- Email: [email protected]
Selective detection of uric acid in the presence of ascorbic acid based on electrochemiluminescence quenching
Available online at www.sciencedirect.com Journal of
Electroanalytical Chemistry Journal of Electroanalytical Chemistry 612 (2008) 151–155 www.elsevier.com/locate/jelechem
Short Communication
Selective detection of uric acid in the presence of ascorbic acid based on electrochemiluminescence quenching Zuofeng Chen, Yanbing Zu
*
Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong, China Received 14 May 2007; received in revised form 11 September 2007; accepted 18 September 2007 Available online 22 September 2007
Abstract The direct detection of uric acid (UA) using electrochemical method is usually significantly interfered by the presence of other electroactive species such as ascorbic acid (AA) in biological samples. Here, we describe a new method of UA determination based on the 0 quenching of the electrochemiluminescence (ECL). It was found that the emission of the RuðbpyÞ2þ 3 (bpy = 2,2 -bipyridine)/tri-n-propylamine (TPrA) system could be influenced much more significantly by UA than by AA, particularly when the ECL was produced via the low-oxidation-potential (LOP) route. In the presence of 10 mM TPrA, the Stern–Volmer constant (KSV) of the LOP ECL quenching could be as high as 9.1 · 104 M1 for UA, while the influence of AA on the LOP ECL signal was not obvious. Other possible interfering biological species, such as cysteine, oxalate, purine, glucose, and urea, exhibited much less influence on the LOP ECL as compared with UA. The unique quenching effects led to sensitive and selective detection of UA in human urine samples. 2007 Elsevier B.V. All rights reserved. Keywords: Uric acid; Ascorbic acid; Electrochemiluminescence; Tris(2,2 0 -bipyridine)ruthenium(II); Low-oxidation-potential emission
ðE0 0:9 V vs: SCEÞ
1. Introduction
TPrA e ! TPrAþ
The last few decades have seen intensive electrochemiluminescence (ECL) studies on fundamentals and applica2þ tions, and the ECL of RuðbpyÞ3 (bpy = 2,2 0 -bipyridine) with tri-n-propylamine (TPrA) as the coreactant has now been widely used in clinical analysis (immunoassay and DNA probes) [1,2]. Meanwhile, two emission routes have been proposed on the basis of the coreactant oxidation pathway [3–7]. When RuðbpyÞ2þ 3 concentration is relatively high, TPrA oxidation mainly proceeds via the catalytic route:
The product of the coreactant oxidation, TPrA cation radical, will undergo a rapid decomposition, which leads to the formation of a highly reducing ECL intermediate, TPrA free radical; and then the excited state is produced 3þ by the energetic electron transfer between RuðbpyÞ3 and TPrA :
3þ ðE0 1:02 V vs: SCEÞ RuðbpyÞ2þ 3 e ! RuðbpyÞ3
ð1aÞ
2þ þ RuðbpyÞ3þ 3 þ TPrA ! RuðbpyÞ3 þ TPrA
ð1bÞ 2þ
But, in the presence of dilute RuðbpyÞ3 and concentrated TPrA, TPrA oxidation alters to mainly proceed following the direct oxidation route: *
Corresponding author. Tel.: +852 28598023; fax: +852 28571586. E-mail address: [email protected] (Y. Zu).
0022-0728/$ - see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jelechem.2007.09.018
TPrAþ ! TPrA þ Hþ 3þ RuðbpyÞ3
þ TPrA !
ð2Þ
ð3Þ 2þ RuðbpyÞ3
þ P1
ð4Þ
where TPrA+ = Pr3N+, TPrA = Pr2NCHCH2CH3, and P1 = Pr2NC+HCH2CH3. Recently, a new ECL route, which involves the intermediacy of the TPrA cation radical, was proposed [8]: þ RuðbpyÞ2þ 3 þ TPrA ! RuðbpyÞ3 þ P1
ð5Þ
RuðbpyÞþ 3
ð6Þ
þ TPrA
þ
!
RuðbpyÞ2þ 3
þ TPrA
152
Z. Chen, Y. Zu / Journal of Electroanalytical Chemistry 612 (2008) 151–155
Since no oxidation of RuðbpyÞ2þ 3 is required in this ECL route, the emission can be generated at a potential below 1.0 V versus SCE. Evident low-oxidation-potential (LOP) ECL signals were observed at a freshly polished glassy carbon (GC) electrode and at a nonionic fluorosurfactantmodified gold electrode [4,8–12]. In above ECL routes, the production of the highly reducing intermediates, such as TPrA and RuðbpyÞþ 3 , is crucial for the generation of the excited state. In the presence of reducible quencher species (Q), the intermediate radicals might be consumed by their side reactions with the quencher molecules, resulting in the drop of ECL intensity [11–13]: TPrA þ Q ! P1 þ P2 RuðbpyÞþ 3
þQ!
RuðbpyÞ2þ 3
ð7Þ þ P2
ð8Þ
where P2 represents the reduction product of the quencher molecule. The significance of the intermediate-radical quenching would depend on the competition between the ECL pathways and the side reactions. Our recent study shows that the LOP ECL route is less efficient than the conventional ones and, therefore, the LOP emission may be much more efficiently suppressed by the quenchers [11,12]. Uric acid (UA) is the primary product of purine nucleotide metabolism in human system and has often been regarded as a key biomarker in evaluation of physiological wellbeing [14]. Abnormal levels of UA are related to a number of diseases including gout, hyperuricemia, LeschNyan, cardiovascular disease, kidney diseases, etc [15]. The detection of UA in physiological samples by using electrochemical techniques has been a subject of intensive study [16–20]. The major problem comes from the interference of ascorbic acid (AA) because of their overlapping oxidation peaks [21]. A variety of modified electrodes and electrode pretreatment methods have been proposed with the aim of resolving the anodic processes and sensing UA in the presence of an excess of AA [18,20,22–24]. In this report, we describe a new method of UA determination based on the quenching of the ECL of the RuðbpyÞ2þ 3 =TPrA system. Recently, it has been reported that some molecules, such as oxygen and phenolic compounds, could effectively quench the ECL of the 2þ RuðbpyÞ3 =TPrA system [11,12,25], and a new mechanism involving the interception of the ECL intermediate radicals by quencher molecules has been proposed [11,12]. The intermediate-radical quenching route may lead to much more significant decrease of ECL intensity as compared with the excited-state quenching route. Thus, we examined the influences of UA and AA on the ECL signals generated via different routes, and revealed that the LOP ECL of the 2þ RuðbpyÞ3 =TPrA system could be quenched efficiently by UA, while it was much less sensitive to AA, suggesting the possibility of selective detection of UA in the presence of AA. The influences of some other possible interfering biological species on the LOP ECL emission have also been examined, and the results showed that the detection of UA
would not be affected significantly by these species. This allowed for the development of a sensitive and selective method based on ECL quenching for the analysis of UA in biological samples. 2. Experimental 2.1. Chemicals Tris(2,2 0 -bipyridyl)ruthenium(II) dichloride hexahydrate (Ru(bpy)3Cl2 Æ 6H2O, min 98%), tri-n-propylamine (TPrA, 98%), and Zonyl FSO-100 (F(CF2CF2)17CH2CH2O(CH2CH2O)015H) were purchased from Sigma–Aldrich. Other chemicals were analytical reagent graded and used as received. All solutions were prepared with deionized water (Milli Q, Millipore). The pH of the phosphate buffer solution (PBS, 0.15 M) containing TPrA was adjusted with concentrated NaOH or phosphoric acid. 2.2. Apparatus Photoluminescence (PL) experiments were performed on a Spex Fluorolog 111 spectrofluorometer. Cyclic voltammetry (CV) was performed with the model 600A electrochemical workstation (CH Instruments, Austin, TX). The three-electrode system consisted of a working electrode, a coiled Pt wire counter electrode, and a saturated calomel reference electrode (SCE) separated from the working cell by a salt bridge. The ECL signal was measured with a photomultiplier tube (PMT, Hamamatsu R928) installed under the electrochemical cell. A voltage of 800 V (or 600 V when 1 mM RuðbpyÞ2þ was used) was supplied to the 3 PMT with the Sciencetech PMH-02 instrument (Sciencetech Inc., Hamilton, Ontario, Canada). 2.3. Procedures CV was performed with a scan rate of 100 mV/s. GC, platinum, and gold electrodes were polished with 0.05-lm alumina slurry to obtain a mirror surface and then were sonicated and thoroughly rinsed with Milli-Q water. Before each experiment, the gold and platinum working electrodes were subjected to repeated scanning in the potential ranges from 0.5 to 1.4 or 0.65 to 1.2 V, respectively, in 0.15 M PBS (pH 7.5) until reproducible voltammograms were obtained. The modification of the gold electrode with FSO-100 (FSO-Au) was conducted by immersing the electrode in 5 wt.% FSO aqueous solution for 2 min, followed by thoroughly rinsing with distilled water. Solution pH was adjusted to 7.5 to obtain intense LOP ECL signal [10]. In order to eliminate the influence of oxygen [11], solutions were deaerated by bubbling high purity (99.995%) of N2, and a constant flow of N2 was maintained over the solution during the measurements. All electrochemical measurements were referenced with respect to a SCE. All experiments were performed at room temperature 20 ± 1 C. Reported values for ECL intensity are based on the aver-
Z. Chen, Y. Zu / Journal of Electroanalytical Chemistry 612 (2008) 151–155
age of at least three scans with a relative standard deviation (RSD) of ±5%. Freshly collected human urine sample from adult people was diluted 200 times with 0.15 M PBS (pH 7.5) containing 2þ 1 lM RuðbpyÞ3 and 10 mM TPrA, without either purification or filtration. The UA concentration in the urine sample was determined by standard addition method using the LOP ECL signal at the FSO-Au electrode. 3. Results and discussion 3.1. Quenching effects of UA and AA on photoluminescence and ECL 2þ
The effects of UA and AA on PL of RuðbpyÞ3 were examined firstly in 0.15 M PBS (pH 7.5). The excited-state quenching behavior can be described by the Stern–Volmer equation: I 0 =I ¼ 1 þ K SV ½Q 0
ð9Þ
where I and I represent the emission intensities in the absence and presence of the quencher, respectively. The Stern–Volmer constant KSV defines the efficiency of quenching. No quenching effect of PL was observed in the presence of UA up to 1 mM, while AA exhibited weak quenching effect with a KSV 1.3 · 10 M1, close to the value reported previously (1.2 · 10 M1) [26]. As demonstrated in previous reports [11,12], the pre2þ dominant ECL route of the RuðbpyÞ3 =TPrA system can be altered by changing the solution conditions and the electrode materials; therefore, the effects of quenchers (i.e. UA and AA) on each ECL route can be examined separately. For the catalytic ECL emission, a platinum electrode was 2þ used to oxidize 1 mM RuðbpyÞ3 in the presence of 1 mM TPrA. TPrA was oxidized mainly by the electrogen3þ erated RuðbpyÞ3 , while its direct oxidation was inhibited by the growth of the surface oxide layer at the Pt electrode [4]. On the other hand, when the ECL was produced at a 2þ GC electrode in the presence of trace RuðbpyÞ3 (1 lM) and relatively high concentration of TPrA (10– 100 mM), the coreactant direct oxidation route would be predominant [4]. A nonionic fluorosurfactant-modified gold electrode was used to generate the LOP ECL [9,10]. The adsorption of the fluorosurfactant molecules (i.e. Zonyl FSO) at the gold electrode would facilitate TPrA oxidation by rendering the electrode surface more hydrophobic and inhibiting the growth of the surface oxides in the potential region below 1.0 V, which resulted in a strong LOP ECL signal. 2þ Unlike RuðbpyÞ3 PL which occurred under optical excitation, the ECL was preceded by electrochemical reactions. In contrast to the weak PL quenching, more significant quenching effects on ECL have been observed (Table 1). It was found that the ECL quenching behavior might also be described by the Stern–Volmer equation. Obviously, the excited-state quenching mechanism could not solely account for the larger KSV of the ECL quenching.
153
Table 1 The quenching effects of UA and AA on RuðbpyÞ2þ 3 photoluminescence and ECL via different routes Emission route
Photoluminescence ECL-catalytic routea ECL-direct coreactant oxidation routeb ECL-LOP routec
KSV (M1) UA
AA
0 5.85 · 103 1.68 · 104 9.10 · 104
1.30 · 10 4.91 · 102 2.80 · 102 0
ECL experimental conditions: a 1 mM RuðbpyÞ2þ 3 , 1 mM TPrA, Pt elec2þ c trode; b 1 lM RuðbpyÞ2þ 3 , 10 mM TPrA, GC electrode; 1 lM RuðbpyÞ3 , 10 mM TPrA, FSO-modified gold electrode, [AA] < 100 lM.
As suggested by previous studies [11–13], the ECL process may be remarkably influenced by the side reactions between the ECL reducing intermediate radicals (such as TPrA free radical and RuðbpyÞþ 3 ) and the quencher species. In the potential regions where ECL occurred, both UA and AA would be oxidized. Their oxidation products could act as ECL quenchers. It has been reported that the oxidation of UA would result in the formation of reactive dimine species [16]. The reducible species could probably react with ECL intermediate radicals, and act as efficient ECL quenchers. Although the oxidation product of AA has been found to undergo rapid reaction with water that leads to an electro-inactive species [27], it might still be able to intercept ECL intermediate radicals before the hydrolysis. In addition, the ECL intensity could decrease due to the con3þ sumption of RuðbpyÞ3 by its reactions with AA and UA (the LOP ECL would not be influenced by these reactions 3þ since RuðbpyÞ3 was not involved in the LOP emission route). Overall, because of the complicated ECL quenching processes (including the excited-state quenching and the intermediate-radical quenching), both UA and AA exhibited very different influences on the ECL than on the PL. In various ECL routes, the predominant reaction steps were different, and the effects of UA and AA on the emission intensities changed, leading to the diverse values of KSV in Table 1. It can be seen that the LOP ECL signals were most greatly quenched by UA among the three ECL routes. Fig. 1 shows the quenching effects of UA and AA on the LOP ECL and the corresponding Stern– Volmer plots. When 10 mM TPrA was used, a KSV value of 9.10 · 104 M1 was obtained in the presence of UA, which allowed for the sensitive detection of UA. Under our experimental condition using conventional ECL cell, the detection limit of UA was 1 lM. This sensitivity is comparable to those reported in the literatures obtained via flow injection ECL of ruthenium complex [28–30]. It is interesting to note that the influence of AA on the ECL intensity was generally much less significant as compared to that of UA. When the ECL signal was predominantly produced via the catalytic or the direct coreactant oxidation routes, the KSV values of UA were about 12 and 60 times larger than that of AA, respectively. Fig. 1 shows that, when the emission was produced via the LOP ECL route, the intensity of the ECL signal was nearly
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Z. Chen, Y. Zu / Journal of Electroanalytical Chemistry 612 (2008) 151–155
would provide a distinct advantage over the conventional routes in selective detection of UA in the presence of AA. 3.2. Quenching effects of other possible interfering biological compounds on the LOP ECL Besides AA, other common interfering biological species encountered in UA detection include cysteine, oxalate, purine, glucose, urea etc. Their KSV values for the LOP ECL quenching were also examined (Table 2). The influences of these compounds on the LOP ECL were much less significant than that of UA. The value of KSV obtained for UA was at least several tens of times larger. Moreover, considering that these compounds exist in biological fluids (especially in urine samples) at a much lower level than UA [16,17], their interference on UA detection could be negligible. Our previous study [11,12] revealed that the LOP ECL quenching efficiency would be higher at low coreactant concentration. It was found that, when 2 mM TPrA was used, a KSV value as high as 2.90 · 105 M1 could be obtained for UA. However, the influence of other biological species on the emission intensity became severe too in this case. To achieve higher selectivity as well as reasonably good sensitivity, TPrA concentration of 10 mM was found appropriate for the detection of UA in biological samples.
Fig. 1. (a) Quenching effects of UA and AA on the LOP ECL of the RuðbpyÞ2þ 3 =TPrA system. (b) Stern–Volmer plots. Electrode, FSO-modified gold electrode. Electrolyte solution, 0.15 M PBS (pH 7.5) containing 1 lM RuðbpyÞ2þ 3 þ 10 mM TPrA. Potential scan rate, 100 mV/s.
unchanged upon the addition of a small amount of AA (up to 100 lM). As compared with the conventional ECL routes, the unique features of the LOP ECL include (a) no oxidation of RuðbpyÞ2þ 3 is required, (b) the formation of the excited state involves the intermediacy of the TPrA cation radical (TPrA+) [8]. The little influence of AA on the LOP ECL is unusual. Although the complicated emission processes and their interactions with AA and its oxidation products make it difficult to interpret clearly the effect of AA at this stage, the result indicates that the LOP ECL quenching
Fig. 2. The LOP ECL profiles of the RuðbpyÞ2þ 3 (1 lM)/TPrA (10 mM) system in 0.15 M PBS (pH 7.5). Solid line: no analyte; dashed line: in the presence of 200-fold diluted urine sample; dashed–dotted line: after 15 lM UA spiking; dotted line: after subsequent 25 lM AA spiking. Potential scan rate, 100 mV/s.
Table 2 The quenching effects of possible interfering biological compounds on the LOP ECL of the RuðbpyÞ2þ 3 =TPrA system Compound 1
KSV (M ) KSV(UA)/KSV(other)
Cysteine
Oxalate 3
2.90 · 10 31
Purine 2
2.20 · 10 414
3
1.28 · 10 71
Glucose
Urea
AA
0 1
0 1
0 1
Z. Chen, Y. Zu / Journal of Electroanalytical Chemistry 612 (2008) 151–155
155
Table 3 Assay results of a human urine sample based on the LOP ECL quenching of the RuðbpyÞ2þ 3 /TPrA system
Republic of China (Projects HKU 7061/04P and HKU 7059/05P) is gratefully acknowledged.
UA in diluted samplea (lM)
UA added (lM)
UA measured (lM)
Recovery (%)
AA added (lM)
AA influenceb (%)
References
14.60
15
29.10
96.70
25
3.10
a
Sample dilution factor, 200. AA influence was obtained by comparing the LOP ECL intensity before and after AA spiking. b
3.3. Determination of UA in a human urine sample To demonstrate the validity of the proposed method for the determination of UA in biological samples, the analysis of a human urine sample has been carried out. Fig. 2 shows the LOP ECL profiles obtained in the presence of 200-fold diluted urine sample before and after UA and AA spiking. The assay results are shown in Table 3. The recovery for UA determination was reasonably good, and the influence of added AA on the analysis was insignificant. Note the urine sample was diluted by 200-fold, 25 lM AA spiking represented 5 mM AA in the original urine sample. 4. Conclusions The quenching effects of UA and AA on different ECL routes of the RuðbpyÞ2þ 3 =TPrA system have been examined. UA exhibited much more remarkable influence on the ECL than AA, especially when the emission was generated predominantly via the LOP ECL route. Other possible interfering biological species were found to exhibit negligible influence on the LOP ECL as comparing with UA. The LOP ECL quenching effect may lead to the development of a new method for the detection of UA in biological samples. Acknowledgement Financial support from the Research Grants Council of the Hong Kong Special Administrative Region, People’s
[1] A.J. Bard (Ed.), Electrogenerated Chemiluminescence, Marcel Dekker, New York, 2004. [2] M.M. Richter, Chem. Rev. 104 (2004) 3003. [3] J.K. Leland, M.J. Powell, J. Electrochem. Soc. 137 (1990) 3127. [4] Y. Zu, A.J. Bard, Anal. Chem. 72 (2000) 3223. [5] F. Kanoufi, Y. Zu, A.J. Bard, J. Phys. Chem. B 105 (2001) 210. [6] Y. Zu, A.J. Bard, Anal. Chem. 73 (2001) 3960. [7] K. Honda, M. Yoshimura, T.N. Rao, A. Fujishima, J. Phys. Chem. B 107 (2003) 1653. [8] W. Miao, J.-P. Choi, A.J. Bard, J. Am. Chem. Soc. 124 (2002) 14478. [9] F. Li, Y. Zu, Anal. Chem. 76 (2004) 1768. [10] Y. Zu, F. Li, Anal. Chim. Acta 550 (2005) 47. [11] H. Zheng, Y. Zu, J. Phys. Chem. B 109 (2005) 12049. [12] H. Zheng, Y. Zu, J. Phys. Chem. B 109 (2005) 16047. [13] E.M. Gross, P. Pastore, R.M. Wightman, J. Phys. Chem. B 105 (2001) 8732. [14] G. Dryhurst, Electrochemistry of Biological Molecules, Academic Press, New York, 1977. [15] H.A. Harper, V.W. Rodwell, P.A. Mayes, Review of Physiological Chemistry, 16th ed., Lange Medical Publications, California, 1977. [16] J.S.N. Dutt, M.F. Cardosi, C. Livingstone, J. Davis, Electroanalysis 17 (2005) 1233. [17] E. Popa, Y. Kubota, D.A. Tryk, A. Fujishima, Anal. Chem. 72 (2000) 1724. [18] R. Aguilar, M.M. Da´vila, M.P. Elizalde, J. Mattusch, R. Wennrich, Electrochim. Acta 49 (2004) 851. [19] T. Nakaminami, S.I. Ito, S. Kuwabata, H. Yoneyama, Anal. Chem. 71 (1999) 1928. [20] C.R. Raj, T. Ohsaka, J. Electroanal. Chem. 540 (2003) 69. [21] Z. Gao, H. Huang, Chem. Commun. 19 (1998) 2107. [22] Z. Chen, Y. Zu, J. Electroanal. Chem. 603 (2007) 281. [23] G.M. Greenway, P. Ongomo, Analyst 115 (1990) 1297. [24] E.M. Strochkova, Y.I. Tur’yan, I. Kuselman, A. Shenhar, Talanta 44 (1997) 1923. [25] J. McCall, C. Alexander, M.M. Richter, Anal. Chem. 71 (1999) 2523. [26] C. Creutz, N. Sutin, B.S. Brunschwig, J. Am. Chem. Soc. 101 (1979) 1297. [27] R.L. McCreery, in: A.J. Bard (Ed.), Electroanalytical Chemistry, vol. 17, Dekker, New York, 1991. [28] F. Li, H. Cui, X.Q. Lin, Anal. Chim. Acta 471 (2002) 187. [29] F. Li, Y.Q. Pang, X.Q. Lin, H. Cui, Talanta 59 (2003) 627. [30] X.Q. Lin, F. Li, Y.Q. Pang, H. Cui, Anal. Bioanal. Chem. 378 (2004) 2028.
Electroanalytical Chemistry Journal of Electroanalytical Chemistry 612 (2008) 151–155 www.elsevier.com/locate/jelechem
Short Communication
Selective detection of uric acid in the presence of ascorbic acid based on electrochemiluminescence quenching Zuofeng Chen, Yanbing Zu
*
Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong, China Received 14 May 2007; received in revised form 11 September 2007; accepted 18 September 2007 Available online 22 September 2007
Abstract The direct detection of uric acid (UA) using electrochemical method is usually significantly interfered by the presence of other electroactive species such as ascorbic acid (AA) in biological samples. Here, we describe a new method of UA determination based on the 0 quenching of the electrochemiluminescence (ECL). It was found that the emission of the RuðbpyÞ2þ 3 (bpy = 2,2 -bipyridine)/tri-n-propylamine (TPrA) system could be influenced much more significantly by UA than by AA, particularly when the ECL was produced via the low-oxidation-potential (LOP) route. In the presence of 10 mM TPrA, the Stern–Volmer constant (KSV) of the LOP ECL quenching could be as high as 9.1 · 104 M1 for UA, while the influence of AA on the LOP ECL signal was not obvious. Other possible interfering biological species, such as cysteine, oxalate, purine, glucose, and urea, exhibited much less influence on the LOP ECL as compared with UA. The unique quenching effects led to sensitive and selective detection of UA in human urine samples. 2007 Elsevier B.V. All rights reserved. Keywords: Uric acid; Ascorbic acid; Electrochemiluminescence; Tris(2,2 0 -bipyridine)ruthenium(II); Low-oxidation-potential emission
ðE0 0:9 V vs: SCEÞ
1. Introduction
TPrA e ! TPrAþ
The last few decades have seen intensive electrochemiluminescence (ECL) studies on fundamentals and applica2þ tions, and the ECL of RuðbpyÞ3 (bpy = 2,2 0 -bipyridine) with tri-n-propylamine (TPrA) as the coreactant has now been widely used in clinical analysis (immunoassay and DNA probes) [1,2]. Meanwhile, two emission routes have been proposed on the basis of the coreactant oxidation pathway [3–7]. When RuðbpyÞ2þ 3 concentration is relatively high, TPrA oxidation mainly proceeds via the catalytic route:
The product of the coreactant oxidation, TPrA cation radical, will undergo a rapid decomposition, which leads to the formation of a highly reducing ECL intermediate, TPrA free radical; and then the excited state is produced 3þ by the energetic electron transfer between RuðbpyÞ3 and TPrA :
3þ ðE0 1:02 V vs: SCEÞ RuðbpyÞ2þ 3 e ! RuðbpyÞ3
ð1aÞ
2þ þ RuðbpyÞ3þ 3 þ TPrA ! RuðbpyÞ3 þ TPrA
ð1bÞ 2þ
But, in the presence of dilute RuðbpyÞ3 and concentrated TPrA, TPrA oxidation alters to mainly proceed following the direct oxidation route: *
Corresponding author. Tel.: +852 28598023; fax: +852 28571586. E-mail address: [email protected] (Y. Zu).
0022-0728/$ - see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jelechem.2007.09.018
TPrAþ ! TPrA þ Hþ 3þ RuðbpyÞ3
þ TPrA !
ð2Þ
ð3Þ 2þ RuðbpyÞ3
þ P1
ð4Þ
where TPrA+ = Pr3N+, TPrA = Pr2NCHCH2CH3, and P1 = Pr2NC+HCH2CH3. Recently, a new ECL route, which involves the intermediacy of the TPrA cation radical, was proposed [8]: þ RuðbpyÞ2þ 3 þ TPrA ! RuðbpyÞ3 þ P1
ð5Þ
RuðbpyÞþ 3
ð6Þ
þ TPrA
þ
!
RuðbpyÞ2þ 3
þ TPrA
152
Z. Chen, Y. Zu / Journal of Electroanalytical Chemistry 612 (2008) 151–155
Since no oxidation of RuðbpyÞ2þ 3 is required in this ECL route, the emission can be generated at a potential below 1.0 V versus SCE. Evident low-oxidation-potential (LOP) ECL signals were observed at a freshly polished glassy carbon (GC) electrode and at a nonionic fluorosurfactantmodified gold electrode [4,8–12]. In above ECL routes, the production of the highly reducing intermediates, such as TPrA and RuðbpyÞþ 3 , is crucial for the generation of the excited state. In the presence of reducible quencher species (Q), the intermediate radicals might be consumed by their side reactions with the quencher molecules, resulting in the drop of ECL intensity [11–13]: TPrA þ Q ! P1 þ P2 RuðbpyÞþ 3
þQ!
RuðbpyÞ2þ 3
ð7Þ þ P2
ð8Þ
where P2 represents the reduction product of the quencher molecule. The significance of the intermediate-radical quenching would depend on the competition between the ECL pathways and the side reactions. Our recent study shows that the LOP ECL route is less efficient than the conventional ones and, therefore, the LOP emission may be much more efficiently suppressed by the quenchers [11,12]. Uric acid (UA) is the primary product of purine nucleotide metabolism in human system and has often been regarded as a key biomarker in evaluation of physiological wellbeing [14]. Abnormal levels of UA are related to a number of diseases including gout, hyperuricemia, LeschNyan, cardiovascular disease, kidney diseases, etc [15]. The detection of UA in physiological samples by using electrochemical techniques has been a subject of intensive study [16–20]. The major problem comes from the interference of ascorbic acid (AA) because of their overlapping oxidation peaks [21]. A variety of modified electrodes and electrode pretreatment methods have been proposed with the aim of resolving the anodic processes and sensing UA in the presence of an excess of AA [18,20,22–24]. In this report, we describe a new method of UA determination based on the quenching of the ECL of the RuðbpyÞ2þ 3 =TPrA system. Recently, it has been reported that some molecules, such as oxygen and phenolic compounds, could effectively quench the ECL of the 2þ RuðbpyÞ3 =TPrA system [11,12,25], and a new mechanism involving the interception of the ECL intermediate radicals by quencher molecules has been proposed [11,12]. The intermediate-radical quenching route may lead to much more significant decrease of ECL intensity as compared with the excited-state quenching route. Thus, we examined the influences of UA and AA on the ECL signals generated via different routes, and revealed that the LOP ECL of the 2þ RuðbpyÞ3 =TPrA system could be quenched efficiently by UA, while it was much less sensitive to AA, suggesting the possibility of selective detection of UA in the presence of AA. The influences of some other possible interfering biological species on the LOP ECL emission have also been examined, and the results showed that the detection of UA
would not be affected significantly by these species. This allowed for the development of a sensitive and selective method based on ECL quenching for the analysis of UA in biological samples. 2. Experimental 2.1. Chemicals Tris(2,2 0 -bipyridyl)ruthenium(II) dichloride hexahydrate (Ru(bpy)3Cl2 Æ 6H2O, min 98%), tri-n-propylamine (TPrA, 98%), and Zonyl FSO-100 (F(CF2CF2)17CH2CH2O(CH2CH2O)015H) were purchased from Sigma–Aldrich. Other chemicals were analytical reagent graded and used as received. All solutions were prepared with deionized water (Milli Q, Millipore). The pH of the phosphate buffer solution (PBS, 0.15 M) containing TPrA was adjusted with concentrated NaOH or phosphoric acid. 2.2. Apparatus Photoluminescence (PL) experiments were performed on a Spex Fluorolog 111 spectrofluorometer. Cyclic voltammetry (CV) was performed with the model 600A electrochemical workstation (CH Instruments, Austin, TX). The three-electrode system consisted of a working electrode, a coiled Pt wire counter electrode, and a saturated calomel reference electrode (SCE) separated from the working cell by a salt bridge. The ECL signal was measured with a photomultiplier tube (PMT, Hamamatsu R928) installed under the electrochemical cell. A voltage of 800 V (or 600 V when 1 mM RuðbpyÞ2þ was used) was supplied to the 3 PMT with the Sciencetech PMH-02 instrument (Sciencetech Inc., Hamilton, Ontario, Canada). 2.3. Procedures CV was performed with a scan rate of 100 mV/s. GC, platinum, and gold electrodes were polished with 0.05-lm alumina slurry to obtain a mirror surface and then were sonicated and thoroughly rinsed with Milli-Q water. Before each experiment, the gold and platinum working electrodes were subjected to repeated scanning in the potential ranges from 0.5 to 1.4 or 0.65 to 1.2 V, respectively, in 0.15 M PBS (pH 7.5) until reproducible voltammograms were obtained. The modification of the gold electrode with FSO-100 (FSO-Au) was conducted by immersing the electrode in 5 wt.% FSO aqueous solution for 2 min, followed by thoroughly rinsing with distilled water. Solution pH was adjusted to 7.5 to obtain intense LOP ECL signal [10]. In order to eliminate the influence of oxygen [11], solutions were deaerated by bubbling high purity (99.995%) of N2, and a constant flow of N2 was maintained over the solution during the measurements. All electrochemical measurements were referenced with respect to a SCE. All experiments were performed at room temperature 20 ± 1 C. Reported values for ECL intensity are based on the aver-
Z. Chen, Y. Zu / Journal of Electroanalytical Chemistry 612 (2008) 151–155
age of at least three scans with a relative standard deviation (RSD) of ±5%. Freshly collected human urine sample from adult people was diluted 200 times with 0.15 M PBS (pH 7.5) containing 2þ 1 lM RuðbpyÞ3 and 10 mM TPrA, without either purification or filtration. The UA concentration in the urine sample was determined by standard addition method using the LOP ECL signal at the FSO-Au electrode. 3. Results and discussion 3.1. Quenching effects of UA and AA on photoluminescence and ECL 2þ
The effects of UA and AA on PL of RuðbpyÞ3 were examined firstly in 0.15 M PBS (pH 7.5). The excited-state quenching behavior can be described by the Stern–Volmer equation: I 0 =I ¼ 1 þ K SV ½Q 0
ð9Þ
where I and I represent the emission intensities in the absence and presence of the quencher, respectively. The Stern–Volmer constant KSV defines the efficiency of quenching. No quenching effect of PL was observed in the presence of UA up to 1 mM, while AA exhibited weak quenching effect with a KSV 1.3 · 10 M1, close to the value reported previously (1.2 · 10 M1) [26]. As demonstrated in previous reports [11,12], the pre2þ dominant ECL route of the RuðbpyÞ3 =TPrA system can be altered by changing the solution conditions and the electrode materials; therefore, the effects of quenchers (i.e. UA and AA) on each ECL route can be examined separately. For the catalytic ECL emission, a platinum electrode was 2þ used to oxidize 1 mM RuðbpyÞ3 in the presence of 1 mM TPrA. TPrA was oxidized mainly by the electrogen3þ erated RuðbpyÞ3 , while its direct oxidation was inhibited by the growth of the surface oxide layer at the Pt electrode [4]. On the other hand, when the ECL was produced at a 2þ GC electrode in the presence of trace RuðbpyÞ3 (1 lM) and relatively high concentration of TPrA (10– 100 mM), the coreactant direct oxidation route would be predominant [4]. A nonionic fluorosurfactant-modified gold electrode was used to generate the LOP ECL [9,10]. The adsorption of the fluorosurfactant molecules (i.e. Zonyl FSO) at the gold electrode would facilitate TPrA oxidation by rendering the electrode surface more hydrophobic and inhibiting the growth of the surface oxides in the potential region below 1.0 V, which resulted in a strong LOP ECL signal. 2þ Unlike RuðbpyÞ3 PL which occurred under optical excitation, the ECL was preceded by electrochemical reactions. In contrast to the weak PL quenching, more significant quenching effects on ECL have been observed (Table 1). It was found that the ECL quenching behavior might also be described by the Stern–Volmer equation. Obviously, the excited-state quenching mechanism could not solely account for the larger KSV of the ECL quenching.
153
Table 1 The quenching effects of UA and AA on RuðbpyÞ2þ 3 photoluminescence and ECL via different routes Emission route
Photoluminescence ECL-catalytic routea ECL-direct coreactant oxidation routeb ECL-LOP routec
KSV (M1) UA
AA
0 5.85 · 103 1.68 · 104 9.10 · 104
1.30 · 10 4.91 · 102 2.80 · 102 0
ECL experimental conditions: a 1 mM RuðbpyÞ2þ 3 , 1 mM TPrA, Pt elec2þ c trode; b 1 lM RuðbpyÞ2þ 3 , 10 mM TPrA, GC electrode; 1 lM RuðbpyÞ3 , 10 mM TPrA, FSO-modified gold electrode, [AA] < 100 lM.
As suggested by previous studies [11–13], the ECL process may be remarkably influenced by the side reactions between the ECL reducing intermediate radicals (such as TPrA free radical and RuðbpyÞþ 3 ) and the quencher species. In the potential regions where ECL occurred, both UA and AA would be oxidized. Their oxidation products could act as ECL quenchers. It has been reported that the oxidation of UA would result in the formation of reactive dimine species [16]. The reducible species could probably react with ECL intermediate radicals, and act as efficient ECL quenchers. Although the oxidation product of AA has been found to undergo rapid reaction with water that leads to an electro-inactive species [27], it might still be able to intercept ECL intermediate radicals before the hydrolysis. In addition, the ECL intensity could decrease due to the con3þ sumption of RuðbpyÞ3 by its reactions with AA and UA (the LOP ECL would not be influenced by these reactions 3þ since RuðbpyÞ3 was not involved in the LOP emission route). Overall, because of the complicated ECL quenching processes (including the excited-state quenching and the intermediate-radical quenching), both UA and AA exhibited very different influences on the ECL than on the PL. In various ECL routes, the predominant reaction steps were different, and the effects of UA and AA on the emission intensities changed, leading to the diverse values of KSV in Table 1. It can be seen that the LOP ECL signals were most greatly quenched by UA among the three ECL routes. Fig. 1 shows the quenching effects of UA and AA on the LOP ECL and the corresponding Stern– Volmer plots. When 10 mM TPrA was used, a KSV value of 9.10 · 104 M1 was obtained in the presence of UA, which allowed for the sensitive detection of UA. Under our experimental condition using conventional ECL cell, the detection limit of UA was 1 lM. This sensitivity is comparable to those reported in the literatures obtained via flow injection ECL of ruthenium complex [28–30]. It is interesting to note that the influence of AA on the ECL intensity was generally much less significant as compared to that of UA. When the ECL signal was predominantly produced via the catalytic or the direct coreactant oxidation routes, the KSV values of UA were about 12 and 60 times larger than that of AA, respectively. Fig. 1 shows that, when the emission was produced via the LOP ECL route, the intensity of the ECL signal was nearly
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would provide a distinct advantage over the conventional routes in selective detection of UA in the presence of AA. 3.2. Quenching effects of other possible interfering biological compounds on the LOP ECL Besides AA, other common interfering biological species encountered in UA detection include cysteine, oxalate, purine, glucose, urea etc. Their KSV values for the LOP ECL quenching were also examined (Table 2). The influences of these compounds on the LOP ECL were much less significant than that of UA. The value of KSV obtained for UA was at least several tens of times larger. Moreover, considering that these compounds exist in biological fluids (especially in urine samples) at a much lower level than UA [16,17], their interference on UA detection could be negligible. Our previous study [11,12] revealed that the LOP ECL quenching efficiency would be higher at low coreactant concentration. It was found that, when 2 mM TPrA was used, a KSV value as high as 2.90 · 105 M1 could be obtained for UA. However, the influence of other biological species on the emission intensity became severe too in this case. To achieve higher selectivity as well as reasonably good sensitivity, TPrA concentration of 10 mM was found appropriate for the detection of UA in biological samples.
Fig. 1. (a) Quenching effects of UA and AA on the LOP ECL of the RuðbpyÞ2þ 3 =TPrA system. (b) Stern–Volmer plots. Electrode, FSO-modified gold electrode. Electrolyte solution, 0.15 M PBS (pH 7.5) containing 1 lM RuðbpyÞ2þ 3 þ 10 mM TPrA. Potential scan rate, 100 mV/s.
unchanged upon the addition of a small amount of AA (up to 100 lM). As compared with the conventional ECL routes, the unique features of the LOP ECL include (a) no oxidation of RuðbpyÞ2þ 3 is required, (b) the formation of the excited state involves the intermediacy of the TPrA cation radical (TPrA+) [8]. The little influence of AA on the LOP ECL is unusual. Although the complicated emission processes and their interactions with AA and its oxidation products make it difficult to interpret clearly the effect of AA at this stage, the result indicates that the LOP ECL quenching
Fig. 2. The LOP ECL profiles of the RuðbpyÞ2þ 3 (1 lM)/TPrA (10 mM) system in 0.15 M PBS (pH 7.5). Solid line: no analyte; dashed line: in the presence of 200-fold diluted urine sample; dashed–dotted line: after 15 lM UA spiking; dotted line: after subsequent 25 lM AA spiking. Potential scan rate, 100 mV/s.
Table 2 The quenching effects of possible interfering biological compounds on the LOP ECL of the RuðbpyÞ2þ 3 =TPrA system Compound 1
KSV (M ) KSV(UA)/KSV(other)
Cysteine
Oxalate 3
2.90 · 10 31
Purine 2
2.20 · 10 414
3
1.28 · 10 71
Glucose
Urea
AA
0 1
0 1
0 1
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155
Table 3 Assay results of a human urine sample based on the LOP ECL quenching of the RuðbpyÞ2þ 3 /TPrA system
Republic of China (Projects HKU 7061/04P and HKU 7059/05P) is gratefully acknowledged.
UA in diluted samplea (lM)
UA added (lM)
UA measured (lM)
Recovery (%)
AA added (lM)
AA influenceb (%)
References
14.60
15
29.10
96.70
25
3.10
a
Sample dilution factor, 200. AA influence was obtained by comparing the LOP ECL intensity before and after AA spiking. b
3.3. Determination of UA in a human urine sample To demonstrate the validity of the proposed method for the determination of UA in biological samples, the analysis of a human urine sample has been carried out. Fig. 2 shows the LOP ECL profiles obtained in the presence of 200-fold diluted urine sample before and after UA and AA spiking. The assay results are shown in Table 3. The recovery for UA determination was reasonably good, and the influence of added AA on the analysis was insignificant. Note the urine sample was diluted by 200-fold, 25 lM AA spiking represented 5 mM AA in the original urine sample. 4. Conclusions The quenching effects of UA and AA on different ECL routes of the RuðbpyÞ2þ 3 =TPrA system have been examined. UA exhibited much more remarkable influence on the ECL than AA, especially when the emission was generated predominantly via the LOP ECL route. Other possible interfering biological species were found to exhibit negligible influence on the LOP ECL as comparing with UA. The LOP ECL quenching effect may lead to the development of a new method for the detection of UA in biological samples. Acknowledgement Financial support from the Research Grants Council of the Hong Kong Special Administrative Region, People’s
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