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Vitamin C derivatives as new coreactants for tris(2,2′-bipyridine)ruthenium(II) electrochemiluminescence
Analytica Chimica Acta 701 (2011) 169–173
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Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Vitamin C derivatives as new coreactants for tris(2,2 -bipyridine)ruthenium(II) electrochemiluminescence Yali Yuan a,b , Haijuan Li a,b , Shuang Han a,b , Lianzhe Hu a,b , Saima Parveen a,b,c , Guobao Xu a,∗ a b c
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China Graduate University of the Chinese Academy of Sciences, Beijing 100864, China Department of Chemistry, Baghdad-ul-Jadeed Campus, The Islamia University of Bahawalpur, Bahawalpur, Pakistan
a r t i c l e
i n f o
Article history: Received 19 May 2011 Received in revised form 22 June 2011 Accepted 27 June 2011 Available online 5 July 2011 Keywords: Tris(2,2 -bipyridine)ruthenium Electrochemiluminescence Vitamin C derivatives Ascorbyl palmitate Ascorbyl phosphate
a b s t r a c t Vitamin C derivatives (VCDs) have been widely used as the alternative and stable sources of vitamin C, and accordingly exhibit many new applications, such as anti-tumor and central nervous system drug delivery. In this study, their Ru(bpy)3 2+ electrochemiluminescence (ECL) properties have been investigated for the first time using well-known ascorbyl phosphate and ascorbyl palmitate as representative VCDs. Ascorbyl phosphate and ascorbyl palmitate are VCDs with different substituted positions. Both of them increase Ru(bpy)3 2+ ECL, indicating that other VCDs may also enhance Ru(bpy)3 2+ ECL signal. The calibration plot for ascorbyl phosphate is linear from 3 × 10−6 to 1.0 × 10−3 M with a detection limit of 1.4 × 10−6 M at a signal-to-noise ratio of 3. The relative standard deviation is 3.6% for six replicate measurements of 0.01 mM ascorbyl 2-phosphate solution. The proposed method is about one order of magnitude more sensitive than electrochemical and UV–vis methods for the determination of ascorbyl phosphate, and is used successfully for the determination of ascorbyl phosphate in whitening and moisturising body wash. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Vitamin C is an essential nutrient of life. It is required in many reactions involved in body processes, such as collagen synthesis, carnitine synthesis, tyrosine synthesis catabolism, and neurotransmitter synthesis. It acts as an antioxidant and radical scavenger. Moreover, it plays a crucial role in the protection of cellular components against oxidative damage by free radicals and oxidants that are involved in the development and exacerbation of many diseases such as cancer, heart disease, brain malfunction, aging, rheumatism, inflammation, stroke emphysema, and AIDS. It is one of the relatively few topical agents whose effectiveness against wrinkles and fine lines is backed by a fair amount of reliable scientific evidence, thus is a very popular skin-whitening agent used in cosmetics. Therefore, it is one of the most important biomolecules, and is perhaps the most popular vitamin [1–4]. However, the practical use of vitamin C, particularly in skin care and antioxidant food additives, presents some difficulties due to its labile oxidative properties. Being able to be transformed to vitamin C by enzymes in body, many vitamin C derivatives (VCDs) with better stability have been designed through chemical modification of hydroxyl groups of ascorbic acid, such as ascorbyl 2-phosphate,
∗ Corresponding author. Tel.: +86 431 85262747; fax: +86 431 85262747. E-mail address: [email protected] (G. Xu). 0003-2670/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2011.06.051
ascorbyl 6-palmitate, ascorbyl tetra-isopalmitoyl, tetrahexyldecyl ascorbate, ascorbic acid 2-sulfate, ascorbyl stearate, ascorbyl isostearate, ascorbyl oleate, ascorbyl glucoside, aminopropyl ascorbyl phosphate, ascorbyl laurate, and disodium ascorbyl phytostanol phosphate. In addition to their use as a source of vitamin C, VCDs have also been found to exhibit many new applications, such as anti-tumor and central nervous system drug delivery [5–7]. Electrogenerated chemiluminescence (ECL) is light-emitting phenomena involving electrochemical reactions [8–15]. There are many ECL systems, such as tris(2,2 -bipyridyl)ruthenium (II), peroxyoxalate, luminol, acridinium esters, lucigenin, graphene, carbon nanodots, nanocrystals, and metal nanoclusters [16–24]. Tris(2,2 bipyridyl)ruthenium(II) ECL is one of the most widely used ECL systems. On the one hand, it has been widely used in immunoassays [25,26], DNA probe assays [27], and aptasensors [28]. On the other hand, it has been used for the determination of numerous coreactants, such as oxalate, alkylamines, NADH, amino acids, dopamine, ascorbic acid, and so on [29–36]. Previous studies show that Ru(bpy)3 2+ ECL properties depend significantly on the structure of coreactants [9,14,18,24]. For example, no ECL can be observed when alkylamines are chemically transformed to amides or NADH is enzymatically transformed to NAD. Vitamin C has been investigated with different ECL luminophores, such as Ru(bpy)3 2+ [37], lucigenin [38], and luminol [39,40]. For example, Chen et al. [41] have developed an interesting luminol ECL method for the detection of vitamin
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Fig. 1. Molecular structure of ascorbic acid, ascorbyl 2-phosphate and ascorbyl 6-palmitate.
C using an electrically heated ionic-liquid/multi-walled carbon nanotube composite electrode. Chen et al. [33,42] have demonstrated that vitamin C is an effective coreactant for Ru(bpy)3 2+ ECL for the first time. Zorzi et al. [34] have found that vitamin C and dehydroascorbic acid show the same Ru(bpy)3 2+ ECL behavior and can be detected with same calibration graph. Takahashi and Jin [43] have reported that ECL of the Ru(bpy)3 2+ /vitamin C system can be quenched by excess vitamin C or upon ultrasonic irradiation. However, the effect of derivation of ascorbic acid on ECL is unknown. In this study, Ru(bpy)3 2+ ECL of VCDs has been reported for the first time. Ascorbyl 2-phosphate and ascorbyl 6-palmitate were selected as representatives because of three reasons. Firstly, both compounds have found their way into the broad skin care market. Secondly, their substituted positions are different as shown in Fig. 1. The substituted positions for ascorbyl 2-phosphate and ascorbyl 6palmitate are C2–OH position and C6–OH position, respectively. It allows the investigation of the effect of substituted positions on ECL. Finally, pure ascorbyl 2-phosphate and ascorbyl 6-palmitate are commercially available. The study shows that ascorbyl 2-phosphate and ascorbyl 6-palmitate can increase Ru(bpy)3 2+ ECL. Moreover, the ECL method was successfully applied to the determination of ascorbyl 2-phosphate in commercial bleaching cosmetic products. 2. Experimental
mode. It is sensitive to photons with wavelengths ranging from 200 to 800 nm. Unless noted otherwise, the PMT was biased at 700 V. The ECL cell was located in a light-tight box of the luminescent analyzer. 2.3. Procedures Prior to each measurement, the glassy carbon electrode was first polished with alumina powder, and then cleaned by ultrasonication in deionized water. Ascorbyl 2-phosphate and ascorbyl 6-palmitate solutions were freshly prepared in degassed deionized water before use. These newly prepared VCDs solutions were later mixed with 0.25 mM Ru(bpy)3 2+ at different ratio for electrochemical characterization and calibration. 3. Results and discussion 3.1. Electrochemistry of ascorbyl 2-phosphate and ascorbyl 6-palmitate Fig. 2 shows cyclic voltammograms of ascorbic acid and ascorbyl 2-phosphate at glassy carbon electrodes. Both ascorbic acid and ascorbyl 2-phosphate have irreversible anodic waves. The anodic peak potentials for ascorbic acid and ascorbyl 2-phosphate are about +0.18 V and +0.7 V, respectively. By comparison, the derivation positively shifts the anodic peak potential by about 0.5 V,
2.1. Materials Tris(2,2 -bipyridyl)ruthenium(II) chloride was purchased from Aldrich. 2-phospho-l-ascorbic acid trisodium salt was obtained from Fluka. l-Ascorbic acid 6-palmitate (99%) was purchased from Alfa Aesar. All other reagents were of reagent grade. Solutions were prepared using water, which was deionized and further purified in a Millipore system. Whitening and moisturising body wash was bought from Watsons (Hongkong, China). 2.2. Instrumentation Electrochemical measurements were performed in a conventional three-electrode cell (volume 0.6 mL) with a CHI 800B potentiostat (CH Instruments, Shanghai, China). The threeelectrode system included a glassy carbon electrode (diameter 3 mm), an Ag/AgCl (saturated KCl) reference electrode, and a Pt wire auxiliary electrode. The Teflon gasket (2 mm in thickness) was sandwiched between the electrode block and a Plexiglas window. ECL intensities were measured through the bottom of the electrochemical cell with a BPCL Ultra-Weak luminescence analyzer, which was purchased from Institute of Biophysics, Chinese Academy of Sciences. The photomultiplier tube (PMT) used in the BPCL Ultra-Weak luminescence analyzer was operated in current
Fig. 2. Cyclic voltammograms at glassy carbon electrodes in 0.05 M pH 8.0 phosphate buffer solutions containing (a) 0.2 mM ascorbic acid; (b) 0.2 mM ascorbyl 2-phosphate; (c) 0.25 mM Ru(bpy)3 2+ and 0.2 mM ascorbyl 2-phosphate; (d) 0.25 mM Ru(bpy)3 2+ , scan rate: 0.1 V s−1 .
Y. Yuan et al. / Analytica Chimica Acta 701 (2011) 169–173
171
Fig. 3. Cyclic voltammograms at glassy carbon electrodes in 0.05 M pH 8.0 phosphate buffer solutions containing ethanol at a volume ratio of 1:6 with the addition of (a) 0.05 M pH 8.0 PBS; (b) 0.2 mM ascorbyl 6-palmitate; (c) 0.25 mM Ru(bpy)3 2+ , (d) 0.25 mM Ru(bpy)3 2+ and ascorbyl 6-palmitate, scan rate: 0.1 V s−1 .
indicating that ascorbyl 2-phosphate has better stability than ascorbic acid. The better stability is ascribed to the esterification of an unstable hydroxyl group of ascorbic acid with phosphate. Fig. 2c and d shows cyclic voltammograms of Ru(bpy)3 2+ in the absence and presence of ascorbyl 2-phosphate, respectively. A typical pair of redox waves is observed for Ru(bpy)3 2+ solution, which was attributed to the one-electron redox reaction of Ru(bpy)3 2+ . A comparison of Fig. 2c and d demonstrates that Ru(bpy)3 2+ catalyzes the oxidation of ascorbyl 2-phosphate, since the anodic current of Ru(bpy)3 2+ increases and the cathodic current of Ru(bpy)3 2+ decreases in the presence of ascorbyl 2-phosphate, and the oxidation potential of Ru(bpy)3 2+ is much larger than the oxidation potential of ascorbyl 2-phosphate. To investigate the effect of substituted position, the electrochemistry of another VCD, ascorbyl 6-palmitate, was also studied. Since ascorbyl 6-palmitate is insoluble in water, all electrochemical experiments of ascorbyl 6-palmitate were carried out in 0.05 M phosphate buffer solution containing ethanol at a volume ratio of 1:6. As shown in Fig. 3, an irreversible broad anodic wave of ascorbyl 6-palmitate was observed at around +1.05 V. The anodic peak potential of ascorbyl 6-palmitate is much more positive than that of ascorbic acid, indicating that the substitution of ascorbic acid at C6–OH position also improves the stability. 3.2. ECL of ascorbyl 2-phosphate and ascorbyl 6-palmitate Fig. 4 described the ECL-potential profile of ascorbyl 2phosphate and ascorbyl 6-palmitate. The onset of ECL occurred near 1.0 V and the ECL intensity reached a maximum at 1.15 V, consistent with the oxidation potential of Ru(bpy)3 2+ . It indicates that the electrochemical oxidation of Ru(bpy)3 2+ is necessary for ECL reaction. The ECL mechanism is similar to that of oxidative-reductive ECL systems [33,34]. Ru(bpy)3 2+ is electrochemically oxidized to generate Ru(bpy)3 3+ (Eq. (1)). VCD is oxidized directly on the electrode and by electrogenerated Ru(bpy)3 3+ to generate a strong reductant VCD• (Eqs. (2) and (3)). Then electrogenerated Ru(bpy)3 3+ reacts with VCD• to generate the excited state Ru(bpy)3 2+* (Eq. (4)) and emit light subsequently (Eq. (5)). By comparison, the ECL intensities increase about 16 times and 6 times upon the addition of ascorbyl 2-phosphate and ascorbyl 6-palmitate, respectively. These results suggest that C2–OH substituted VCDs and C6–OH substituted VCDs
Fig. 4. (A) ECL-potential profile at glassy carbon electrodes in 0.05 M pH 11.0 phosphate buffer solutions containing (a) 0.25 mM Ru(bpy)3 2+ and 0.2 mM ascorbyl 2-phosphate; (b) 0.25 mM Ru(bpy)3 2+ . (B) ECL-potential profile at glassy carbon electrodes in 0.05 M pH 11.0 phosphate buffer solutions containing ethanol at a volume ratio of 1:6 with the presence of (c) 0.25 mM Ru(bpy)3 2+ and 0.2 mM ascorbyl 6-palmitate; (d) 0.25 mM Ru(bpy)3 2+ .
may enhance Ru(bpy)3 2+ ECL signal. Since ascorbyl 2-phosphate has good solubility in aqueous solutions, it was used as a representative VCD to demonstrate the analytical applications of Ru(bpy)3 2+ ECL for the determination of VCDs. Ru(bpy)3 2+ − e → Ru(bpy)3 3+ VCD − e →
(1)
VCD•
(2)
Ru(bpy)3
3+
+ VCD → Ru(bpy)3
Ru(bpy)3
3+
+ VCD•
Ru(bpy)3
2+∗
2+∗
→ Ru(bpy)3
→ Ru(bpy)3
2+
+ VCD•
2+∗
+ h
(3) (4) (5)
3.3. ECL determination of ascorbyl 2-phosphate Fig. 5 shows the dependence of ECL intensity on pH in Ru(bpy)3 2+ solutions in the absence or presence of ascorbyl 2phosphate. A comparison of Fig. 5a and b indicates that the ECL reaction between ascorbyl 2-phosphate and Ru(bpy)3 2+ occurs at pH higher than 10. The ECL intensities resulting from the ECL reaction between ascorbyl 2-phosphate and Ru(bpy)3 2+ increase sharply with increasing pH from 10.0 to 11.0 and then decrease slightly at pH higher than 11.0. Therefore, a pH of 11.0 was used for the subsequent measurements. The strong pH dependence of ECL
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Table 1 Assay results for the ECL analysis of ascorbyl 2-phosphate in whitening and moisturising body wash.
Whitening and moisturising body wash samplea Whitening and moisturising body wash sample + 10 M ascorbyl 2-phosphate Whitening and moisturising body wash sample + 20 M ascorbyl 2-phosphate a
Number of detection
Coefficient of variation (%)
Detection results (M)
Recovery (%)
Real sample content (wt.%)
6 6 6
4.83 3.91 4.16
3.72 14.59 24.72
– 106 105
0.76 – –
Dilution factor: 1/15,000.
may mainly result from the pH dependence of deprotonation of ascorbyl 2-phosphate and its radical cation, and the reduced availability of Ru(bpy)3 3+ due to the competitive reaction with the OH− ion at higher pHs [44–52]. Fig. 6 shows the dependence of ECL intensity on potential. The ECL intensities increase sharply from 1.0 to 1.15 V, and then decrease at potentials above 1.15 V. The electrochemical oxidation of OH− at these high potentials could also be responsible for the overall ECL intensity decrease [31]. To get good reproducibility and sensitivity, subsequent experiments were conducted at a potential of 1.15 V for ECL excitation. The log–log calibration plot for ascorbyl 2-phosphate is linear from 3 × 10−6 to 1.0 × 10−3 mol L−1 (slope = 0.779; inter-
cept = 7.101; n = 7; correlation coefficient = 0.9959). The detection limit is 1.4 × 10−6 M at a signal-to-noise ratio of 3. The relative standard deviation is 3.6% for six replicate measurements of 0.01 mM ascorbyl 2-phosphate. The total analysis time was less than 2 min. The proposed method is about one order of magnitude more sensitive than electrochemical method and UV–vis methods [2–7]. 3.4. Analysis of whitening and moisturising body wash sample Appropriate amount of whitening and moisturising body wash was first diluted with water at the ratio of 1:300, and then hexane was added to extract the organic ingredients in the mixture. After extraction, the obtained aqueous solution was again diluted at the ratio of 1:50 with 0.05 M pH 11.0 phosphate buffer solution to prepare the sample solution. Under optimum conditions, the ECL determination of ascorbyl 2-phosphate in cosmetic product was successfully achieved, as shown in Table 1. The calculated content of ascorbyl 2-phosphate in this cosmetic product was 0.76%, which was comparable with its content. The standard addition method was used for testing recovery. The recovery was 106% and 105% for 10 M and 20 M ascorbyl 2-phosphate, respectively. 4. Conclusions
Fig. 5. Effect of pH on ECL of 0.25 mM Ru(bpy)3 2+ solutions in the (a) absence of and (b) presence of 0.2 mM ascorbyl 2-phosphate. Applied potential, 1.15 V. Each point represents the average of three measurements.
VCDs have been demonstrated as new coreactants for Ru(bpy)3 2+ ECL for the first time using ascorbyl phosphate and ascorbyl palmitate as representative VCDs. Both ascorbyl 2-phosphate and ascorbyl 6-palmitate increase Ru(bpy)3 2+ ECL significantly. These results suggest that C2–OH substituted VCDs and C6–OH substituted VCDs may also enhance Ru(bpy)3 2+ ECL signal. Ru(bpy)3 2+ ECL method is one order of magnitude more sensitive than electrochemical and UV–vis methods for the determination of ascorbyl 2-phosphate. Moreover, the ECL method was successfully applied to the determination of ascorbyl 2-phosphate in commercial bleaching cosmetic products with excellent recovery. This study is helpful for understanding ECL properties of VCDs, and provides a new way to detect VCDs. It has been demonstrated in Barnett’s excellent reviews on Ru(bpy)3 2+ chemiluminescence that most coreactants of Ru(bpy)3 2+ ECL can react with Ru(bpy)3 3+ to generate chemiluminescence [11,16]. Therefore, our study indicates that VCDs may also react with Ru(bpy)3 3+ to generate chemiluminescence and may also be detected through other chemiluminescent systems that have been used to detect vitamin C [53,54]. Acknowledgements
Fig. 6. Effect of potential on ECL at glassy carbon electrodes in 0.05 M pH 11.0 phosphate buffer solutions containing 0.25 mM Ru(bpy)3 2+ and 0.2 mM ascorbyl 2-phosphate. Each point represents the average of three measurements.
This project was kindly supported by the National Natural Science Foundation of China (No. 20875086), the Ministry of Science and Technology of the People’s Republic of China (No. 2006BAE03B08), Chinese Academy of Sciences (CAS), the Academy of Sciences for the Developing World (TWAS), the Department of Sciences & Technology of Jilin Province (No. 20070108 and 20082104), and Changchun Institute of Applied Chemistry.
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References [1] A. Chisvert, A. Balaguer, A. Salvador, Analysis of Cosmetics Products, Elsevier Science B.V., Amsterdam, 2007. [2] A. Balaguer, A. Chisvert, A. Salvador, J. Sep. Sci. 31 (2008) 229–236. [3] A. Varvaresou, E. Tsirivas, K. Iakovou, E. Gikas, Z. Papathomas, A. Vonaparti, I. Panderi, Anal. Chim. Acta 573 (2006) 284–290. [4] N.M. Pauli, W. Schuep, J. Chromatogr. B 715 (1998) 369–377. [5] S.C. Huang, C.C. Lin, M.C. Huang, K.C. Wen, J. Food Drug Anal. 12 (2004) 13–18. [6] M.L. Chang, C.M. Chang, J. Pharm. Biomed. Anal. 33 (2003) 617–626. [7] M.L. Chang, C.M. Chang, J. Food Drug Anal. 13 (2005) 205–211. [8] A.J. Bard, Electrogenerated Chemiluminescence, Marcel Dekker, New York, 2004. [9] A.W. Knight, G.M. Greenway, Analyst 121 (1996) R101–R106. [10] W.Y. Lee, Mikrochim. Acta 127 (1997) 19–39. [11] R.D. Gerardi, N.W. Barnett, P. Jones, Anal. Chim. Acta 388 (1999) 1–10. [12] K.A. Fahnrich, M. Pravda, G.G. Guilbault, Talanta 54 (2001) 531–559. [13] W.J. Miao, A.J. Bard, Anal. Chem. 76 (2004) 5379–5386. [14] M.M. Richter, Chem. Rev. 104 (2004) 3003–3036. [15] X.B. Yin, S.J. Dong, E. Wang, Trends Anal. Chem. 23 (2004) 432–441. [16] B.A. Gorman, P.S. Francis, N.W. Barnett, Analyst 131 (2006) 616–639. [17] C.A. Marquette, L.J. Blum, Anal. Bioanal. Chem. 390 (2008) 155–168. [18] W.J. Miao, Chem. Rev. 108 (2008) 2506–2553. [19] J.F. Rusling, E.G. Hvastkovs, D.O. Hull, J.B. Schenkman, Chem. Commun. 2 (2008) 141–154. [20] H. Wei, E.K. Wang, Trends Anal. Chem. 27 (2008) 447–459. [21] P. Bertoncello, R.J. Forster, Biosens. Bioelectron. 24 (2009) 3191–3200. [22] R.J. Forster, P. Bertoncello, T.E. Keyes, Annu. Rev. Anal. Chem. 2 (2009) 359–385. [23] H.J. Li, S. Han, L.Z. Hu, G.B. Xu, Chin. J. Anal. Chem. 37 (2009) 1557–1565. [24] L.Z. Hu, G.B. Xu, Chem. Soc. Rev. 39 (2010) 3275–3304. [25] F. Deiss, C.N. LaFratta, M. Symer, T.M. Blicharz, N. Sojic, D.R. Walt, J. Am. Chem. Soc. 131 (2009) 6088–6089. [26] R. Kurita, K. Arai, K. Nakamoto, D. Kato, O. Niwa, Anal. Chem. 82 (2010) 1692–1697. [27] L. Dennany, R.J. Forster, J.F. Rusling, J. Am. Chem. Soc. 125 (2003) 5213–5218.
173
[28] X.Q. Liu, L.H. Shi, W.X. Niu, H.J. Li, G.B. Xu, Angew. Chem. Int. Ed. 46 (2007) 421–424. [29] Z.J. Lin, X.M. Chen, T.T. Jia, X.D. Wang, Z.X. Xie, M. Oyama, X. Chen, Anal. Chem. 81 (2009) 830–833. [30] I.S. Shin, S.W. Bae, H. Kim, J.I. Hong, Anal. Chem. 82 (2010) 8259–8265. [31] Y.W. Chi, Y.Q. Dong, G.N. Chen, Anal. Chem. 79 (2007) 4521–4528. [32] I. Rubinstein, A.J. Bard, J. Am. Chem. Soc. 102 (1980) 6641–6642. [33] X. Chen, M. Sato, Anal. Sci. 11 (1995) 749–754. [34] M. Zorzi, P. Pastore, F. Magno, Anal. Chem. 72 (2000) 4934–4939. [35] H.N. Choi, S.H. Cho, W.Y. Lee, Anal. Chem. 75 (2003) 4250–4256. [36] K.F. Chow, F. Mavré, J.A. Crooks, B.Y. Chang, R.M. Crooks, J. Am. Chem. Soc. 131 (2009) 8364–8365. [37] X.M. Sun, Y. Niu, S. Bi, S.S. Zhang, Electrophoresis 29 (2008) 2918–2924. [38] J.H. Chen, Z.Y. Lin, G.N. Chen, Electrochim. Acta 52 (2007) 4457–4462. [39] H. Dai, X.P. Wu, Y.M. Wang, W.C. Zhou, G.N. Chen, Electrochim. Acta 53 (2008) 5113–5117. [40] J.F. Rong, Y.W. Chi, Y.J. Zhang, G.N. Chen, Electrochem. Commun. 12 (2010) 270–273. [41] Y.T. Chen, X.P. Chen, Z.Y. Lin, Electrochem. Commun. 11 (2009) 1142–1145. [42] X. Chen, L. Jia, X.R. Wang, G.L. Hu, M. Sato, Anal. Sci. 13 (1997) 71–75. [43] F. Takahashi, J.Y. Jin, Luminescence 23 (2008) 121–125. [44] J.K. Leland, M.J. Powell, J. Electrochem. Soc. 137 (1990) 3127–3131. [45] Y.B. Zu, A.J. Bard, Anal. Chem. 72 (2000) 3223–3232. [46] S. Workman, M.M. Richter, Anal. Chem. 72 (2000) 5556–5561. [47] F. Kanoufi, Y. Zu, A.J. Bard, J. Phys. Chem. B 105 (2001) 210–216. [48] Y.B. Zu, A.J. Bard, Anal. Chem. 73 (2001) 3960–3964. [49] B. Factor, B. Muegge, S. Workman, E. Bolton, J. Bos, M.M. Richter, Anal. Chem. 73 (2001) 4621–4624. [50] E.M. Gross, P. Pastore, R.M. Wightman, J. Phys. Chem. B 105 (2001) 8732–8738. [51] W. Miao, J.P. Choi, A.J. Bard, J. Am. Chem. Soc. 124 (2002) 14478–14485. [52] R.M. Wightman, S.P. Forry, R. Maus, D. Badocco, P. Pastore, J. Phys. Chem. B 108 (2004) 19119–19125. [53] N. Anastos, N.W. Barnett, B.J. Hindson, Talanta 64 (2004) 130–134. [54] A.J. Brown, P.S. Francis, J.L. Adcock, N.W. Barnett, Anal. Chim. Acta 624 (2008) 175–183.
Contents lists available at ScienceDirect
Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Vitamin C derivatives as new coreactants for tris(2,2 -bipyridine)ruthenium(II) electrochemiluminescence Yali Yuan a,b , Haijuan Li a,b , Shuang Han a,b , Lianzhe Hu a,b , Saima Parveen a,b,c , Guobao Xu a,∗ a b c
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China Graduate University of the Chinese Academy of Sciences, Beijing 100864, China Department of Chemistry, Baghdad-ul-Jadeed Campus, The Islamia University of Bahawalpur, Bahawalpur, Pakistan
a r t i c l e
i n f o
Article history: Received 19 May 2011 Received in revised form 22 June 2011 Accepted 27 June 2011 Available online 5 July 2011 Keywords: Tris(2,2 -bipyridine)ruthenium Electrochemiluminescence Vitamin C derivatives Ascorbyl palmitate Ascorbyl phosphate
a b s t r a c t Vitamin C derivatives (VCDs) have been widely used as the alternative and stable sources of vitamin C, and accordingly exhibit many new applications, such as anti-tumor and central nervous system drug delivery. In this study, their Ru(bpy)3 2+ electrochemiluminescence (ECL) properties have been investigated for the first time using well-known ascorbyl phosphate and ascorbyl palmitate as representative VCDs. Ascorbyl phosphate and ascorbyl palmitate are VCDs with different substituted positions. Both of them increase Ru(bpy)3 2+ ECL, indicating that other VCDs may also enhance Ru(bpy)3 2+ ECL signal. The calibration plot for ascorbyl phosphate is linear from 3 × 10−6 to 1.0 × 10−3 M with a detection limit of 1.4 × 10−6 M at a signal-to-noise ratio of 3. The relative standard deviation is 3.6% for six replicate measurements of 0.01 mM ascorbyl 2-phosphate solution. The proposed method is about one order of magnitude more sensitive than electrochemical and UV–vis methods for the determination of ascorbyl phosphate, and is used successfully for the determination of ascorbyl phosphate in whitening and moisturising body wash. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Vitamin C is an essential nutrient of life. It is required in many reactions involved in body processes, such as collagen synthesis, carnitine synthesis, tyrosine synthesis catabolism, and neurotransmitter synthesis. It acts as an antioxidant and radical scavenger. Moreover, it plays a crucial role in the protection of cellular components against oxidative damage by free radicals and oxidants that are involved in the development and exacerbation of many diseases such as cancer, heart disease, brain malfunction, aging, rheumatism, inflammation, stroke emphysema, and AIDS. It is one of the relatively few topical agents whose effectiveness against wrinkles and fine lines is backed by a fair amount of reliable scientific evidence, thus is a very popular skin-whitening agent used in cosmetics. Therefore, it is one of the most important biomolecules, and is perhaps the most popular vitamin [1–4]. However, the practical use of vitamin C, particularly in skin care and antioxidant food additives, presents some difficulties due to its labile oxidative properties. Being able to be transformed to vitamin C by enzymes in body, many vitamin C derivatives (VCDs) with better stability have been designed through chemical modification of hydroxyl groups of ascorbic acid, such as ascorbyl 2-phosphate,
∗ Corresponding author. Tel.: +86 431 85262747; fax: +86 431 85262747. E-mail address: [email protected] (G. Xu). 0003-2670/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2011.06.051
ascorbyl 6-palmitate, ascorbyl tetra-isopalmitoyl, tetrahexyldecyl ascorbate, ascorbic acid 2-sulfate, ascorbyl stearate, ascorbyl isostearate, ascorbyl oleate, ascorbyl glucoside, aminopropyl ascorbyl phosphate, ascorbyl laurate, and disodium ascorbyl phytostanol phosphate. In addition to their use as a source of vitamin C, VCDs have also been found to exhibit many new applications, such as anti-tumor and central nervous system drug delivery [5–7]. Electrogenerated chemiluminescence (ECL) is light-emitting phenomena involving electrochemical reactions [8–15]. There are many ECL systems, such as tris(2,2 -bipyridyl)ruthenium (II), peroxyoxalate, luminol, acridinium esters, lucigenin, graphene, carbon nanodots, nanocrystals, and metal nanoclusters [16–24]. Tris(2,2 bipyridyl)ruthenium(II) ECL is one of the most widely used ECL systems. On the one hand, it has been widely used in immunoassays [25,26], DNA probe assays [27], and aptasensors [28]. On the other hand, it has been used for the determination of numerous coreactants, such as oxalate, alkylamines, NADH, amino acids, dopamine, ascorbic acid, and so on [29–36]. Previous studies show that Ru(bpy)3 2+ ECL properties depend significantly on the structure of coreactants [9,14,18,24]. For example, no ECL can be observed when alkylamines are chemically transformed to amides or NADH is enzymatically transformed to NAD. Vitamin C has been investigated with different ECL luminophores, such as Ru(bpy)3 2+ [37], lucigenin [38], and luminol [39,40]. For example, Chen et al. [41] have developed an interesting luminol ECL method for the detection of vitamin
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Fig. 1. Molecular structure of ascorbic acid, ascorbyl 2-phosphate and ascorbyl 6-palmitate.
C using an electrically heated ionic-liquid/multi-walled carbon nanotube composite electrode. Chen et al. [33,42] have demonstrated that vitamin C is an effective coreactant for Ru(bpy)3 2+ ECL for the first time. Zorzi et al. [34] have found that vitamin C and dehydroascorbic acid show the same Ru(bpy)3 2+ ECL behavior and can be detected with same calibration graph. Takahashi and Jin [43] have reported that ECL of the Ru(bpy)3 2+ /vitamin C system can be quenched by excess vitamin C or upon ultrasonic irradiation. However, the effect of derivation of ascorbic acid on ECL is unknown. In this study, Ru(bpy)3 2+ ECL of VCDs has been reported for the first time. Ascorbyl 2-phosphate and ascorbyl 6-palmitate were selected as representatives because of three reasons. Firstly, both compounds have found their way into the broad skin care market. Secondly, their substituted positions are different as shown in Fig. 1. The substituted positions for ascorbyl 2-phosphate and ascorbyl 6palmitate are C2–OH position and C6–OH position, respectively. It allows the investigation of the effect of substituted positions on ECL. Finally, pure ascorbyl 2-phosphate and ascorbyl 6-palmitate are commercially available. The study shows that ascorbyl 2-phosphate and ascorbyl 6-palmitate can increase Ru(bpy)3 2+ ECL. Moreover, the ECL method was successfully applied to the determination of ascorbyl 2-phosphate in commercial bleaching cosmetic products. 2. Experimental
mode. It is sensitive to photons with wavelengths ranging from 200 to 800 nm. Unless noted otherwise, the PMT was biased at 700 V. The ECL cell was located in a light-tight box of the luminescent analyzer. 2.3. Procedures Prior to each measurement, the glassy carbon electrode was first polished with alumina powder, and then cleaned by ultrasonication in deionized water. Ascorbyl 2-phosphate and ascorbyl 6-palmitate solutions were freshly prepared in degassed deionized water before use. These newly prepared VCDs solutions were later mixed with 0.25 mM Ru(bpy)3 2+ at different ratio for electrochemical characterization and calibration. 3. Results and discussion 3.1. Electrochemistry of ascorbyl 2-phosphate and ascorbyl 6-palmitate Fig. 2 shows cyclic voltammograms of ascorbic acid and ascorbyl 2-phosphate at glassy carbon electrodes. Both ascorbic acid and ascorbyl 2-phosphate have irreversible anodic waves. The anodic peak potentials for ascorbic acid and ascorbyl 2-phosphate are about +0.18 V and +0.7 V, respectively. By comparison, the derivation positively shifts the anodic peak potential by about 0.5 V,
2.1. Materials Tris(2,2 -bipyridyl)ruthenium(II) chloride was purchased from Aldrich. 2-phospho-l-ascorbic acid trisodium salt was obtained from Fluka. l-Ascorbic acid 6-palmitate (99%) was purchased from Alfa Aesar. All other reagents were of reagent grade. Solutions were prepared using water, which was deionized and further purified in a Millipore system. Whitening and moisturising body wash was bought from Watsons (Hongkong, China). 2.2. Instrumentation Electrochemical measurements were performed in a conventional three-electrode cell (volume 0.6 mL) with a CHI 800B potentiostat (CH Instruments, Shanghai, China). The threeelectrode system included a glassy carbon electrode (diameter 3 mm), an Ag/AgCl (saturated KCl) reference electrode, and a Pt wire auxiliary electrode. The Teflon gasket (2 mm in thickness) was sandwiched between the electrode block and a Plexiglas window. ECL intensities were measured through the bottom of the electrochemical cell with a BPCL Ultra-Weak luminescence analyzer, which was purchased from Institute of Biophysics, Chinese Academy of Sciences. The photomultiplier tube (PMT) used in the BPCL Ultra-Weak luminescence analyzer was operated in current
Fig. 2. Cyclic voltammograms at glassy carbon electrodes in 0.05 M pH 8.0 phosphate buffer solutions containing (a) 0.2 mM ascorbic acid; (b) 0.2 mM ascorbyl 2-phosphate; (c) 0.25 mM Ru(bpy)3 2+ and 0.2 mM ascorbyl 2-phosphate; (d) 0.25 mM Ru(bpy)3 2+ , scan rate: 0.1 V s−1 .
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Fig. 3. Cyclic voltammograms at glassy carbon electrodes in 0.05 M pH 8.0 phosphate buffer solutions containing ethanol at a volume ratio of 1:6 with the addition of (a) 0.05 M pH 8.0 PBS; (b) 0.2 mM ascorbyl 6-palmitate; (c) 0.25 mM Ru(bpy)3 2+ , (d) 0.25 mM Ru(bpy)3 2+ and ascorbyl 6-palmitate, scan rate: 0.1 V s−1 .
indicating that ascorbyl 2-phosphate has better stability than ascorbic acid. The better stability is ascribed to the esterification of an unstable hydroxyl group of ascorbic acid with phosphate. Fig. 2c and d shows cyclic voltammograms of Ru(bpy)3 2+ in the absence and presence of ascorbyl 2-phosphate, respectively. A typical pair of redox waves is observed for Ru(bpy)3 2+ solution, which was attributed to the one-electron redox reaction of Ru(bpy)3 2+ . A comparison of Fig. 2c and d demonstrates that Ru(bpy)3 2+ catalyzes the oxidation of ascorbyl 2-phosphate, since the anodic current of Ru(bpy)3 2+ increases and the cathodic current of Ru(bpy)3 2+ decreases in the presence of ascorbyl 2-phosphate, and the oxidation potential of Ru(bpy)3 2+ is much larger than the oxidation potential of ascorbyl 2-phosphate. To investigate the effect of substituted position, the electrochemistry of another VCD, ascorbyl 6-palmitate, was also studied. Since ascorbyl 6-palmitate is insoluble in water, all electrochemical experiments of ascorbyl 6-palmitate were carried out in 0.05 M phosphate buffer solution containing ethanol at a volume ratio of 1:6. As shown in Fig. 3, an irreversible broad anodic wave of ascorbyl 6-palmitate was observed at around +1.05 V. The anodic peak potential of ascorbyl 6-palmitate is much more positive than that of ascorbic acid, indicating that the substitution of ascorbic acid at C6–OH position also improves the stability. 3.2. ECL of ascorbyl 2-phosphate and ascorbyl 6-palmitate Fig. 4 described the ECL-potential profile of ascorbyl 2phosphate and ascorbyl 6-palmitate. The onset of ECL occurred near 1.0 V and the ECL intensity reached a maximum at 1.15 V, consistent with the oxidation potential of Ru(bpy)3 2+ . It indicates that the electrochemical oxidation of Ru(bpy)3 2+ is necessary for ECL reaction. The ECL mechanism is similar to that of oxidative-reductive ECL systems [33,34]. Ru(bpy)3 2+ is electrochemically oxidized to generate Ru(bpy)3 3+ (Eq. (1)). VCD is oxidized directly on the electrode and by electrogenerated Ru(bpy)3 3+ to generate a strong reductant VCD• (Eqs. (2) and (3)). Then electrogenerated Ru(bpy)3 3+ reacts with VCD• to generate the excited state Ru(bpy)3 2+* (Eq. (4)) and emit light subsequently (Eq. (5)). By comparison, the ECL intensities increase about 16 times and 6 times upon the addition of ascorbyl 2-phosphate and ascorbyl 6-palmitate, respectively. These results suggest that C2–OH substituted VCDs and C6–OH substituted VCDs
Fig. 4. (A) ECL-potential profile at glassy carbon electrodes in 0.05 M pH 11.0 phosphate buffer solutions containing (a) 0.25 mM Ru(bpy)3 2+ and 0.2 mM ascorbyl 2-phosphate; (b) 0.25 mM Ru(bpy)3 2+ . (B) ECL-potential profile at glassy carbon electrodes in 0.05 M pH 11.0 phosphate buffer solutions containing ethanol at a volume ratio of 1:6 with the presence of (c) 0.25 mM Ru(bpy)3 2+ and 0.2 mM ascorbyl 6-palmitate; (d) 0.25 mM Ru(bpy)3 2+ .
may enhance Ru(bpy)3 2+ ECL signal. Since ascorbyl 2-phosphate has good solubility in aqueous solutions, it was used as a representative VCD to demonstrate the analytical applications of Ru(bpy)3 2+ ECL for the determination of VCDs. Ru(bpy)3 2+ − e → Ru(bpy)3 3+ VCD − e →
(1)
VCD•
(2)
Ru(bpy)3
3+
+ VCD → Ru(bpy)3
Ru(bpy)3
3+
+ VCD•
Ru(bpy)3
2+∗
2+∗
→ Ru(bpy)3
→ Ru(bpy)3
2+
+ VCD•
2+∗
+ h
(3) (4) (5)
3.3. ECL determination of ascorbyl 2-phosphate Fig. 5 shows the dependence of ECL intensity on pH in Ru(bpy)3 2+ solutions in the absence or presence of ascorbyl 2phosphate. A comparison of Fig. 5a and b indicates that the ECL reaction between ascorbyl 2-phosphate and Ru(bpy)3 2+ occurs at pH higher than 10. The ECL intensities resulting from the ECL reaction between ascorbyl 2-phosphate and Ru(bpy)3 2+ increase sharply with increasing pH from 10.0 to 11.0 and then decrease slightly at pH higher than 11.0. Therefore, a pH of 11.0 was used for the subsequent measurements. The strong pH dependence of ECL
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Table 1 Assay results for the ECL analysis of ascorbyl 2-phosphate in whitening and moisturising body wash.
Whitening and moisturising body wash samplea Whitening and moisturising body wash sample + 10 M ascorbyl 2-phosphate Whitening and moisturising body wash sample + 20 M ascorbyl 2-phosphate a
Number of detection
Coefficient of variation (%)
Detection results (M)
Recovery (%)
Real sample content (wt.%)
6 6 6
4.83 3.91 4.16
3.72 14.59 24.72
– 106 105
0.76 – –
Dilution factor: 1/15,000.
may mainly result from the pH dependence of deprotonation of ascorbyl 2-phosphate and its radical cation, and the reduced availability of Ru(bpy)3 3+ due to the competitive reaction with the OH− ion at higher pHs [44–52]. Fig. 6 shows the dependence of ECL intensity on potential. The ECL intensities increase sharply from 1.0 to 1.15 V, and then decrease at potentials above 1.15 V. The electrochemical oxidation of OH− at these high potentials could also be responsible for the overall ECL intensity decrease [31]. To get good reproducibility and sensitivity, subsequent experiments were conducted at a potential of 1.15 V for ECL excitation. The log–log calibration plot for ascorbyl 2-phosphate is linear from 3 × 10−6 to 1.0 × 10−3 mol L−1 (slope = 0.779; inter-
cept = 7.101; n = 7; correlation coefficient = 0.9959). The detection limit is 1.4 × 10−6 M at a signal-to-noise ratio of 3. The relative standard deviation is 3.6% for six replicate measurements of 0.01 mM ascorbyl 2-phosphate. The total analysis time was less than 2 min. The proposed method is about one order of magnitude more sensitive than electrochemical method and UV–vis methods [2–7]. 3.4. Analysis of whitening and moisturising body wash sample Appropriate amount of whitening and moisturising body wash was first diluted with water at the ratio of 1:300, and then hexane was added to extract the organic ingredients in the mixture. After extraction, the obtained aqueous solution was again diluted at the ratio of 1:50 with 0.05 M pH 11.0 phosphate buffer solution to prepare the sample solution. Under optimum conditions, the ECL determination of ascorbyl 2-phosphate in cosmetic product was successfully achieved, as shown in Table 1. The calculated content of ascorbyl 2-phosphate in this cosmetic product was 0.76%, which was comparable with its content. The standard addition method was used for testing recovery. The recovery was 106% and 105% for 10 M and 20 M ascorbyl 2-phosphate, respectively. 4. Conclusions
Fig. 5. Effect of pH on ECL of 0.25 mM Ru(bpy)3 2+ solutions in the (a) absence of and (b) presence of 0.2 mM ascorbyl 2-phosphate. Applied potential, 1.15 V. Each point represents the average of three measurements.
VCDs have been demonstrated as new coreactants for Ru(bpy)3 2+ ECL for the first time using ascorbyl phosphate and ascorbyl palmitate as representative VCDs. Both ascorbyl 2-phosphate and ascorbyl 6-palmitate increase Ru(bpy)3 2+ ECL significantly. These results suggest that C2–OH substituted VCDs and C6–OH substituted VCDs may also enhance Ru(bpy)3 2+ ECL signal. Ru(bpy)3 2+ ECL method is one order of magnitude more sensitive than electrochemical and UV–vis methods for the determination of ascorbyl 2-phosphate. Moreover, the ECL method was successfully applied to the determination of ascorbyl 2-phosphate in commercial bleaching cosmetic products with excellent recovery. This study is helpful for understanding ECL properties of VCDs, and provides a new way to detect VCDs. It has been demonstrated in Barnett’s excellent reviews on Ru(bpy)3 2+ chemiluminescence that most coreactants of Ru(bpy)3 2+ ECL can react with Ru(bpy)3 3+ to generate chemiluminescence [11,16]. Therefore, our study indicates that VCDs may also react with Ru(bpy)3 3+ to generate chemiluminescence and may also be detected through other chemiluminescent systems that have been used to detect vitamin C [53,54]. Acknowledgements
Fig. 6. Effect of potential on ECL at glassy carbon electrodes in 0.05 M pH 11.0 phosphate buffer solutions containing 0.25 mM Ru(bpy)3 2+ and 0.2 mM ascorbyl 2-phosphate. Each point represents the average of three measurements.
This project was kindly supported by the National Natural Science Foundation of China (No. 20875086), the Ministry of Science and Technology of the People’s Republic of China (No. 2006BAE03B08), Chinese Academy of Sciences (CAS), the Academy of Sciences for the Developing World (TWAS), the Department of Sciences & Technology of Jilin Province (No. 20070108 and 20082104), and Changchun Institute of Applied Chemistry.
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References [1] A. Chisvert, A. Balaguer, A. Salvador, Analysis of Cosmetics Products, Elsevier Science B.V., Amsterdam, 2007. [2] A. Balaguer, A. Chisvert, A. Salvador, J. Sep. Sci. 31 (2008) 229–236. [3] A. Varvaresou, E. Tsirivas, K. Iakovou, E. Gikas, Z. Papathomas, A. Vonaparti, I. Panderi, Anal. Chim. Acta 573 (2006) 284–290. [4] N.M. Pauli, W. Schuep, J. Chromatogr. B 715 (1998) 369–377. [5] S.C. Huang, C.C. Lin, M.C. Huang, K.C. Wen, J. Food Drug Anal. 12 (2004) 13–18. [6] M.L. Chang, C.M. Chang, J. Pharm. Biomed. Anal. 33 (2003) 617–626. [7] M.L. Chang, C.M. Chang, J. Food Drug Anal. 13 (2005) 205–211. [8] A.J. Bard, Electrogenerated Chemiluminescence, Marcel Dekker, New York, 2004. [9] A.W. Knight, G.M. Greenway, Analyst 121 (1996) R101–R106. [10] W.Y. Lee, Mikrochim. Acta 127 (1997) 19–39. [11] R.D. Gerardi, N.W. Barnett, P. Jones, Anal. Chim. Acta 388 (1999) 1–10. [12] K.A. Fahnrich, M. Pravda, G.G. Guilbault, Talanta 54 (2001) 531–559. [13] W.J. Miao, A.J. Bard, Anal. Chem. 76 (2004) 5379–5386. [14] M.M. Richter, Chem. Rev. 104 (2004) 3003–3036. [15] X.B. Yin, S.J. Dong, E. Wang, Trends Anal. Chem. 23 (2004) 432–441. [16] B.A. Gorman, P.S. Francis, N.W. Barnett, Analyst 131 (2006) 616–639. [17] C.A. Marquette, L.J. Blum, Anal. Bioanal. Chem. 390 (2008) 155–168. [18] W.J. Miao, Chem. Rev. 108 (2008) 2506–2553. [19] J.F. Rusling, E.G. Hvastkovs, D.O. Hull, J.B. Schenkman, Chem. Commun. 2 (2008) 141–154. [20] H. Wei, E.K. Wang, Trends Anal. Chem. 27 (2008) 447–459. [21] P. Bertoncello, R.J. Forster, Biosens. Bioelectron. 24 (2009) 3191–3200. [22] R.J. Forster, P. Bertoncello, T.E. Keyes, Annu. Rev. Anal. Chem. 2 (2009) 359–385. [23] H.J. Li, S. Han, L.Z. Hu, G.B. Xu, Chin. J. Anal. Chem. 37 (2009) 1557–1565. [24] L.Z. Hu, G.B. Xu, Chem. Soc. Rev. 39 (2010) 3275–3304. [25] F. Deiss, C.N. LaFratta, M. Symer, T.M. Blicharz, N. Sojic, D.R. Walt, J. Am. Chem. Soc. 131 (2009) 6088–6089. [26] R. Kurita, K. Arai, K. Nakamoto, D. Kato, O. Niwa, Anal. Chem. 82 (2010) 1692–1697. [27] L. Dennany, R.J. Forster, J.F. Rusling, J. Am. Chem. Soc. 125 (2003) 5213–5218.
173
[28] X.Q. Liu, L.H. Shi, W.X. Niu, H.J. Li, G.B. Xu, Angew. Chem. Int. Ed. 46 (2007) 421–424. [29] Z.J. Lin, X.M. Chen, T.T. Jia, X.D. Wang, Z.X. Xie, M. Oyama, X. Chen, Anal. Chem. 81 (2009) 830–833. [30] I.S. Shin, S.W. Bae, H. Kim, J.I. Hong, Anal. Chem. 82 (2010) 8259–8265. [31] Y.W. Chi, Y.Q. Dong, G.N. Chen, Anal. Chem. 79 (2007) 4521–4528. [32] I. Rubinstein, A.J. Bard, J. Am. Chem. Soc. 102 (1980) 6641–6642. [33] X. Chen, M. Sato, Anal. Sci. 11 (1995) 749–754. [34] M. Zorzi, P. Pastore, F. Magno, Anal. Chem. 72 (2000) 4934–4939. [35] H.N. Choi, S.H. Cho, W.Y. Lee, Anal. Chem. 75 (2003) 4250–4256. [36] K.F. Chow, F. Mavré, J.A. Crooks, B.Y. Chang, R.M. Crooks, J. Am. Chem. Soc. 131 (2009) 8364–8365. [37] X.M. Sun, Y. Niu, S. Bi, S.S. Zhang, Electrophoresis 29 (2008) 2918–2924. [38] J.H. Chen, Z.Y. Lin, G.N. Chen, Electrochim. Acta 52 (2007) 4457–4462. [39] H. Dai, X.P. Wu, Y.M. Wang, W.C. Zhou, G.N. Chen, Electrochim. Acta 53 (2008) 5113–5117. [40] J.F. Rong, Y.W. Chi, Y.J. Zhang, G.N. Chen, Electrochem. Commun. 12 (2010) 270–273. [41] Y.T. Chen, X.P. Chen, Z.Y. Lin, Electrochem. Commun. 11 (2009) 1142–1145. [42] X. Chen, L. Jia, X.R. Wang, G.L. Hu, M. Sato, Anal. Sci. 13 (1997) 71–75. [43] F. Takahashi, J.Y. Jin, Luminescence 23 (2008) 121–125. [44] J.K. Leland, M.J. Powell, J. Electrochem. Soc. 137 (1990) 3127–3131. [45] Y.B. Zu, A.J. Bard, Anal. Chem. 72 (2000) 3223–3232. [46] S. Workman, M.M. Richter, Anal. Chem. 72 (2000) 5556–5561. [47] F. Kanoufi, Y. Zu, A.J. Bard, J. Phys. Chem. B 105 (2001) 210–216. [48] Y.B. Zu, A.J. Bard, Anal. Chem. 73 (2001) 3960–3964. [49] B. Factor, B. Muegge, S. Workman, E. Bolton, J. Bos, M.M. Richter, Anal. Chem. 73 (2001) 4621–4624. [50] E.M. Gross, P. Pastore, R.M. Wightman, J. Phys. Chem. B 105 (2001) 8732–8738. [51] W. Miao, J.P. Choi, A.J. Bard, J. Am. Chem. Soc. 124 (2002) 14478–14485. [52] R.M. Wightman, S.P. Forry, R. Maus, D. Badocco, P. Pastore, J. Phys. Chem. B 108 (2004) 19119–19125. [53] N. Anastos, N.W. Barnett, B.J. Hindson, Talanta 64 (2004) 130–134. [54] A.J. Brown, P.S. Francis, J.L. Adcock, N.W. Barnett, Anal. Chim. Acta 624 (2008) 175–183.