对比三种纳米金的催化活性

DOI:10.1002/cphc.201100906Comparison of the Peroxidase-Like Activity of Unmodified, Amino-Modified,and Citrate-Capped Gold Nanoparticles Sheng Wang,Wei Chen,*Ai-Lin Liu,Lei Hong,Hao-Hua Deng,and Xin-Hua Lin*[a]1.IntroductionRecently,increasing attention has been paid to nanoscale per-oxidase mimetics and their potential applications,since the discovery that iron oxide magnetic nanoparticles(NPs)have in-trinsic enzyme-mimetic activity similar to that of natural peroxi-dases.[1]FeS nanosheets,[2]graphene oxide,[3]bimetallic alloy nanoparticles,[4]V2O5nanowires,[5]single-walled carbon nano-tubes,[6]cubic Pt nanocrystals,[7]Au@Pt nanostructures,[8]and cupric oxide nanoparticles[9]have been shown to catalyze the oxidation of peroxidase substrates by hydrogen peroxide.This attractive property has made these enzyme nanomimetics can-didates for applications in bioanalysis[10–18]and environmental chemistry.[19–23]Gold,which is one of the most ancient subjects of investiga-tion in science,is now attracting renewed attention as an in-dispensable part of nanoscience and nanotechnology because of its appealing features.The unique electronic and optical properties of gold nanoparticles have made them model sys-tems for exploring a wide range of phenomena such as self-as-sembly,[24]near-field plasmon coupling,[25]crystal growth,[26] and electron-transfer theories.[27]In addition,gold nanoparti-cles offer good biocompatibility,facile synthesis,and conjuga-tion to a variety of biomolecular ligands,antibodies,and other targeting moieties,making them suitable for use in biosens-ing,[28,29]biological imaging,[30]medical diagnostics,[31,32]and therapeutic applications.[33]Although Au0is traditionally considered to be catalytically inert,catalytic activity in the form of nanoparticles on metal oxide supports has been clearly established recently.[34]Many attempts have been made to utilize gold nanoparticles sup-ported on various matrices as catalysts in reactions such as low-temperature CO oxidation,[35]selective oxidation of ole-fins,[36,37]and oxidative dehydrogenation of alcohols.[38]Howev-er,there are few reports on the catalytic activity and related applications of unsupported gold nanoparticles.The most suc-cessful application of unsupported gold nanoparticles as cata-lysts in the liquid phase may be a series of chemiluminescence reactions.[39–41]Recently,Jv et al.made the surprising discovery that cysteamine-modified positively charged gold nanoparti-cles and citrate-capped negatively charged gold nanoparticles have different peroxidase-like activities.[42]They considered the surface charge properties of gold nanoparticles to be the main factor which affects their catalytic activity.It is well known that, in heterogeneous catalysis,chemical bonds of the reacting molecules adsorbed on the catalytically active solid surface are broken and formed,and eventually the products are released back into the liquid or gas phase.Therefore,occupation of the gold surface by cysteamine or citrate should influence the cat-alytic activity of gold nanoparticles.To elucidate the origin of the peroxidase-like activity,unmodified gold nanoparticles were fabricated and investigated in this pared to cysteamine-or citrate-modified gold nanoparticles,unmodified gold nanoparticles show significantly high catalytic activity toward peroxidase substrates.Furthermore,some influencing factors,such as fabrication process,redox property of the modifier,and charge property of the substrate,were studied.2.Results and DiscussionAs shown in Figure1,HAuCl4can directly oxidize3,3’,5,5’-tetra-methylbenzidine(TMB)to form a blue product,which is the same as that produced in the catalytic oxidation of TMB byhy-[a]S.Wang,Prof.Dr.W.Chen,Prof.Dr.A.-L.Liu,L.Hong,H.-H.Deng,Prof.X.-H.LinDepartment of Pharmaceutical AnalysisFujian Medical UniversityFuzhou350004(China)E-mail:chenandhu@xhl1963@Supporting information for this article is available on the WWW under /10.1002/cphc.201100906.drogen peroxide in the presence of peroxidase or gold nano-particles.Therefore,the fabrication processes of gold nanopar-ticles should be checked carefully to avoid the influence of re-maining HAuCl 4in investigations on peroxidase-like activity.The unmodified and citrate-capped gold nanoparticles were formed rapidly in the presence of excess reductant,either sodium borohydride or trisodium citrate.[43]No direct oxidation effect of HAuCl 4can be observed after 1h in these two reac-tion systems,that is,depletion of HAuCl 4in the reaction solu-tions is rapid.Gold nanoparticles formed by the reduction of HAuCl 4by sodium borohydride and trisodium citrate exhibited a red color with absorption at 516and 518nm,respectively (Figure 2).This feature is commonly known as the surface plas-mon resonance (SPR)peak and is dependent on particle size.In the fabrication of amino-modified gold nanoparticles,a small amount of sodium borohydride was used to initiate the reaction.[44]The formation of gold nanoparticles can be di-vided into two stages.In the first stage,the solution was opaque and brownish soon after the addition of NaBH 4.Ac-cording to the reaction stoichiometry,a large amount of HAuCl 4should remain after depletion of sodium borohydride (over 10min).In the second stage,further consumption of HAuCl 4could be observed in the presence of 2-aminoethane-thiol at a relatively slow rate.The brownish solution slowly changed to clear wine red in this period.As shown in Figure 3,an absorption peak was observed at 530nm within the first hour.This peak shifted slightly to a shorter wavelength of 523nm and intensified with time until a maximum was ach-ieved after 6h.At the same time,the concentration of HAuCl 4in the reaction system decreased.As shown in Figure 4,the direct oxidation effect of HAuCl 4can be neglected after 6h,which matches well with the result mentioned above.This fabrication process of amino-modified gold nanoparti-cles in the presence of 2-aminoethanethiol was proposed by Niidome et al.,and then applied for DNA transfection and col-orimetric determination of glucose.[42,44]However,a detailed mechanism was not reported.According to the phenomena observed in the fabrication process,2-aminoethanethiol should be not only a modifier but also a reductant in the formation of gold nanoparticles.However,gold nanoparticles can not form without initiation by NaBH 4.No SPR peak of gold nanoparticles can be observed even after 7h for a mixture of 2-aminoetha-nethiol and HAuCl 4.A reasonable explanation is that the ad-sorption of 2-aminoethanethiol on gold nanoparticles can result in formation of Au ÀS bonds and produce free hydrogen atoms,which should be reductive.[45]To verify this explanation,a small amount of unmodified gold nanoparticles was then added to the reaction solution,and formation of gold nano-particles could be initiated in this case.As shown in Figure 5,a clear SPR peak of gold nanoparticles then can be observed.The average sizes of unmodified,amino-modified,andcitrate-Figure 1.Typical absorption curves of TMB a)before and b)after oxidation by HAuCl 4.The concentrations of TMB and HAuCl 4were 1.0m m and21.2m m ,respectively.Inset:TMB solution before (left)and after (right)oxida-tion by HAuCl 4.Figure 2.UV/Vis absorption spectra of a)unmodified and b)citrate-capped goldnanoparticles.Figure 3.UV/Vis absorption spectra of amino-modified gold nanoparticles in the fabrication process:a)20min,b)1h,c)2h,d)3h,and e)6h.Figure 4.Time evolution of the direct oxidizing effect of HAuCl 4during the synthesis of amino-modified gold nanoparticles.W.Chen,X.-H.Lin et al.capped gold nanoparticles were determined by TEM imaging to be 10,30,and 13nm,respectively (Figure 6).The peroxidase-like behavior of the synthesized gold nano-particles was examined by using 3,3’,5,5’-tetramethylbenzidine (TMB)and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS)as chromogenic substrates.ABTS and TMB were chosen because they have opposite charge charac-teristics.As a commonly used peroxidase substrate,TMB con-tains two amino groups,which likely result in stronger affinity to a negatively charged nanoparticle surface.[46]TMB can be oxidized by peroxidase in the presence of H 2O 2,and a bluecharge-transfer complex (chromogen)is quickly formed and shows a maximum absorbance at 652nm (Scheme 1A).[47]Figure 7shows that all gold nanoparticles display catalytic ac-tivity toward oxidation of TMB in the presence of hydrogen peroxide,yet the peroxidase-like activity varies with thetypeof surfacemodification.Cationic amino-modified gold nano-particles show weak catalytic activity toward TMB due to theirlow affinity toward the positively charged substrate.In con-trast,citrate-capped gold nanoparticles,which are negatively charged,tend to attract amino groups of TMB electrostatically.These anionic nanoparticles will exhibit strong affinity withTMB as reaction substrate.Experimental data are in strong sup-port of this claim.Citrate-capped gold nanoparticles exhibit higher catalytic activity than amino-modified gold nanoparti-cles.Since the related standard derivation of each detection is less than 3%,this result is exact opposite to that of Jv et al.[42]different results may be ascribed to the concentration of peroxide used in the experiments.For citrate-gold nanoparticles,the intrinsic redox property of cit-rate,which has a negative effect on the peroxidase-like activityof gold nanoparticles,is an important factor that should betaken into account.The reduction activity of citrate could be observed when HAuCl 4was added to the citrate-capped gold nanoparticles.The SPR peak of the gold nanoparticles intensi-fied and shifted to longer wavelength (Figure 8).In addition,the peroxidase-like activity of the gold nanoparticles dimin-ished in the presence of excesscitrate (Figure 9).Therefore,the fabrication process of citrate-capped gold nanoparticles should be carefully controlled to duplicate the catalytic activity of the products.Although electrostatic affinity exerted by superficial charge could enhance catalytic activity,the physical presence of a coat-ing could also shield active do-mains on the surface of particles,weakening catalytic activity.[46]This effect can be observed when comparing the unmodified Figure 5.UV/Vis absorption spectra of the solution of 2-aminoethanethiol and HAuCl 4a)0and b)7h after adding unmodified gold nanoparticles.Figure 6.TEM images of unmodified (left),amino-modified (middle),and cit-rate-capped (right)gold nanoparticles.Scheme 1.Gold nanoparticle catalyzed reaction of A)TMB and B)ABTS with hydrogen peroxide.Figure parison of the peroxidase-like activity of a)amino-modified,b)citrate-capped,and c)unmodified gold nanoparticles toward TMB as per-oxidase substrate.The concentration of TMB was 1m m and the H 2O 2con-centration was varied.Inset (left to right):images of the catalysis product in the presence of amino-modified,citrate-capped,and unmodified gold nano-particles.The concentration of TMB was 1m m and the H 2O 2concentration was 750m m .Peroxidase-Like Activity of Gold Nanoparticlesand modified particles.In support of our claim,the unmodified gold nanoparticles exhibit much higher catalytic activity than both citrate-capped and amino-modified gold nanoparticles (Figure 7).This indicates that the superficial gold atoms are a contributing factor to observed peroxidase-like activity.To further confirm this,ABTS was chosen as another peroxi-dase substrate for activity comparison.In contrast to TMB,ABTS,which has two sulfo groups per molecule,is a negatively charged chromogenic substrate.ABTS can also be catalytically oxidized by H 2O 2,and an oxidized colored product is formed with an absorbance maximum at 420nm (Scheme 1B).The catalytic activities of the three types of gold nanoparticles are compared in Figure 10.The highest catalytic activity is again observed for unmodified gold pared to the TMB system,the amino-modified gold nanoparticles show higher catalytic activity than citrate-capped gold nanoparticles in the ABTS system,that is,charge characteristics of the nano-particles and the substrate also play an important role in the catalytic reactions.When mercaptoacetic acid modified gold nanoparticles are used instead of citrate-capped gold nanoparticles,similar re-sults can be obtained (see the Supporting Information).The catalytic activity of mercaptoacetic acid modified gold nano-particles is lower than that of unmodified gold nanoparticles but higher than that of amino-modified gold nanoparticles when TMB is used as substrate.However,the catalytic activity of mercaptoacetic acid modified gold nanoparticles is lowerthan that of amino-modified gold nanoparticles when ABTS is used as substrate.3.ConclusionsUnmodified gold nanoparticles have been shown to have in-trinsic peroxidase-like pared to amino-modified or citrate-capped gold nanoparticles,unmodified gold nanoparti-cles show significantly higher catalytic activity toward both TMB and ABTS,that is,the superficial gold atoms are a contri-buting factor to the observed peroxidase-like activity.Due to the absence of surface modifiers,unmodified gold nanoparti-cles could be conveniently labeled by biological molecules such as antigens,antibodies,and oligonucleotides.The differ-ent catalytic activities of amino-modified and citrate-capped gold nanoparticles toward TMB and ABTS show that the charge characteristics of the nanoparticles and the substrate also play an important role in the catalytic reactions.The influ-ence of superficial modifiers on the number of active gold atoms and the superficial charge is worthy of further studies to control the peroxidase-like activity of gold nanoparticles for different applications.Experimental SectionChemicals and Reagents:HAuCl 4·3H 2O,30%H 2O 2,and sodium cit-rate were purchased from Sinopharm Chemical Reagent Co.Ltd.3,3’,5,5’-Tetramethylbenzidine (TMB),sodium borohydride,2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS),and 2-aminoethanethiol were purchased from Aladdin Re-agent Company (Shanghai).Other reagents and chemicals were of at least analytical reagent grade.Double-distilled water was used throughout experiments.Instrumentation:The UV/Vis spectra of gold nanoparticles were re-corded on a Shimadzu UV/2450spectrophotometer (Shimadzu,Japan).The TEM images were recorded on a FEI Tecnai-12trans-mission electron microscope with an accelerating voltage of 120kV.Figure 8.UV/Vis absorption spectra of citrate-capped gold nanoparticles a)0and b)7h after adding HAuCl 4.Figure 9.Influence of citrate ions on the peroxidase-like activity of unmodi-fied gold nanoparticles.Concentration of sodium citrate:a)0,b)0.5,c)0.75,d)1.25m m.Figure parison of the peroxidase-like activity of a)citrate-capped,b)amino-modified,and c)unmodified gold nanoparticles toward ABTS as peroxidase substrate.The concentration of TMB was 6m m and the H 2O 2concentration was varied.Inset (left to right):Solutions of the catalysis prod-uct in the presence of citrate-capped,amino-modified,and unmodified gold nanoparticles.The concentration of ABTS was 6m m and the H 2O 2concentra-tion was 2m .W.Chen,X.-H.Lin et al.Preparation of Gold Nanoparticles:Unmodified gold nanoparticles were synthesized by reduction of HAuCl4with NaBH4as sole reduc-ing agent.Briefly,HAuCl4solution(500m L,1%)was diluted with water(39.5mL),and NaBH4solution(1mL,1%)was injected under vigorous stirring(the injection time was about5min).An immedi-ate color change of the solution from pale yellow to a wine red was observed,indicating formation of gold nanoparticles.The un-modified gold nanoparticles can remain stable against aggregation in aqueous solution for at least two months.When stored in a re-frigerator at48C for two months,no change in catalytic activity could be found.Amino-modified gold nanoparticles were synthesized in aqueous solution by following a previous publication with some modifica-tions.[44]Briefly,HAuCl4solution(500m L,1%)was diluted with water(39.5mL),and2-aminoethanethiol solution(85m L,213m m) was added to the gold solution.After stirring for20min at room temperature,NaBH4solution(2.1m L,10m m)was added,and the mixture was stirred for more than10h in the dark.The final solu-tion was stored at48C in refrigerator.Citrate-capped gold nanoparticles were prepared according to pre-viously published protocols.[48]Briefly,HAuCl4solution(1mL,1%) was dissolved in water(100mL)and boiled.Trisodium citrate solu-tion(3mL,1%)was quickly added to the refluxing HAuCl4solution, and the resulting color change from pale yellow to deep red indi-cated formation of gold nanoparticles.After a continuously reflux-ing for an additional15min,the solution was slowly cooled to room temperature.The final solution was stored at48C in a refrig-erator.Influence of Citrate Ions on the Catalytic Activity of Gold Nanopar-ticles:A TMB solution was used to detect the influence of citric acid ions on the catalytic activity of gold nanoparticles.Experi-ments were carried out with280m L of unmodified gold nanoparti-cles in a reaction volume of4.0mL with different amounts of citric acid sodium(0.01m,from150to600m L)in the presence of1m m TMB as substrate.H2O2concentration was750m m.The mixed so-lution was incubated in a378C water bath for10min and used for adsorption spectroscopic measurements at652nm.Comparison of Catalytic Activity of Three Types of Gold Nanoparti-cles:Experiments on the TMB–H2O2catalytic reaction were carried out with280m L of unmodified and amino-modified gold nanopar-ticles or315m L of citrate-capped gold nanoparticles in a reaction volume of4.0mL in the presence of varying concentrations of H2O2(from25m m to2m).The concentration of TMB was5m m. The mixed solution was incubated in a378C water bath for10min and used for absorption spectroscopic measurements at652nm. The catalytic activity was quantified as the absorbance normalized by the total surface area of the gold nanoparticles.Experiments on the ABTS–H2O2catalytic reaction were carried out with280m L of unmodified and amino-modified gold nanoparticles or315m L of citrate-capped gold nanoparticles in a reaction volume of4.0mL in the presence of varying concentrations of H2O2(from25m m to2m).The concentration of ABTS was6m m. The mixed solution was incubated in a378C water bath for5min and used for absorption spectroscopic measurements at420nm. The catalytic activity was quantified as the absorbance normalized by the total surface area of the gold nanoparticles.AcknowledgementsWe acknowledge the financial support of the National Natural Science Foundation of China(21175023),the Medical Innovation Project of Fujian Province(2009-CX-17),the Natural Science Foundation of Fujian Province(2011J01034),Science and Technol-ogy Planning Project of Fujian Province(2011Y0030),and the Program for New Century Excellent Talents in Fujian Province University(W.C.,JA11102).Keywords:catalytic activity·enzyme mimics·gold·heterogeneous catalysis·nanoparticles[1]L.Gao,J.Zhuang,L.Nie,J.Zhang,Y.Zhang,N.Gu,T.Wang,J.Feng,D.Yang,S.Perrett,X.Yan,Nat.Nanotechnol.2007,2,577–583.[2]Z.Dai,S.Liu,J.Bao,H.Ju,Chem.Eur.J.2009,15,4321–4326.[3]Y.Song,K.Qu,C.Zhao,J.Ren,X.Qu,Adv.Mater.2010,22,2206–2210.[4]W.He,X.Wu,J.Liu,X.Hu,K.Zhang,S.Hou,W.Zhou,S.Xie,Chem.Mater.2010,22,2988–2994.[5]R.AndrØ,F.Natµlio,M.Humanes,J.Leppin,K.Heinze,R.Wever,H.C.Schrçder,W.E.G.Müller,W.Tremel,Adv.Funct.Mater.2011,21,501–509.[6]Y.Song,X.Wang,C.Zhao,K.Qu,J.Ren,X.Qu,Chem.Eur.J.2010,16,3617–3621.[7]M.Ma,Y.Zhang,N.Gu,Colloids Surf.A2011,373,6–10.[8]W.W.He,Y.Liu,J.S.Y,J.J.Yin,X.C.Wu,X.N.Hu,K.Zhang,J.B.Liu,C.Y.Chen,Y.L.Ji,Y.T.Guo,Biomaterials2011,32,1139–1147.[9]W.Chen,J.Chen,A.L.Liu,L.M.Wang,G.W.Li,X.H.Lin,ChemCatChem2011,3,1151–1154.[10]H.Wei,E.Wang,Anal.Chem.2008,80,2250–2254.[11]X.Q.Zhang,S.W.Gong,Y.Zhang,T.Yang,C.Y.Wang,N.Gu,J.Mater.Chem.2010,20,5110–5116.[12]W.Shi,Q.Wang,Y.Long,Z.Cheng,S.Chen,H.Zheng,Y.Huang,Chem.Commun.2011,47,6695–6697.[13]F.Qu,T.Li,M.Yang,Biosens.Bioelectron.2011,26,3927–3931.[14]N.Ding,N.Yan,C.Ren,X.Chen,Anal.Chem.2010,82,5897–5899.[15]L.Gao,J.Wu,S.Lyle,K.Zehr,L.Cao,D.Gao,J.Phys.Chem.C2008,112,17357–17361.[16]R.Cui,Z.Han,J.J.Zhu,Chem.Eur.J.2011,17,9377–9384.[17]J.Li,J.D.Qiu,J.J.Xu,H.Y.Chen,X.H.Xia,Adv.Funct.Mater.2007,17,1574–1580.[18]Y.Ding,G.Gu,X.H.Xia,J.Solid State Electrochem.2008,12,553–558.[19]X.Zuo,C.Peng,Q.Huang,S.Song,L.Wang,D.Li,C.Fan,Nano Res.2009,2,617–623.[20]W.Luo,L.Zhu,N.Wang,H.Tang,M.Cao,Y.She,Environ.Sci.Technol.2010,44,1786–1791.[21]H.Niu,D.Zhang,S.Zhang,X.Zhang,Z.Meng,Y.Cai,J.Hazard.Mater.2011,190,559–565.[22]H.Wang,Y.Huang,J.Hazard.Mater.2011,191,163–169.[23]J.Zhang,J.Zhuang,L.Gao,Chemosphere2008,73,1524–1528.[24]A.N.Shipway,hav,I.Willner,Adv.Mater.2000,12,993–998.[25]Q.H.Wei,K.H.Su,S.Durant,X.Zhang,Nano Lett.2004,4,1067–1071.[26]J.Becker,O.Schubert,C.Sçnnichsen,Nano Lett.2007,7,1664–1669.[27]K.Weber,L.Hockett,S.Creager,J.Phys.Chem.B1997,101,8286–8291.[28]R.Elghanian,J.J.Storhoff,R.C.Mucic,R.L.Letsinger,C.A.Mirkin,Sci-ence1997,277,1078–1081.[29]J.M.Nam,C.S.Thaxton,C.A.Mirkin,Science2003,301,1884–1886.[30]P.K.Jain,K.S.Lee,I.H.El-Sayed,M.A.El-Sayed,J.Phys.Chem.B2006,110,7238–7248.[31]X.Qian,X.H.Peng,D.O.Ansari,Q.Yin-Goen,G.Z.Chen,D.M.Shin,L.Yang,A.N.Young,M.D.Wang,S.Nie,Nat.Biotechnol.2008,26,83–90.[32]I.H.El-Sayed,X.Huang,M.A.El-Sayed,Nano Lett.2005,5,829–834.[33]Y.Cheng,A.C.Samia,J.D.Meyers,I.Panagopoulos,B.Fei,C.Burda,J.Am.Chem.Soc.2008,130,10643–10647.[34]B.K.Min,C.M.Friend,Chem.Rev.2007,107,2709–2724.[35]otti,W.C.Li,B.Spliethoff,F.Schüth,J.Am.Chem.Soc.2006,128,917–924.Peroxidase-Like Activity of Gold Nanoparticles[36]M.Turner,V.B.Golovko,O.P.H.Vaughan,P.Abdulkin,A.Berenguer-Murcia,M.S.Tikhov,B.F.G.Johnson,mbert,Nature2008,454, 981–983.[37]M.D.Hughes,Y.J.Xu,P.Jenkins,P.McMorn,ndon,D.I.Enache,A.F.Carley,G.A.Attard,G.J.Hutchings,F.King,E.H.Stitt,P.Johnston,K.Griffin,C.J.Kiely,Nature2005,437,1132–1135.[38]S.Rousseau,O.Marie,P.Bazin,M.Daturi,S.Verdier,V.Harle,J.Am.Chem.Soc.2010,132,10832–10841.[39]Z.F.Zhang,H.Cui,i,L.J.Liu,Anal.Chem.2005,77,3324–3329.[40]A.Safavi,G.Absalan,F.Bamdad,Anal.Chim.Acta2008,610,243–248.[41]H.Cui,Z.F.Zhang,M.J.Shi,J.Phys.Chem.B2005,109,3099–3103.[42]Y.Jv,B.Li,R.Cao,mun.2010,46,8017–8019.[43]G.Frens,Nat.Phys.Sci.1973,241,20–22.[44]T.Niidome,K.Nakashima,H.Takahashi,Y.Niidome,mun.2004,1978–1979.[45]J.Z.Guo,H.Cui,J.Phys.Chem.C2007,111,12254–12259.[46]F.Yu,Y.Huang,A.J.Cole,V.C.Yang,Biomaterials2009,30,4716–4722.[47]P.D.Josephy,T.E.Eling,R.P.Mason,J.Biol.Chem.1982,257,3669–3675.[48]C.P.Jia,X.Q.Zhong,B.Hua,M.Y.Liu,F.X.Jing,X.H.Lou,S.H.Yao,J.Q.Xiang,Q.H.Jin,J.L.Zhao,Biosens.Bioelectron.2009,24,2836–2841. Received:November11,2012Revised:January19,2012Published online on March2,2012W.Chen,X.-H.Lin et al.。