sensitivity of inhibitors (analytes) can be improved using a solution of relatively low salt concentration. Now, it is possible to explain the great difference in ECL inhibition efficiencies between static ECL and FIA-ECL as previously mentioned in the introduction by above-mentioned model. Generally, the uncompensated resistance in static ECL mode (i.e., ECL in bulk solution) is about several hundreds of ohms,18 whereas in FIA-ECL mode (i.e., ECL in thin-layer cell) it is about tens of kiloohms.16b If we omit the influence of flow rate on mass transfer, then eq 18 holds for both static ECL and FIA-ECL. It is assumed that, in both cases, the catalytic oxidation current of Ru(bpy)32+, I0Ru, is 10 µA; optimum potentials are applied, i.e., H/H0 ) 0.9; and half-inhibition of ECL occurs, i.e., h/H0 ) 0.5. Moreover, the uncompensated resistances (R) in static ECL and FIA are assigned to be100 and 10 kΩ, respectively. By use of eq 18, the concentration factors, K ) k(C*I /C*Ru), were calculated to be 67.2 and 1.17 for static ECL and FIA-ECL, respectively. Herein, (C*Ru/C*I ) at half ECL inhibition is defined as inhibition efficiency, then the inhibition efficiency in FIA-ECL, (C*Ru/C*I )FIA, is nearly 100 times as much as that in static ECL, (C*Ru/C*I )static. Until now,
Figure 10. Plots of K vs inhibited ECL intensity (h/H0) at different H/H0: H/H0 ) (a) 0.1, (b) 0.5, (c) 0.9, (d) 0.99, BS (e) 0.999. I0RuR is 0.1 V.
the present ECL inhibition model not only had succeeded in explaining the great difference in ECL inhibition efficiencies between static ECL and FIA-ECL but also suggested that the inhibition of Ru(bpy)32+/tertiary amines ECL by phenols and catechol derivatives in FIA most likely resulted from the electrochemical oxidation of these inhibitors rather than the formation of quenching products, such as quinones. Effect of Inhibitor Concentration. It can be known from Figure 6 that when K is high enough, the inhibited ECL height, h/H0, has the same value as the ECL background, H/H0; i.e., Ru(bpy)32+/TEA ECL is completely inhibited (see linear lines with 9 dots in these figures). Herein, we note K as Klim when complete ECL inhibition happens. Obviously, at Klim, complete ECL inhibition happens at any value of H/H0; i.e., it is independent of applied potential, V. By comparison of these figures (Figure 6A-C), it can be concluded that the value of Klim is dependent on the IR drop of thin-layer flow-through cell; the higher IR is, the smaller is the Klim found. The values of Klim at I0RuR values of 0.1, 0.2, and 0.5 V are estimated to be about 4.0, 2.5, and 1.5, respectively. The presence of complete ECL inhibition was experimentally confirmed by using a high concentration of inhibitors (data not shown). Apparently, the study on the effect of inhibitor concentration on inhibition of ECL intensity is of great significance for analytical application. Although Figure 6 has given us the information that the inhibition of ECL intensity (h) is influenced by the concentration factor, K, it is necessary to know the linear response range and detection sensitivity, which are both important for detection of inhibitors. To obtain this information, plots of K versus h/H0 were made and analyzed. First, the K ∼ h/H0 plots were made for an IR drop of 0.1 V and different H/H0 (0.1-0.999), and the results are shown in Figure 10. It can be known from the figure that both the linear response range and sensitivity vary with H/H0 (see curves a-c and e in Figure 10). When H/H0 is changed from 0.1 to 0.9, detection sensitivity (slope of linear portion of curves a-c) is basically kept unchanged, but linear response range is enlarged with increasing H/H0. When further increasing H/H0 (from 0.99 to 0.999), both detection sensitivity and linear response range are reduced (see curves d and e). By comparison of these plots, the ideal linear response range and sensitivity can be obtained when H/H0 is ∼0.9 (see cure c in Figure 10). Similar results were found for other IR drops, such as 0.2 and 0.5 V. Plots
Figure 11. Plots of K vs inhibited ECL intensity (h/H0) at different IR drops: I0RuR ) (a) 0.1, (b) 0.2, and (c) 0.5 V. Data were calculated at H/H0 ) 0.9. Table 2. Optimum Linear Response Ranges at Various IR Drop IR drop (V)
linear range (h/H0)
regression equation
correlation coefficient (r)
0.1 0.2 0.5
0-0.8 0-0.8 0-0.8
K ) 2.215h/H0 +0.077 K ) 1.607h/H0 +0.038 K ) 1.243h/H0 +0.015
0.9989 0.9995 0.9999
of K ∼ h/H0 at some typical IR drops and corresponding regression equation parameters are compared in Figure 11 and Table 2, respectively. It is evident that at all these IR drop conditions the linear response range is around 0 -0.8 h/H0. This indicates that the FIA method based on this type of inhibited ECL mechanism has a wide linear response range and a low detection limit, implying its good application perspective in analytical chemistry. Table 2 shows that a higher IR drop results in a better linear response and detection sensitivity. Effect of Solution pH. Although pH factor is not included in our proposed ECL inhibition model (see eq 18), this does not means that inhibition intensity is pH independent. Equation 7 implies that Ru(bpy)32+/TEA ECL intensity is dependent on pH, as does the Ru(bpy)32+/TPA ECL system.1 Therefore, the effect of solution pH on the ECL intensity of Ru(bpy)32+/TEA in the absence and presence of hypoxanthine was investigated. Both the ECL intensity of Ru(bpy)32+/TEA (ECL background, i.e., H) and the ECL inhibition intensity, h, are pH-dependent. The shape of h-pH curve is very similar to that of the H-pH curve (data not shown). Both H and h have a maximum value at pH 10.0. Hence, to obtain a maximum detection sensitivity, we suggest using pH 10 for analytical application of the Ru(bpy)32+/TEA/inhibitor ECL system. CONCLUSIONS Ru(bpy)32+/TEA ECL has been found to be inhibited strongly by many compounds with electrochemical oxidation activities, covering inorganic compounds such as NO2-, NO, [Fe(CN)6]4-, I-, and H2O2, and organic compounds such as phenol-containing Analytical Chemistry, Vol. 79, No. 12, June 15, 2007
4527
compounds (phenol, tyrosine, catechol, hydroquinoe, homogentisic acid), indoles (indole, indole-3-acetic acid), aniline, imidazoles (imidazole, methimazole), purines (xanthine, hypoxanthine, adenine, guanine, allopurinol, oxypurinol), and pyrimidines (thymine, cytosine, uracil, thiouracils). A new electrochemical oxidation inhibiting mechanism has been proposed via a model to explain the inhibition of Ru(bpy)32+/TEA ECL by these electroactive inhibitors. The model suggests that the oxidation of this type of inhibitors whose oxidation potentials are lower than that of Ru(bpy)32+ results in an increase in IR drop, thus an decrease in effective potential of working electrode, and ultimately the inhibition of ECL intensity. The effects of applied potential, uncompensated resistance, and concentration of inhibitor on inhibited ECL intensity predicted by the model have been well verified by designed experiments. The new ECL mechanism can be use to
explain many kinds of inhibited ECL found, including systems of Ru(bpy)32+/TEA/inhibitor, Ru(bpy)32+/TPA/inhibitor,11-15 Ru(bpy)32+/C2O42-/inhibitor,13 Ru(bpy)32+/OH-/inhibitor,5,9 and even luminol/OH-/inhibitor.19-21 This study is envisioned to result in finding of more and more new inhibitors of this type and establishment of sensitive detection methods for them.
(19) He, C.; Cui, H.; Zhao, G. Anal. Chim. Acta 1997, 351, 241-246. (20) Cui, H.; Li, Q.; Meng, R.; Zhao, H.; He, C. Anal. Chim. Acta 1998, 362, 151-155. (21) Cui, H.; He, C.; Zhao, G. J. Chromatogr., A 1999, 895, 171-179.
Received for review February 5, 2007. Accepted April 5, 2007.
4528
Analytical Chemistry, Vol. 79, No. 12, June 15, 2007
ACKNOWLEDGMENT This study was financially supported by the National Nature Sciences Funding of China (20575011, 20377007), the Specialized Research Fund for the Doctoral Program of Higher Education (20040386002), the Key Project of Chinese Ministry of Education (207052), the Natural Science Foundation of Fujian Province of China (D0610009), the Project Sponsored by SRF for ROCS, SEM, and Fuzhou University (XRC-0528).
AC0702443
