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A ruthenium(II) complex based turn-on electrochemiluminescence probe for the detection of nitric oxide

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Electrochemiluminescence (ECL) detection technique using bipyridine-ruthenium(II) complexes as probes is a highly sensitive and widely used method for the detection of various biological and bioactive molecules. In this work, the spectral,
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  A ruthenium( II ) complex based turn-on electrochemiluminescence probe forthe detection of nitric oxide Wenzhu Zhang, a Dan Zhao, b Run Zhang, a Zhiqiang Ye, a Guilan Wang, a Jingli Yuan * a and Mei Yang * b Received 15th December 2010, Accepted 22nd February 2011 DOI: 10.1039/c0an01003k Electrochemiluminescence (ECL) detection technique using bipyridine-ruthenium( II ) complexes asprobes is a highly sensitive and widely used method for the detection of various biological and bioactivemolecules. In this work, the spectral, electrochemical and ECL properties of a chemically modifiedbipyridine-ruthenium( II ) complex, [Ru(bpy) 2 (dabpy)] 2+ (bpy: 2,2 0 -bipyridine; dabpy: 4-(3,4-diaminophenoxy)-2,2 0 -bipyridine), were investigated and compared with those of its nitric oxide(NO)-reaction derivative [Ru(bpy) 2 (T-bpy)] 2+ (T-bpy: 4-triazolephenoxy-2,2 0 -bipyridine) and [Ru(bpy) 3 ] 2+ . Itwasfound thatthe ECL intensity of[Ru(bpy) 2 (dabpy)] 2+ couldbeselectively andsensitivelyenhanced by NO due to the formation of [Ru(bpy) 2 (T-bpy)] 2+ in the presence of tri-n-propylamine. Byusing [Ru(bpy) 2 (dabpy)] 2+ as a probe, a sensitive and selective ECL method with a wide linear range(0.55 to 220.0  m M) and a low detection limit (0.28  m M) was established for the detection of NO inaqueous solutions and living cells. The results demonstrated the utility and advantages of the new ECLprobe for the detection of NO in complicated biological samples. 1. Introduction Nitric oxide (NO) is a ubiquitous and short-lived free radical thatcan be biosynthesized in animal and plant organisms. Earlystudies on NO only focused on its toxicity as an air pollutant.Subsequently, it was gradually recognized that a number of diseases were implicated with the NO imbalances. To date, it hasbeen known that NO plays an important role in many funda-mental biochemical processes including blood pressure control,neurotransmission and immune response. 1–3 As an intra- andinter-cellular messenger, NO transfers signals in the cardiovas-cular and nervous systems to regulate the immune balance at lowconcentrations. However, at high concentration levels it canrapidly react with other reactive oxygen species (ROS) to formreactive nitrogen species (RNS) to cause the damage of the cells. 4 Therefore, the detection of NO is an attractive work forunderstanding the complicated functions of NO in livingsystems, and a number of methods, such as fluorescence, 5–10 chemiluminescence, 11 electron paramagnetic resonance spec-troscopy, 3,12–14 and electrochemiluminescence, 15 have beendeveloped for the detection of NO.Electrochemiluminescence or electrogenerated chem-iluminescence (ECL) represents a marriage between electro-chemical and spectroscopic methods. 16 Because ECL combinesthe electrochemical and photoluminescent properties withoutany extra source of light, it can provide high sensitivity, selec-tivity, and stability over other spectroscopic detectionmethods. 16,17 The ECL researches on theoretical and applicationaspects have been rapidly developed over the past two decades.Since Tokel and Bard reported the ECL property of tris(2,2 0 -bipyridine)ruthenium( II ) ([Ru(bpy) 3 ] 2+ ) in 1972, 18 this complexhas become one of the most extensively investigated ECL lumi-nophores for highly sensitive immunoassay and DNA assayapplications 19–26 due to its high ECL efficiency and good solu-bility in aqueous solutions. Currently, a series of derivatives of [Ru(bpy) 3 ] 2+ has been designed and synthesized for improvingthe light-emitting efficiency or detection selectivity. 27–33 It hasbeen also known that different functions of Ru( II ) complexes canbe realized by different ligand modifications. Muegge andRichter reported that the Ru( II ) complexes conjugated withcrown ether moieties could be used as ECL sensors for metalions. 34 Beer’s group demonstrated the utilities of the function-alized bipyridine-Ru( II ) complexes for the ECL sensing of cationsand anions. 35–37 More recently, Berni  et al.  described the appli-cation of a guanidinium group modified bipyridine-Ru( II )complex for the ECL detection of anions. 38 It can be considered that the bipyridine-Ru( II ) complexes withsuitable ligand modifications can offer more extensive ECLapplications for detecting different analytes. In a previous work,we demonstrated that an  o -diaminophenyl-modified bipyridine-Ru( II ) complex, [Ru(bpy) 2 (dabpy)] 2+ (bpy: 2,2 0 -bipyridine;dabpy: 4-(3,4-diaminophenoxy)-2,2 0 -bipyridine), could be usedas a luminescence probe for the detection of NO. 39 In this work,the spectral, electrochemical and ECL properties of [Ru a State Key Laboratory of Fine Chemicals, School of Chemistry, DalianUniversity of Technology, Dalian 116024, China. E-mail: jingliyuan@ yahoo.com.cn; Fax: +0086-411-84986041; Tel: +0086-411-84986041 b School of Chemistry and Chemical Engineering, Liaoning Normal University, Dalian 116029, China. E-mail: yangmeils@163.com; Fax:+0086-411-84258088; Tel: +0086-411-84258088 This journal is  ª  The Royal Society of Chemistry 2011  Analyst  , 2011,  136 , 1867–1872 | 1867 Dynamic Article Links C < Analyst  Cite this:  Analyst  , 2011,  136 , 1867www.rsc.org/analyst  PAPER  (bpy) 2 (dabpy)] 2+ and its NO-reaction derivative [Ru(bpy) 2 -(T-bpy)] 2+ (T-bpy: 4-triazolephenoxy-2,2 0 -bipyridine) wereinvestigated. It was found that the ECL signal of [Ru(bpy) 2 -(dabpy)] 2+ in the presence of tri-n-propylamine (TPrA) was veryweak. However, after NO was added, the ECL intensity could beremarkably enhanced (Fig. 1). These results indicate that [Ru(bpy) 2 (dabpy)] 2+ itself is ECL low active, while its NO-reactionderivative [Ru(bpy) 2 (T-bpy)] 2+ is ECL highly active. The ‘off–on’ECL behaviors of [Ru(bpy) 2 (dabpy)] 2+ in the absence and pres-ence of NO enable it to beused as an ECL probe for the detectionof NO. Furthermore, it was found that the ECL response of thecomplex was highly specific to NO even in the presence of variousROS and RNS. Thus, a highly selective and sensitive ECLdetection method for NO was established by using [Ru(bpy) 2 -(dabpy)] 2+ as a probe, and the utility of the method for thedetection of NO in aqueous and biological samples (living plantcells) was demonstrated. 2. Experimental 2.1 Materials and instrumentation Tris(2,2 0 -bipyridine)dichlororuthenium( II ) hexahydrate ([Ru(bpy) 3 ]Cl 2 $ 6H 2 O), and tri-n-propylamine (TPrA) werepurchased from Sigma-Aldrich. [Ru(bpy) 2 (dabpy)][PF 6 ] 2  and[Ru(bpy) 2 (T-bpy)][PF 6 ] 2  were synthesized according to ourprevious method. 39 Gardenia  cells were obtained from theDepartment of Biotechnology, Dalian Institute of ChemicalPhysics, Chinese Academy of Sciences. The NO solution wasprepared by bubbling NO gas (passing through a degassed KOHsolution to remove trace impurities) for 3 h into a 0.1 M argondeoxidized phosphate buffer at pH 7.4. The concentration of NOwas measured by using the Griess method. 40 Unless otherwisestated, all chemical materials were purchased from commercialsources and used without further purification.UV-vis spectra were measured on a Perkin-Elmer Lambda 35UV-vis spectrometer. Luminescence spectra were measured ona Perkin-Elmer LS 50B luminescence spectrometer. The cyclicvoltammograms of the Ru( II ) complexes were measured on anLK2005 electrochemical analyzer (Tianjin Lanlike Chemical andElectron High Technology Co., Ltd., China). The glassy carbonand Ag/AgCl (KCl saturated) electrodes were used as workingelectrode and reference electrode, respectively, and a platinumwire was used as the auxiliary electrode. All the ECL experimentswere carried out on a laboratory-use ECL instrument consistingof a syringe pump, an ECL flow cell, a three-electrode system,and an ECL detector system on an IFFM-D chemiluminescenceanalyzer (Xi’An Remax Science & Technology Co. Ltd.,China). 41 The platinum electrode, Ag/AgCl (KCl saturated)electrode and stainless steel electrode were used as the workingelectrode, the reference electrode, and the auxiliary electrode,respectively. 2.2 Reactions of [Ru(bpy) 2 (dabpy)] 2+ with different ROS andRNS All the reactions were carried out in 0.1 M phosphate buffer atpH 7.4 containing 5.0 mM TPrA and 10 m M [Ru(bpy) 2 (dabpy)] 2+ for 0.5 h at room temperature. Hydroxyl radicals ( _ OH) weregenerated in the Fenton system from ferrous ammonium sulfateand hydrogen peroxide. 42 Singlet oxygen was generated from the  OCl–H 2 O 2  system. 43 Peroxynitrite was synthesized from sodiumnitrite (0.6 M) and H 2 O 2  (0.65 M) in a quenched-flow reactor(excess H 2 O 2  was used to minimize nitrite contamination). Afterthe reaction, the solution was treated with MnO 2  to eliminate theexcess H 2 O 2 . The concentration of the ONOO  stock solutionwas determined by measuring the absorbance at 302 nm witha molar extinction coefficient of 1670 M  1 cm  1 . 44 Hydrogenperoxide (H 2 O 2 ) was diluted immediately from a stabilized 30%solution, and was assayed by using its molar absorption coeffi-cient of 43.6 M  1 cm  1 at 240 nm. 45 The freshly prepared aqueoussolutions of NaOCl, KO 2 ,NaNO 2  andKNO 3  were usedas ClO  ,O 2  , NO 2  and NO 3  sources, respectively. 2.3 ECL detection of NO in living plant cells Fresh  Gardenia  cells (1.5 g) cultured in Murashige and Skoog(MX) medium 46 were harvested by filtration of the cell suspen-sion through a 200-mesh nickel screen and rinsed twice withdistilled water. The cells were suspended in 5.0 mL of 0.1 Mphosphate buffer at pH 7.4 containing 100  m M [Ru(bpy) 2 -(dabpy)] 2+ . The cell suspension was divided into 5 parts (1.0 mLper part), and then incubated at room temperature with slowshaking. At different times (0, 1, 2, 3 and 4 h), the cells wereseparated by centrifugation for 5 min at 10 000 rpm, washed3 times with the phosphate buffer, and then added to 1.0 mL of 5.0 mM TPrA–0.1 M phosphate buffer at pH 7.4 for the ECLdetection. 3. Results and discussion 3.1 Spectral and electrochemical properties of the Ru( II )complexes The UV-vis absorption and luminescence spectra as well as cyclicvoltammograms of the Ru( II ) complexes, [Ru(bpy) 2 (dabpy)] 2+ ,[Ru(bpy) 2 (T-bpy)] 2+ and [Ru(bpy) 3 ] 2+ , were measured in 0.1 Mphosphate buffer at pH 7.4. Although three Ru( II ) complexeshave the same UV-vis absorption pattern (Fig. 2A,  p / p * Fig. 1  ECL behaviors of [Ru(bpy) 2 (dabpy)] 2+ (10 m M) in the absence (a)and presence (b) of NO (220  m M) in 5.0 mM TPrA–0.1 M phosphatebuffer at pH 7.4. The inset shows the reaction of [Ru(bpy) 2 (dabpy)] 2+ with NO under aerobic conditions. 1868 |  Analyst  , 2011,  136 , 1867–1872 This journal is  ª  The Royal Society of Chemistry 2011  transition of the ligand at 288 nm and metal-to-ligand chargetransfer at 455 nm), their luminescence behaviors are drasticallydifferent (Fig. 2B). Both [Ru(bpy) 3 ] 2+ and [Ru(bpy) 2 (T-bpy)] 2+ show a strong emission band at 616 nm under the excitation of 455 nm, whereas [Ru(bpy) 2 (dabpy)] 2+ is almost non-luminescent.This difference is caused by the fact that the  o -diaminophenylgroup in [Ru(bpy) 2 (dabpy)] 2+ can strongly quench the excitedstate of the complex by a photo-induced electron transfermechanism, while the same electron transfer cannot occur in [Ru(bpy) 2 (T-bpy)] 2+ and [Ru(bpy) 3 ] 2+ . 39 The cyclic voltammogramsof the three Ru( II ) complexes (Fig. 2C) indicate that the oxida-tion potentials of [Ru(bpy) 2 (dabpy)] 2+ and [Ru(bpy) 2 (T-bpy)] 2+ arealittlehigherthan thatof[Ru(bpy) 3 ] 2+ ,which mightbecausedby the electron-rich phenoxy group in [Ru(bpy) 2 (dabpy)] 2+ and[Ru(bpy) 2 (T-bpy)] 2+ . 3.2 ECL behaviors of the Ru( II ) complexes The ECL behaviors of the three Ru( II ) complexes (10  m M) weredetermined in 0.1 M phosphate buffer at pH 7.4. According tothe literatures, 16,17 the ECL system involving [Ru(bpy) 3 ] 2+ usuallyrequires a suitable co-reactant, such as TPrA, to improve theefficiency of light emission. In our experiments, it was also foundthat no ECL signals could be observed from the solutions(10  m M) of [Ru(bpy) 2 (dabpy)] 2+ and [Ru(bpy) 2 (T-bpy)] 2+ in theabsence of TPrA. However, in the presence of TPrA (5.0 mM),the ECL signal of the [Ru(bpy) 2 (dabpy)] 2+ solution was still veryweak, but a strong ECL signal was observed from the [Ru(bpy) 2 (T-bpy)] 2+ solution (Fig. 3A).In the ECL system, it has been known that the excited state of the Ru( II ) complex, [RuL 3 ] 2+ *, is produced by the reduction of [RuL 3 ] 3+ with the TPrA _ radicals ([RuL 3 ] 3+ + TPrA _ / [RuL 3 ] 2+ *).After [RuL 3 ] 2+ is oxidized to [RuL 3 ] 3+ on the electrode, theproductisfurther reduced byTPrA _ radicalstoproduce [RuL 3 ] 2+ *,and then the luminescence is emitted due to the occurrence of the[RuL 3 ] 2+ * / [RuL 3 ] 2+ energy transfer process. To understand thestructure-dependent ECL properties of the Ru( II ) complexes, themolecular orbital energy levels of [Ru(bpy) 2 (dabpy)] 3+ , [Ru(bpy) 2 (T-bpy)] 3+ and [Ru(bpy) 3 ] 3+ were calculated based on thedensity functional theory (DFT) using a Gaussian 03 software, 47 because they directly corresponded to the ECL efficiency of theRu( II ) complexes. Fig. 3B shows the molecular orbitals and the Fig. 2  Absorption (A) and luminescence (B) spectra as well as cyclicvoltammograms (C) of the three Ru( II ) complexes (10  m M) in 0.1 Mphosphate buffer at pH 7.4: (a) [Ru(bpy) 2 (dabpy)] 2+ ; (b) [Ru(bpy) 2 -(T-bpy)] 2+ ; (c) [Ru(bpy) 3 ] 2+ . Fig. 3  ECL behaviors (A) and the molecular orbitals (B, the insets showthe energy levels of the orbitals) of [Ru(bpy) 2 (dabpy)] 3+ (a), [Ru(bpy) 2 -(T-bpy)] 3+ (b) and [Ru(bpy) 3 ] 3+ (c). This journal is  ª  The Royal Society of Chemistry 2011  Analyst  , 2011,  136 , 1867–1872 | 1869  calculation results of energy levels for the three Ru( III )complexes. It can be observed that [Ru(bpy) 3 ] 3+ has the lowestenergy level among the complexes, which indicates that [Ru(bpy) 3 ] 2+ * can be formed easiest so that [Ru(bpy) 3 ] 2+ has thehighest ECL efficiency than the other two Ru(II) complexes. Theenergy levels of the three Ru( III ) complexes show an order of [Ru(bpy) 3 ] 3+ < [Ru(bpy) 2 (T-bpy)] 3+ < [Ru(bpy) 2 (dabpy)] 3+ , which isin good agreement with the ECL experimental results. 3.3 ECL detection of NO in aqueous solution Before the detection, the effects of pH and the TPrA concen-tration on the ECL intensities of the Ru( II ) complexes wereinvestigated. As shown in Fig. 4A, the ECL intensities of [Ru(bpy) 2 (dabpy)] 2+ and [Ru(bpy) 2 (T-bpy)] 2+ are graduallyincreased with the increase of pH (from 6.0 to 10). Thisphenomenon is owing to that higher pH is beneficial to theproduction of TPrA _  radicals. Although the maximum signal-to-background (S/N) ratio (the intensity ratio of [Ru(bpy) 2 (T-bpy)] 2+ to [Ru(bpy) 2 (dabpy)] 2+ ) was observed at pH 9.0, since this workaimed at the detection of NO in biological systems, a buffersolution of pH 7.4 was used in the following experiments. Fig. 4Bshows the effect of the TPrA concentration on the ECL inten-sities of [Ru(bpy) 2 (dabpy)] 2+ and [Ru(bpy) 2 (T-bpy)] 2+ in 0.1 Mphosphate buffer of pH 7.4. It can be observed that the higherTPrA concentration offers the higher ECL intensity, and thehighest S/N ratio is reached when the TPrA concentration is5.0 mM (the ratios at 1.0 mM, 5.0 mM and 10 mM of TPrA are7.3, 13.3 and11.9, respectively). To comprehensively consider theeffects of pH and TPrA concentration on the S/N ratio, the 0.1 Mphosphate buffer at pH 7.4 containing 5.0 mM TPrA wasselected as the optimal medium for the ECL detection of NOusing [Ru(bpy) 2 (dabpy)] 2+ as a probe.To evaluate the performance of the probe for the quantitativeECL detection of NO in aqueous media, an ECL titrationexperiment was carried out in the above optimal medium using[Ru(bpy) 2 (dabpy)] 2+ (10  m M) as a probe and an NO aqueoussolution (2.2 mM) as the NO source. As shown in Fig. 4C, theECL intensity of the probe is sensitively increased with theincrease of NO concentration. The dose-dependent ECLenhancement showsa good linearity that can beexpressed as D I  ¼ 64.94[NO] + 11.76 ( r ¼ 0.995) in the NO concentration range of 0.55 to 220.0  m M (the inset in Fig. 4C). The detection limit forNO, calculated as the concentration corresponding to triplestandard deviations of the background signal, is 0.28  m M. In2007, Chen  et al.  reported the ECL detection of NO using a [Ru(bpy) 3 ] 2+ /TPrA system based on the ECL quenching of NO to thesystem. 15 Compared to this report, our method shows the betterresults including a wider linear range and a lower detection limit. 3.4 ECL response specificity of the probe towards NO Because some ROS and RNS often coexist with NO in bio-systems, to examine the ECL response specificity of [Ru(bpy) 2 (dabpy)] 2+ towards NO, the ECL responses of the probe todifferentROS and RNS weredetermined in 5.0 mMTPrA–0.1Mphosphate buffer at pH 7.4. As shown in Fig. 5, the ECLintensity of [Ru(bpy) 2 (dabpy)] 2+ did not respond noticeably toH 2 O 2 , _ OH, ClO  ,  1 O 2 , O 2  , NO 2  , NO 3  and ONOO  , whereasa significant ECL intensity enhancement was observed after thecomplex was reacted with NO. The above results indicate that[Ru(bpy) 2 (dabpy)] 2+ is a highly specific signal-on ECL probe forNO. In the reported ECL quenching method, 15 the authors onlyinvestigated the effect of NO 2  on the ECL detection of NO, and Fig. 4  (A) Effect of pH on the ECL intensities of [Ru(bpy) 2 (dabpy)]  2+ (10 m M, - ) and [Ru(bpy) 2 (T-bpy)]  2+ (10 m M, B ) in 1.0 mM TPrA–0.1 Mphosphate buffers with different pH values. (B) Effect of the TPrAconcentration on the ECL intensities of [Ru(bpy) 2 (dabpy)] 2+ (10 m M, - )and [Ru(bpy) 2 (T-bpy)] 2+ (10  m M, B ) in 0.1 M phosphate buffer of pH7.4. (C) ECL intensity responses of [Ru(bpy) 2 (dabpy)] 2+ (10  m M) todifferent concentrations of NO in 5.0 mM TPrA–0.1 M phosphate bufferat pH 7.4 (the inset shows the calibration curve for the ECL detection of NO). 1870 |  Analyst  , 2011,  136 , 1867–1872 This journal is  ª  The Royal Society of Chemistry 2011  found that the ECL quenching of NO 2  was   5% compared tothat of NO. In the present method, since the ECL responses of our probe to NO 2  and other ROS/RNS are far weaker than thatto NO, it can be concluded that our ECL enhancement method ismore favorable to be used for the ECL detection of NO incomplicated biological samples. 3.5 ECL detection of the NO generation in living plant cells To investigate the availability of our method for the ECLdetection of NO in biological systems, the generation of endog-enous NO in the cultured  Gardenia  cells was detected by using[Ru(bpy) 2 (dabpy)] 2+ as an intracellular ECL probe since it couldbe easily transferred into the living plant cells by the co-incu-bation method. 39 After the  Gardenia  cells were co-incubated withthe probe, the ECL intensities of the cells were monitored atdifferent incubation times. As shown in Fig. 6, the ECL inten-sities of the cells were gradually increased with the increase of incubation time, which indicates that the NO generation in thecells is a continuous process. The longer the cells are incubated,the more the NO molecules are generated. This result is in goodagreement with the reported results obtained by using the lumi-nescent probes, 39,48 demonstrating that [Ru(bpy) 2 (dabpy)] 2+ isa practically useful ECL probe for the detection of NO in bio-logical samples. 4. Conclusions In summary, we described here a selective and sensitive ECLmethod for the detection of NO in aqueous and biologicalsamples using a modified bipyridine-Ru( II ) complex, [Ru(bpy) 2 (dabpy)] 2+ , as a probe. Compared to the reported ECLquenching method, the new ECL enhancement method has theadvantages of higher selectivity and sensitivity, and wider linearrange, which allows it to be favorably useful for the ECLdetection of NO in complicated biological samples. Additionally,the successful development of this method suggests that thebipyridine-Ru( II ) complexes could be widely used for the rationaldesign of ECL probes for various analytes by appropriate ligandmodifications, which could be a very useful strategy for thedevelopment of the ECL detection technique. Acknowledgements The authors acknowledge financial supports from the NationalNatural Science Foundation of China (Nos. 20835001,20975017, 20923006, 51072074), and the Specialized ResearchFund for the Doctoral Program of Higher Education of China(Nos. 200801410003, 20090041120018). References 1 D. S. Bredt and S. H. Snyder,  Annu. Rev. Biochem. , 1994,  63 , 175.2 F. Murad,  Angew. Chem., Int. Ed. , 1999,  38 , 1857.3 T. Nagano and T. Yoshimura,  Chem. Rev. , 2002,  102 , 1235.4 F. L. M. Fabio Ricciardolo, P. J. Sterk, B. Gaston and G. Folkerts, Physiol. Rev. , 2004,  84 , 731.5 H. Kojima, N. Nakatsubo, K. Kikuchi, S. Kawahara, Y. Kirino,H. Nagoshi, Y. Hirata and T. Nagano,  Anal. Chem. , 1998,  70 , 2446.6 Y. Gabe, Y. Urano, K. Kikuchi, H. Kojima and T. Nagano,  J. Am.Chem. Soc. , 2004,  126 , 3357.7 E. Sasaki, H. Kojima, H. Nishimatsu, Y. Urano, K. Kikuchi,Y. Hirata and T. Nagano,  J. Am. Chem. Soc. , 2005,  127 , 3684.8 M. H. Lim, D. Xu and S. J. Lippard,  Nat. Chem. Biol. , 2006,  2 , 375.9 M. H. Lim, B. A. Wong, W. H. Pitcock, Jr., D. Mokshagundam,M. H. Baik and S. J. Lippard,  J. Am. Chem. Soc. , 2006,  128 , 14364.10 J. H. Kim, D. A. Heller, P. W. Barone, C. Song, J. Zhang,L. J. Trudel, G. N. Wogan, S. R. Tannenbaum and M. S. Strano, Nat. Chem. , 2009,  1 , 473.11 J. K. Robinson, M. J. Bollinger and J. W. Birks,  Anal. Chem. , 1999, 71 , 5131.12 K. Sancier, G. Freeman and I. Mills,  Science , 1962,  137 , 752.13 H. Kosaka, M. Watanabe, H. Yoshihara, N. Harada and T. Shiga, Biochem. Biophys. Res. Commun. , 1992,  184 , 1119.14 Y. Katayama, N. Soh and M. Maeda,  ChemPhysChem , 2001,  2 , 655.15 J. Chen, M. Miyake, Y. Chi, T. Nishiumi and K. Aoki, Electroanalysis , 2007,  19 , 181.16 W. Miao,  Chem. Rev. , 2008,  108 , 2506.17 M. M. Richter,  Chem. Rev. , 2004,  104 , 3003.18 N. Tokel and A. J. Bard,  J. Am. Chem. Soc. , 1972,  94 , 2862.19 G. F. Blackburn, H. P. Shah, J. H. Kenten, J. Leland, R. A. Kamin,J. Link, J. Peterman, M. J. Powell, A. Shah, D. B. Talley, S. K. Tyagi,E. Wilkins, T.-G. Wu and R. J. Massey,  Clin. Chem. , 1991,  37 , 1534.20 N. Gassler, T. Peuschel and R. Pankau,  Clin. Lab. , 2000,  46 , 553.21 J. DiCesare, B. Grossman, E. Katz, E. Picozza, R. Ragusa andT. Woudenberg,  BioTechniques , 1993,  15 , 152.22 M. Ohlin, M. Silvestri, V. Sundqvist and C. A. K. Borrebaeck,  Clin.Diag. Lab. Immun. , 1997,  4 , 107. Fig. 5  ECL intensities of [Ru(bpy) 2 (dabpy)] 2+ (10 m M) in the presence of different ROS and RNS in 5.0 mM TPrA–0.1 M phosphate buffer at pH7.4. NO: 0.1 mM; H 2 O 2 : 0.1 mM; _ OH: 0.1 mM H 2 O 2  + 0.1 mM ferrousammonium sulfate;  1 O 2 : 0.1 mM H 2 O 2  + 0.1 mM NaOCl; ClO  : 0.1 mMNaOCl; O 2  : 0.1 mM KO 2 ; NO 2  : 0.1 mM NaNO 2 ; NO 3  : 0.1 mMNaNO 3 ; ONOO  : 0.1 mM NaONOO. Fig. 6  ECL intensity changes of the Gardenia cells co-incubated with[Ru(bpy) 2 (dabpy)] 2+ at different incubation times in 5.0 mM TPrA–0.1 Mphosphate buffer at pH 7.4. This journal is  ª  The Royal Society of Chemistry 2011  Analyst  , 2011,  136 , 1867–1872 | 1871
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