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Biosensors and Bioelectronics 31 (2012) 84–89Contents lists available at SciVerse ScienceDirectBiosensors andBioelectronicsj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /b i osMolecularly imprinted polymer anchored on the surface of denatured bovine serum albumin modified CdTe quantum dots as fluorescent artificial receptor for recognition of target proteinWei Zhang a ,Xi-Wen He a ,Yang Chen a ,Wen-You Li a ,∗,Yu-Kui Zhang a ,ba Department of Chemistry,Nankai University,94Weijin Road,Tianjin 300071,ChinabNational Chromatographic Research and Analysis Center,Dalian Institute of Chemical Physics,Chinese Academy of Sciences,Dalian 116011,Chinaa r t i c l ei n f oArticle history:Received 5July 2011Received in revised form 14September 2011Accepted 29September 2011Available online 6 October 2011Keywords:Molecular imprinting Quantum dotsDenatured bovine serum albumin Artificial receptorsSelective protein recognitiona b s t r a c tA new type of molecularly imprinted polymer (MIP)-based fluorescent artificial receptor was developed by anchoring MIP on the surface of denatured bovine serum albumin (dBSA)modified CdTe quantum dots (QDs)using the surface molecular imprinting process.The approach combined the merits of molecular imprinting technology and the fluorescent property of the CdTe QDs.The dBSA was used not only to mod-ify the surface defects of the CdTe QDs,but also as assistant monomer to create effective recognition sites.Three different proteins,namely lysozyme (Lyz),cytochrome c (Cyt)and methylated bovine serum albu-min (mBSA),were tested as the template molecules and then the receptors were synthesized by sol–gel reaction (imprinting process).The results of fluorescence and binding experiments demonstrated the recognition performance of the receptors toward the corresponding template.Under optimum condi-tions,the linear range for Lyz was from 1.4×10−8to 8.5×10−6M,and the detection limit was 6.8nM.Moreover,the new artificial receptors were applied to separate and detect Lyz in real samples.This fluorescent artificial receptor may serve as a starting point in the design of highly effective synthetic fluorescent receptor for recognition of target protein.© 2011 Elsevier B.V. All rights reserved.1.IntroductionMolecular imprinting is a technique providing functional poly-mers able to recognize target molecules of interest.And the process of molecular imprinting involves the formation of polymers under the guidance of a molecular template to produce complementary binding sites with specific recognition ability.In contrast to the biological antibody,the substantial advantages of artificial coun-terparts are their mechanical and chemical stability,low cost of preparation and wide range of operating conditions.To date,MIP has been widely used in the fields of chromatographic separation,as antibody mimetics and artificial receptor,and catalysis (Matsui et al.,2007;Kempe and Mosbach,1995;Yang et al.,2004;Ouyang et al.,2007;Tong et al.,2001;Ye and Mosbach,2001;Bossi et al.,2001;Liu et al.,2011;Zeng et al.,2010).The majority of the MIP has been prepared using small molecules as template,much less attention has been paid to the protein recognition (Rusmini et al.,2007;Gale et al.,2009;Klepamik et al.,2011).This was mainly due to the properties of proteins that are large molecular size,flexi-ble structure,and large number of functional groups available for∗Corresponding author.Tel.:+862223494962;fax:+862223502458.E-mail address:wyli@ (W.-Y.Li).recognition,which makes it impossible to use the same approach as imprinting small molecules for protein imprinting.Therefore,to develop highly selective and efficient analytical methods for identi-fying and quantifying proteins by molecular imprinting technology is of great importance.Recently,QDs as sensing and recognizing element have attracted a prominent attention in the past few years when used as fluorescent labels due to their properties,such as good photosta-bility,high luminescence efficiency and size-dependent emission wavelengths (Bruchez et al.,1998;Chan and Nie,1998).Those prop-erties render these materials suitable for various applications,such as chemical sensor for ion (Tang et al.,2005),small molecules (Tu et al.,2008),and biomacromolecules (Chen et al.,2009a,b;Han et al.,2009;Cao et al.,2009).The molecule imprinting technique is a promising way to improve the selectivity of the QDs through the formation of MIP on the surface of the QDs.Therefore,if we combine the high selectivity of molecular imprinting technology and excellent fluorescent char-acteristics of QDs,a new affinity material,with specific recognition cavity and responding to the binding event with significant fluores-cence intensity change,could be prepared.However,considerable interest has been focused on the development of small molecules imprinted polymer coated QDs,producing protein imprinted poly-mer based QDs was still a challenge (Haupt et al.,1998;Carlson0956-5663/$–see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.bios.2011.09.042W.Zhang et al./Biosensors and Bioelectronics31 (2012) 84–8985Fig.1.Preparative procedures for the fabrication of MIP-coated CdTe QDs asfluorescent artificial receptor.et al.,2006;Li et al.,2010;Lin et al.,2004,2009;Liu et al.,2010; Diltemiz et al.,2008;Wang et al.,2009;Zhang et al.,2011).The objective of the work is to develop a new kind offluores-cent affinity material combining the merits of molecular imprinting technology andfluorescent property of the CdTe QDs for specific recognition of target protein.Herein,the dBSA was used not only to modify the surface of the CdTe QDs,but also as assistant monomer to create effective recognition sites(Kuo et al.,2008;Wang et al., 2006;Guo et al.,2006).Three proteins were chosen as the template molecules and then the receptors were synthesized by sol–gel pro-cess(imprinting process,see Fig.1).To the best of our knowledge, no reports to date have been published on the use of dBSA and QDs for protein imprinting.The performance of the receptors was esti-mated byfluorescence emission spectra,UV–vis absorption spectra and SDS-PAGE analysis.To illustrate the utility of MIP-basedfluo-rescent receptor,Lyz was chosen as the template protein.Lyz exists widely in body tissues and secretions.Its abnormal concentration in serum and urine is related to many diseases,such as renal diseases, leukemia and meningitis.Therefore,development of new methods for the analysis of Lyz in real samples is of considerable impor-tance.In this work,the proposedfluorescent artificial receptor was demonstrated as a simple and selective sensing system for selective separating and detecting of Lyz in real samples.2.Materials and methods2.1.Materials and chemicalsAll chemicals were of analytical grade reagents.Tellurium powder(Beilian Fine Chemical Factory,China),CdCl2·2.5H2O, sodium dodecyl sulfate(SDS)and NaBH4(Guangfu Fine Chem-ical Research Institute,China)were used to prepare CdTe QDs.3-Mercaptopropionic acid(MPA,Alfa Aesar Co.)was used as the capping agent.Tetraethoxysilane(TEOS)and3-aminopropyltriethoxysilane(APTES)were Wuhan University Silicone New Materials Co.,Ltd.(Wuhan,China).Bovine serum albumin(BSA,MW(molecular weight)=67kDa,p I(point iso-electric)=4.9),cytochrome c(MW=12.4kDa,p I=10.2),lysozyme (MW=14.4kDa,p I=11),and methylated bovine serum albumin (MW=68kDa,p I=8.5)were obtained from Sigma–Aldrich Co.(St. Louis,MO).2.2.Preparation of dBSA-coated CdTe QDsThe dBSA was prepared by treating BSA with NaBH4on the basis of a previous literature report(Kuo et al.,2008;Chen et al.,2009a,b). NaBH4was added to the BSA solution with stirring,with aim to reduce the disulfide bonds to sulfhydryl groups(–SH).After being stirred for1h at room temperature,the BSA solution was heated to 60–80◦C in water bath for20min to decompose the excess NaBH4. The water-soluble CdTe QDs were synthesized based on previous publication(Li et al.,2008;Zhang et al.,2011).The solution of CdTe QDs was mixed with the dBSA solution in proper molar ratios.The mixture was heated to60–80◦C in a water bath for15min.The solution was then incubated at room temperature for3days and the results were identified byfluorescence scan and SDS-PAGE.2.3.Synthesis offluorescent artificial receptorIn a typical synthesis of the MIP-based receptor,to a25-mL flask,10mg of template protein,10mL of dBSA coated-CdTe QDs and60␮L of APTES(functional precursor)were added and stirred for30min.Then,0.10mL of TEOS(cross-linker)was added.Next, 0.10mL of the NH3·H2O(25%,w/v)was added and stirred for12h. The resultant MIP-coated QDs were centrifuged and washed with 0.5%SDS,which was repeated several times until no template was detected.Finally,the precipitate of MIP-coated QDs was redis-persed in buffer solution and then stored at4◦C prior to use.The NIP was prepared using the same procedure but without addition of the template molecule.2.4.Protein adsorption experimentsA mass of50mg of the particles was dispersed in certain vol-ume of protein solution and the mixtures were agitated in a shaken bed.At different time intervals,the mixtures were centrifuged and the concentration of protein in the supernatant was measured by a UV–vis spectrophotometer at a wavelength of280nm.The amount of adsorbed protein can be determined by the difference in concen-tration before and after the adsorption.The adsorption capacity(Q,expressed in units of mg/g)of the protein or analogue bound to the MIP-coated QDs is calculated by Q=(C0−C t)VWwhere C0and C t(mg/mL)are the initial concentration and the resid-ual concentration of the protein or analogue,respectively,V(mL) is the volume of the initial solution,and W(g)is the weight of the MIP-coated QDs.2.5.CharacterizationFluorescence measurements were performed on an F-4500 spectrofluorometer(Hitachi,Japan)equipped with a quartz cell (1cm×1cm),the slit widths of the excitation and emission were both5nm,and the excitation wavelength was set at470nm with a recording emission range of490–700nm.The photomultiplier tube voltage was set at950V.UV–vis spectra(200–800nm)were recorded on a UV-2450spectrophotometer(Shimadzu,Japan). Transmission electron microscopy(TEM)was obtained by a Tec-nai G220S-TWIN microscope(Philips,Holland).Fourier transform infrared(FT-IR)spectra(4000–400cm−1)in KBr were recorded using the AVATRA360FT-IR spectrophotometer(Nicolet,Waltham,86W.Zhang et al./Biosensors and Bioelectronics 31 (2012) 84–89Fig.2.Effects of the concentration of the monomer APTES (a)and the cross-linker TEOS (b)on the fluorescence intensity of MIP-coated QDs and the fluorescence change of MIP-coated QDs with template protein.USA).X-ray photoelectron spectroscopy (XPS)measurements were performed with an ESCALAB 250spectrometer (Thermo-VG Scien-tific)with ultra-high vacuum generators.2.6.Analysis of real samplesIn the sample analysis,chicken egg white was separated from a fresh egg and diluted to 50%(v/v)with Tris–HCl buffer (50mM,pH 7.0).After the diluted solution was immersed in an ice bath and centrifuged at 10,000rpm for 30min,the supernatant solu-tion was used as a Lyz source (Qin et al.,2009).The imprinted nanoparticles were applied to purify the Lyz from a 20-fold diluted real sample of egg white at room temperature.The imprinted nanoparticles were then treated with washing procedure to elute the specifically adsorbed protein.The eluate was desalted and concentrated 10-fold,using an ultrafiltration membrane (molec-ular weight cutoff =3000),and 10␮L of each sample was used for SDS-PAGE analysis,using 12.0%polyacrylamide gel (Mini-protean,Bio-Rad,Hercules,CA).3.Results and discussion3.1.Preparation and characterization of MIP-and NIP-coated CdTe QDsThe MIP-based dBSA modified CdTe QDs were prepared via a surface molecular imprinting process similar to a previously reported approach (Wang et al.,2009;Zhang et al.,2011).The 3-aminopropyltriethoxysilane (APTES)was used as a functional monomer which had a noncovalent interaction with the tem-plate protein.The concentrations of the reactants were reduced to obtain a thin MIP layer and to minimize the homogeneous self-condensation of tetraethoxysilane (TEOS)and APTES.To determine the most favorable conditions for synthesizing the receptor,the amounts of the functional monomer APTES and cross-linker TEOS were investigated (Fig.2).It can be seen from Fig.2that the flu-orescence intensity was gradually decreased with increasing the amount of APTES,which may be due to the native structure of the QDs was destroyed by the APTES through forming the silica shell on the surface of the QDs.Considering the effect of the APTES on the fluorescence intensity of the MIP-coated QDs (curve 1of Fig.2a)and fluorescence change of the receptor with template protein (curve 2of Fig.2a),40␮L of the monomer was chosen for the synthesis of the MIP-based receptor.According to the same consideration,60␮L of the cross-linker was selected for producing the receptor (Fig.2b).Moreover,a method to functionalize the QDs by coating with dBSA and the influence of the dBSA on the enhancement of flu-orescence intensity for the QDs were investigated (Fig.S1).In orderto obtain dBSA,the BSA was denatured by NaBH 4.By this reaction,the disulfide bonds of BSA were opened,which made it possible for the stabilizing agent MPA on the surface of CdTe QDs to be substi-tuted by the thiol-group of dBSA through ligand exchange.Because of the high degree of surface defects of the CdTe QDs,dBSA was used to modify the surface via ligand exchange to increase the stability of CdTe QDs (Kuo et al.,2008).And utilizing the dBSA to modify the surface of CdTe QDs could not only improve the chemical stability and photoluminescence quantum yield of the QDs effectively,but also serve as assistant recognition peptide chains as an additional element of monomer to create effective recognition sites.After synthesis of the receptor,the fluorescence uniformity of the receptor or the control particles (non-imprinted polymer (NIP)-coated QDs)was investigated (Fig.S2).It can be seen from Fig.S2that a linear fluorescence intensity (MIP-and NIP-coated QDs)–concentration relationships were attained with a correlation of 0.996and 0.998,respectively.The results shown in Fig.S2indi-cated the stable emission of the MIP-and NIP-coated QDs.And the main reason for the stable emission was that the particles were protected by the silica shell layer (Liu et al.,2010).3.2.Characterization of the nanoparticles3.2.1.Characterization of IRFT-IR spectra of QDs,MIP-coated QDs and NIP-coated QDs are compared in Fig.S3.The strong and broad peak around 1063cm −1indicates the Si–O–Si asymmetric stretching.Other observed bands about 797cm −1showed the Si–O vibration.The presence of the bands around 2966cm −1was the C–H stretching band.The bands at 3426cm −1and 1542cm −1were the N–H stretch,suggesting the copolymer was successfully modified onto the surface of the QDs.All those bands showed that the MIP layer generated from sol–gel condensation was grafted on the surface of the QDs.In addition,the NIP-and MIP-QDs showed similar locations and appearance of the major bands,which was because the main compositions of MIP and NIP were similar.3.2.2.XPS characterization of the MIP-and NIP-coated QDsXPS is a state-of-the-art technique that is well-known as a quantification tool for determining surface elemental compositions (Fig.S4).As shown in Fig.S4,the XPS survey showed intense sig-nals of Si 2p at 103eV,C 1s at 285eV,N 1s at 398eV and O 1s at 531eV,which indicated that the MIP layer generated from sol–gel condensation of APTES and TEOS was copolymerized and anchored onto the surface of the QDs.W.Zhang et al./Biosensors and Bioelectronics31 (2012) 84–8987Fig.3.Fluorescence emission spectra of(a)MIP-and(c)NIP-coated QDs with addition of the indicated concentration of protein Lyz solution.Stern–Volmer plots from(b) MIP-and(d)NIP-coated QDs with protein Lyz.3.2.3.TEM characterization of MIP-and NIP-coated QDsTo characterize the morphology of the nanoparticles,the TEM images of MIP-and NIP-coated QDs were investigated(Fig.S5).As shown in Fig.S5,it can be seen that the particles were nearly uni-form in size,and the mean diameter of the MIP-coated QDs was not distinctly different from that of the NIP-coated QDs.Therefore, the difference of recognition performance between the MIP-coated QDs and NIP-coated QDs in the subsequent study could not be attributed to the morphological difference of the MIP-coated QDs and NIP-coated QDs,but to the imprinting effect.3.3.Optosensing of template protein by MIP-coated QDs3.3.1.Effect of pHThe recognition ability of the receptor was investigated through the changes of thefluorescence signal.The effect of pH on the fluorescence changes of receptor is given in Table S1.As can be seen from Table S1,the change offluorescence intensity for the MIP-coated QDs was larger than that of NIP-coated QDs.The best imprinting effect was observed at pH6.2with an imprinting fac-tor IF1of2.33(Table S1),so a pH of6.2was selected for further experiments.3.3.2.MIP-and NIP-coated QDs with template protein of different concentrationsCompared the response of MIP-coated QDs or control par-ticles with template protein,the specific recognition ability was evaluated(Fig.3).It can be seen from Fig.3that the fluorescence intensity of the MIP-coated QDs was quenched grad-ually with the increasing concentration of template Lyz.Generally, thefluorescence quenching depends on the adsorptive affinity of the particles with the template.In the case of the MIP-coated QDs,thefluorescence quenching was mainly achieved by the affinity of the imprinted cavities with the template due to the specific inter-actions.The imprinting factor(IF),which is the ratio of the K SV,MIP and K SV,NIP(K SV,MIP and K SV,NIP was the linear slopes of Fig.3b and d,respectively),was used to evaluate the selectivity of the mate-rials.Under optimum conditions,the IF(K SV,MIP/K SV,NIP)was2.74, which indicated that the receptor can greatly enhance the quench-ing efficiency offluorescence,enlarging the spectral sensitivity of the MIP-coated QDs to the template protein.Fig.3b and d is plotted by the Stern–Volmer equation analysis for the MIP-and NIP-coated QDs with protein,respectively.F0F=1+K SV[Q]F0and F were thefluorescence intensity of QDs in the absence and presence of template,respectively,K SV was the Stern–Volmer constant,and[Q]was the quencher concentration.In order to show the generality of the imprinting method,the Cyt or mBSA was chosen as the template protein producing Cyt-or mBSA-receptors,respectively.And those results demonstrated the specific recognition ability of the receptors(Figs.S6and S7).3.4.Affinity adsorption experimentThe binding performance of the receptor was investigated through affinity adsorption experiment(Fig.4).Fig.4a shows the UV–vis spectra of the protein solution before and after the adsorption by MIP-or NIP-coated QDs,respectively.And the great difference between MIP-and NIP-coated demonstrates a good recognition ability of the receptor.Fig.4b compares the pro-tein adsorption amounts onto the MIP-or NIP-coated QDs,which directly exhibited the recognition ability of the receptor.88W.Zhang et al./Biosensors and Bioelectronics 31 (2012) 84–89Fig.4.The UV–vis spectra of the protein solution before (curve 1)and after the adsorption by MIP-(curve 3)or NIP (curve 2)-coated QDs,respectively (a)and the adsorption capacity of MIP-and NIP-coated QDs (b).Fig.S8shows the binding performance of the receptor with dif-ferent concentrations of the target protein.A constant amount of MIP-or NIP-coated QDs was incubated with increasing concen-trations of template and allowed to equilibrate for 12h at room temperature.After equilibration,the amount of protein remain-ing in the supernatant was measured by UV–vis analysis.In all of the concentrations studied,the MIP-coated QDs exhibited higher adsorption capacity to Lyz than the NIP-coated QDs.In the lower concentration of Lyz,the amount of Lyz was not enough to saturate the specific binding cavities.However,with the Lyz concentration increasing,almost all the specific imprinted sites were occupied by the Lyz and the adsorption capacity of the receptor was the highest.3.5.Cross-reactivity of the MIP-coated QDsThe specific recognition ability was further investigated through the cross-reactivity test of the receptors (Table 1).And the Cyt-and mBSA-receptors were synthesized in the same way with that of the Lyz receptor.To investigate the recognition ability of the Lyz-,Cyt-and mBSA-receptors,the aforementioned three types of imprinted particles were dedicated to the adsorption of the protein solution of the Lyz,Cyt,mBSA or BSA,respectively.And the results are summarized in Table 1.It can be seen from Table 1that each type of imprinted particles exhibited specific affinity adsorption of the template protein over structurally related proteins,which clearly showed that the receptors displayed selectivity to the template protein and exhibited lower cross-reactivity toward structurally related species,and also demonstrated that the imprinting cavities were discriminating proteins on the basis of molecular shape rather than its size.3.6.Recycling of the MIP-coated QDsRecyclability,with this capability of being used again to bind the template,is one of the important strong points of MIP.In theTable 1Specificity of the bnding of homologous proteins to MIP-coated QDs.a ,bImprinted QDsProtein binding amounts (mg protein/g imprinted QDs)LyzCytmBSABSALyz-imprinted QDs 33.2±1.3 3.7±0.37.2±0.5 4.5±0.2Cyt-imprinted QDs 6.7±0.628.2±2.18.2±1.0 4.7±0.7mBSA-imprinted QDs6.4±0.54.5±0.637.4±2.64.3±0.4aExperiment was conducted by the addition of 50mg of MIP-coated QDs in 0.5mg/mL protein solution at room temperature.bThe results was obtained from three parallel experiments.test of MIP-coated QDs recyclability,the MIP-coated QDs were used to extract Lyz through six extraction/washing cycles.The wash-ing agent used in this experiment was 0.5M NaCl.The results of the recyclability studies are shown in Table S2.It can be seen that the receptor could be repeated many times with minimal binding capacity loss,which indicated that this receptor could be reused for many cycles rather than just as a one-time-use assay.3.7.Detection range and limitThe MIP-based dBSA modified CdTe QDs have distinctly linear fluorescent quenching toward Lyz in the concentration range of 1.4×10−8–8.5×10−6M with a correlation coefficient of 0.992.The detection limit,which was calculated as the concentration of Lyz that quenched three times the standard deviation of the blank sig-nal,was 6.8nM.And the precision for three replicate detections of 0.56␮M Lyz was 2.8%(RSD).Fig.5.SDS-PAGE analysis of the results for selective recognition of lysozyme from egg ne 1:molecular weight standards (markers are in kDa);lane 2:10␮L of 20-fold dilution of egg white before adsorption;lane 3:10␮L of 20-fold dilution of egg white after adsorption by MIP-coated QDs;and lane 4:10␮L of the eluate from the receptor.W.Zhang et al./Biosensors and Bioelectronics31 (2012) 84–89893.8.Application to real sample analysisTo illustrate the utility of the MIP-basedfluorescent receptor to discriminate between Lyz and other co-existence proteins,egg white as a real sample was used to separate the Lyz.The SDS-PAGE analysis(see Fig.5)showed that the eluate of the receptor exhib-ited a single band with a molecular mass of∼14.4kDa,in excellent agreement with that of the template Lyz.All other proteins in the egg white,such as ovalbumin and ovotransferrin,displayed no cross-adsorption by the receptor and did not interfere with the binding of Lyz,which suggested the potential practical applica-tion of the receptor.And this high selectivity of the receptor to the template was attributed to its imprinting effect to the tem-plate.Finally,the optimizedfluorescent artificial receptor has been applied to the analysis of Lyz in egg white samples(Sener et al., 2010;Jing et al.,2010).The average value of the three replicate,was 3.9␮M with a RSD of3.4%.A remarkable merit of using this type of MIP-basedfluorescent receptor for the separation and detection of target molecule in complex biological samples is that expensive antibody and tedious sample treatment procedures can be avoided.4.ConclusionsIn summary,we have demonstrated that the MIP which was anchored on the surface of the dBSA modified CdTe QDs can be used as selective materials for recognition of target protein.The dBSA was used not only to modify the surface defects of the CdTe QDs, but also as assistant monomer to create effective recognition sites. Thefluorescence of the artificial receptors was stronger quenched by the template versus that of control particles,which indicated the receptors could recognize the corresponding template.A series of binding experiments further demonstrated that the materials had good selectivity for template protein over analogues.The results of thefluorescent artificial receptor for egg white analysis further demonstrated its feasibility for target protein recognition.AcknowledgementsThis work was supported by the National Basic Research Program of China(973Program)(Nos.2011CB707703and 2007CB914100),the National Natural Science Foundation of China (Nos.21075069and20875049),and Tianjin Natural Science Foun-dation(No.11JCZDJC21700).Appendix A.Supplementary dataSupplementary data associated with this article can be found,in the online version,at doi:10.1016/j.bios.2011.09.042.ReferencesBossi,A.,Piletsky,S.A.,Piletska,E.V.,Righetti,P.G.,Turner,A.P.F.,2001.Anal.Chem.73,5281–5286.Bruchez Jr.,M.,Moronne,M.,Gin,P.,Weiss,S.,Alivisatos,A.P.,1998.Science281, 2013–2016.Chan,W.C.W.,Nie,S.,1998.Science281,2016–2018.Cao,M.,Cao, C.,Liu,M.G.,Wang,P.,Zhu, C.Q.,2009.Microchim.Acta165, 341–346.Carlson,C.A.,Lloyd,J.A.,Dean,S.L.,Walker,N.R.,Edmiston,P.L.,2006.Anal.Chem.78,3537–3542.Chen,C.,Peng,J.,Xia,H.S.,Yang,G.F.,Wu,Q.S.,Chen,L.D.,Zeng,L.B.,Zhang,Z.L.,Pang,D.W.,Li,Y.,2009a.Biomaterials30,2912–2918.Chen,W.,Xu,D.H.,Liu,L.Q.,Peng,C.F.,Zhu,Y.Y.,Ma,W.,Bian,A.,Li,Z.,Yuan, Y.,Jin,Z.Y.,Zhu,S.F.,Xu, C.L.,Wang,L.B.,2009b.Anal.Chem.81,9194–9198.Diltemiz,S.E.,Say,R.,Buyuktiryaki,S.,Hur,D.,Denizli,A.,Ersoz,A.,2008.Talanta75, 890–896.Gale,D.K.,Gutu,T.,Jiao,J.,Chang,C.H.,Rorrer,G.L.,2009.Adv.Funct.Mater.19, 926–933.Guo,M.J.,Zhao,Z.,Fan,Y.G.,Wang,C.H.,Shi,L.Q.,Xia,J.J.,Long,Y.,Mi,H.F.,2006.Biomaterials27,4381–4387.Han,B.Y.,Yuan,J.P.,Wang,E.K.,2009.Anal.Chem.81,5569–5573.Haupt,K.,Mayes,A.G.,Mosbach,K.,1998.Anal.Chem.70,3936–3939.Jing,T.,Du,H.R.,Dai,Q.,Xia,H.,Niu,J.W.,Hao,Q.L.,Mei,S.R.,Zhou,Y.K.,2010.Biosens.Bioelectron.26,301–306.Kempe,M.,Mosbach,K.,1995.J.Chromatogr.A691,317–323.Klepamik,K.,Voracova,I.,Liskova,M.,Prikryl,J.,Hezinova,V.,Foret,F.,2011.Elec-trophoresis32,1217–1223.Kuo,Y.C.,Wang,Q.,Ruengruglikit,C.,Yu,H.L.,Huang,Q.R.,2008.J.Phys.Chem.C 112,4818–4824.Li,J.,Mei,F.,Li,W.Y.,He,X.W.,Zhang,Y.K.,2008.Spectrochim.Acta A70,811–817.Li,H.B.,Li,Y.L.,Cheng,J.,2010.Chem.Mater.22,2451–2457.Lin,C.I.,Joseph,A.K.,Chang,C.K.,Lee,Y.D.,2004.Biosens.Bioelectron.20,127–131. Lin,H.Y.,Ho,M.S.,Lee,M.H.,2009.Biosens.Bioelectron.25,579–586.Liu,J.X.,Chen,H.,Lin,Z.,Lin,J.M.,2010.Anal.Chem.82,7380.Liu,J.X.,Yang,K.G.,Deng,Q.L.,Zhang,L.H.,Liang,Z.,Zhang,Y.K.,-mun.47,3969–3971.Matsui,J.,Goji,S.,Murashima,T.,Miyoshi,D.,Komai,S.,Shigeyasu,A.,Kushida, T.,Miyazawa,T.,Yamada,T.,Tamaki,K.,Sugimoto,N.,2007.Anal.Chem.79, 1749–1757.Ouyang,R.Z.,Lei,J.P.,Ju,H.X.,Xue,Y.D.,2007.Adv.Funct.Mater.17,3223–3430. Qin,L.,He,X.W.,Zhang,W.,Li,W.Y.,Zhang,Y.K.,2009.Anal.Chem.81,7206–7216.Rusmini,F.,Zhong,Z.,Feijen,J.,2007.Biomacromolecules8,1775–1789.Sener,G.,Ozgur,E.,Yılmaz,E.,Uzun,L.,Say,R.,Denizli,A.,2010.Biosens.Bioelectron.26,815–821.Tang,B.,Niu,J.Y.,Yu,C.G.,Zhuo,L.H.,Ge,J.C.,mun.33,4184–4186. Tong,D.,Hetenyi,Cs.,Bikadi,Zs.,Gao,J.P.,Hjerten,S.,2001.Chromatographia54, 7–14.Tu,R.Y.,Liu,B.H.,Wang,Z.Y.,Gao,D.M.,Wang,F.,Fang,Q.L.,Zhang,Z.P.,2008.Anal.Chem.80,3458–3465.Wang,H.F.,He,Y.,Ji,T.R.,Yan,X.P.,2009.Anal.Chem.81,1615–1621.Wang,Q.,Kuo,Y.,Wang,Y.,Shin,G.,Ruengruglikit,C.,Huang,Q.,2006.J.Phys.Chem.B110,16860–16866.Yang,H.H.,Zhang,S.Q.,Yang,W.,Chen,X.L.,Zhuang,Z.X.,Xu,J.G.,Wang,X.R.,2004.J.Am.Chem.Soc.126,4054–4055.Ye,L.,Mosbach,K.,2001.J.Am.Chem.Soc.123,2901–2902.Zeng,Z.,Hoshino,Y.,Rodriguez,A.,Yoo,H.,Shea,K.J.,2010.ACS Nano4,199–204. Zhang,W.,He,X.W.,Chen,Y.,Li,W.Y.,Zhang,Y.K.,2011.Biosens.Bioelectron.26, 2553–2558.。