A novel lable-free electrochemical immunosensor for carcinoembryonic antigen based on gold nanoparticles–thionine–reduced graphene oxide nanocomposite film modified glassy carbon electrode

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<ul><li><p>Talanta 85 (2011) 2620 2625</p><p>Contents lists available at ScienceDirect</p><p>Talanta</p><p>jo u r n al hom epage: www.elsev ier .com</p><p>A nove sobased o d glm m</p><p>Fen-Ying an Ca State Key Lab , Nanjb Department o</p><p>a r t i c l</p><p>Article history:Received 25 MReceived in reAccepted 13 AAvailable onlin</p><p>Keywords:Label-freeGold nanopartReduced graphene oxideThionineCarcinoembryonic antigen</p><p>inernose</p><p> biocosy carconstwereetry</p><p> pulseof antibodyantigen complexes decreased the peak current of THi in the GNPTHiGR nanocomposites.The decreased currents were proportional to the CEA concentration in the range of 10500 pg/mL with adetection limit of 4 pg/mL. The proposed method was simple, fast and inexpensive for the determinationof CEA at very low levels.</p><p> 2011 Elsevier B.V. All rights reserved.</p><p>1. Introdu</p><p>Carcinoeused clinicamarker of csuch as lungand cystadeCEA is very </p><p>Graphenelectronic pmanufacturrior thermaproperties, ics [1517]actuators [2us is to explThe high suing of the tband gap ar[13]. Graphother to fab</p><p> CorresponE-mail add</p><p>0039-9140/$ doi:10.1016/j.ction</p><p>mbryonic antigen (CEA), is one of the most extensivelyl tumor marker. It was rst described in 1965 as aolon cancer, and was expressed in many malignancies,</p><p> cancer [1,2], ovarian carcinoma [3], breast cancer [46],nocarcinoma [7]. Therefore, sensitive determination ofimportant in clinical research and diagnosis.e has recently attracted much attention for its uniqueroperties, large specic surface area, potentially lowing cost, excellent mechanical properties, and supe-l properties [814]. Owing to all of these exceptionalgraphene nds potential application in microelectron-, large-area optoelectronics [1820], sensors [2123],4] and so forth. Among them, of particular interest forore its application in the eld of electrochemical sensor.rface area is helpful in increasing the surface load-</p><p>arget molecules. The excellent conductivity and smalle favorable for conducting electrons from biomoleculesene-based materials have been reported one after thericate electrochemical sensors, such as electrochemical</p><p>ding author. Tel.: +86 25 83597294; fax: +86 25 83597294.ress: xujj@nju.edu.cn (J.-J. Xu).</p><p>determination of glucose [25,26], -NADH [27], dopamine [28]and cytochrome c [29]. More recently, Lin et al. explored thegraphene-based immunosensor [30]. Gong and co-workers usedgraphene-based nanomaterials for the ultrasensitive detection ofcancer biomarker [31]. Zhangs group used nanogold-enwrappedgraphene nanocomposites as tracer labels to quantify carcinoem-bryonic antigen [32]. These results indicated that graphene-basedmaterials possessed a great potential to fabricate the electrochem-ical sensor.</p><p>In this paper, we proposed a new lable-free electrochemicalimmunosensor based on the graphene material. First we pre-pared the gold nanoparticlethioninereduced graphene oxide(GNPTHiGR) nanocomposites. To fabricate the nanocomposites,THi was initially noncovalently bound to the GR sheet through stacking to form a novel redox active hybrid material (THiGR).Subsequently, GNPs were anchored to the surface of THiGR by thechemisorption of the amine groups of the THi and the opposite-charged adsorption. Herein, THi not only acts as an electrochemicalactive species but also acts as a glue to connect the GNPs to theGR sheets. As shown in Fig. 1. The resulting GNPTHiGR nanocom-posites combined the following advantages: (1) GR possesses fastelectron transfer kinetics and large specic surface area; (2) THihas good electrochemical redox active properties; (3) GNPs canprovide a microenvironment to retain the proteins conformationand make them free in orientation [33]. In addition, the GNPs could</p><p> see front matter 2011 Elsevier B.V. All rights reserved.talanta.2011.08.028l lable-free electrochemical immunosenn gold nanoparticlesthioninereduce</p><p>odied glassy carbon electrode</p><p> Konga,b, Mao-Tian Xub, Jing-Juan Xua,, Hong-Yu of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineeringf Chemistry, Shangqiu Normal University, Shangqiu 476000, Peoples Republic of China</p><p> e i n f o</p><p>ay 2011vised form 10 August 2011ugust 2011e 19 August 2011</p><p>icles</p><p>a b s t r a c t</p><p>In this paper, gold nanoparticlethionprepared to design a label-free immu(CEA). The nanocomposites with goodsurface area were coated onto the glaswas immobilized on the electrode to istry of the formed nanocomposites ultravioletvisible (UVvis) spectromvoltammetry (CV). CV and differential/ locate / ta lanta</p><p>r for carcinoembryonic antigenraphene oxide nanocomposite</p><p>hena</p><p>ing University, Nanjing 210093, Peoples Republic of China</p><p>educed graphene oxide (GNPTHiGR) nanocomposites werensor for the sensitive detection of carcinoembryonic antigenmpatibility, excellent redox electrochemical activity and largebon electrode (GCE) surface and then CEA antibody (anti-CEA)ruct the immunosensor. The morphologies and electrochem-</p><p> investigated by using scanning electron microscopy (SEM),, electrochemical impedance spectroscopy (EIS) and cyclic</p><p> voltammetry (DPV) studies demonstrated that the formation</p></li><li><p>F.-Y. Kong et al. / Talanta 85 (2011) 2620 2625 2621</p><p>effectively promote electronic transfer [34]. So, the immunosensorsbased on the GNPTHiGR nanocomposites signicantly enhancedthe sensitivity and greatly simplied the assay system.</p><p>2. Experimental</p><p>2.1. Reagents</p><p>Graphite powder, trisodium citrate and thionine acetate saltwere obtained from Shanghai Biochemical Reagent Company(Shanghai, China). Chloroauric acid (HAuCl4), lyophilized bovineserum albumin (BSA), CEA and CEA antibody (anti-CEA) were pur-chased from Sigma (St. Louis, USA). GNPs with mean size of 16 nmwere prepared by citrate reduction of HAuCl4 in aqueous solu-tion at 100 C for half an hour [35]. Phosphate buffer solution (PBS,0.1 mol/L, pH 6.0) was used as working solution. All solvents wereof analytical grade. Ultrapure water from a Milli-Q plus system(Millipore Co., &gt;18 M cm) was used in all aqueous solutions andrinsing procedures. The healthy peoples serum specimens suppliedby Central Hospital of Shangqiu were diluted to different concen-trations with PBS of pH 6.0.</p><p>2.2. Apparatus</p><p>Electrochemical measurements were recorded using a CHI660D electrochemical workstation (Shanghai, China) with a con-ventional tas the auxithe referen(GCE, 3 mmtrochemicaperformed K3[Fe(CN)6bias potentthe frequen(UVvis) sptometer (Shimages werscope (TokKQ-100E ulperformed </p><p>2.3. Preparation of GR</p><p>Graphene oxide (GO) was rst synthesized from naturalgraphite powder using a modied Hummers and Offemans method[36]. GR was prepared by the chemical reduction of GO accordingto the literature with a little modication [37]. Briey, the resultingGO dispersion (100 mL) was mixed with certain amount of ascorbicacid solution. Then the mixture was treated by ultrasonication for1 h at 60 C. The obtained black suspension was washed with waterfor several times and dried in vacuum at 60 C overnight.</p><p>2.4. Preparation of GNPTHiGR nanocomposites</p><p>In a typical procedure, the as-prepared GR (1 mg) were dispersedin 1 mL water by ultrasonication, and then mixed with 1 mL of1 mmol/L THi solution at room temperature for at least 12 h. Afterdiscarding the supernatant, the THiGR nanocomposites againwere dispersed in 1 mL water. Subsequently, the THiGR nanocom-posites were added dropwise into 20 mL of the as-prepared GNPs.The mixture was allowed to react at room temperature under stir-ring for 24 h, followed by centrifugation. The resulting GNPTHiGRnanocomposites were washed with water and then re-dispersed in1 mL of PBS (pH 7.4) buffer and stored at 4 C before use.</p><p>brica</p><p>ssy cith 1nicafter</p><p>he suhe obcubare wosen</p><p>emaiashe</p><p> at 4mica</p><p>ompohree electrode system comprised of platinum wireliary electrode, saturated calomel electrode (SCE) asce electrode and modied glassy carbon electrode</p><p> in diameter) as the working electrode. The elec-l impedance spectroscopy (EIS) measurements werein the solution of 0.10 mol/L KCl containing 5.0 mmol/L]/K4[Fe(CN)6] (1:1) mixture as a redox probe at aial of 0.20 V. The alternative voltage was 5 mV andcy range was 0.11.0 105 Hz. The ultravioletvisibleectrometry was acquired with a UV 2300 spectropho-anghai, China). Scanning electron microscopy (SEM)e obtained using an S-3000 N scanning electron micro-yo, Japan). Ultrasonication was performed using atrasonic cleaner (Kunshan, China). All experiments wereat room temperature.</p><p>2.5. Fa</p><p>Glalike wultrasoin air. Aonto tture. Tand inies weimmunsible rAfter wstoredtroche</p><p>Fig. 1. Preparation process of the GNPTHiGR nanoction of the immunosensor</p><p>arbon electrode (GCE) was polished down to mirror-.0 and 0.3 m alumina slurry, followed by successivetion in distilled water and ethanol for 5 min and driedwards, 5 L GNPTHiGR nanocomposites were dippedrface of the GCE, and dried for 2 h at room tempera-tained electrode was immersed in the anti-CEA solutionted for more than 12 h at 4 C. Then, unbound antibod-ashed away with PBS. Subsequently, the as-preparedsor was incubated in 1% BSA for 1 h at 37 C to block pos-ning active sites and avoid the nonspecic adsorption.d carefully for three times with PBS, the electrode was</p><p>C while not in use. The fabrication process of the elec-l immunosensor is schematically illustrated in Fig. 1.</p><p>sites and immunosensor.</p></li><li><p>2622 F.-Y. Kong et al. / Talanta 85 (2011) 2620 2625</p><p>Fig. 2. SEM inanocomposit</p><p>2.6. Electro</p><p>The preption solutio10 min. Aftevoltammetr0.1 mol/L PBthe inhibitinanocompowhich was </p><p>3. Results </p><p>3.1. Charac</p><p>The morposites werGR (Fig. 2a)size. Thesetion of graarea [38,39</p><p>Vvis absorption spectra of GR (a), THiGR (b) and pure THi solution (c).</p><p> on THiGR surface with dense distribution, indicating the interaction between the GNPs and the THiGR nanocom-</p><p> For comparison, we mixed the GR with the GNPs withoutlecules, and observed few GNPs anchored on the GR sheets), indicating the important role of THi molecules play in theg of GNPs to the GR sheet.vis sn GFig. 3. U</p><p>boundstrongposite.THi mo(Fig. 2cbindin</p><p>UVbetweemages of GR (a), GNPTHiGR nanocomposites (b) and GNPGRes (c).</p><p>chemical measurement procedures</p><p>ared immunosensor was incubated in 100 L incuba-n containing different concentration of CEA at 37 C forr the residual was removed with PBS, differential pulsey (DPV) from 0.2 to 0.6 V (vs. SCE) was recorded inS (pH 6.0). The measurement principle was based on</p><p>on of the response current of THi in the GNPTHiGRsites after the formation of antigenantibody complex,directly proportional to the concentration of CEA.</p><p>and discussion</p><p>terization of the GNPTHiGR nanocomposites</p><p>phology of GR, GNPGR and GNPTHiGR nanocom-e characterized by SEM technique. The SEM image of</p><p> showed a wrinkled paper-like structure with irregular wrinkles are important for preventing the aggrega-phene during drying and maintaining a high surface]. From Fig. 2b, it can be clearly seen that GNPs were</p><p>of GR (a), TGR showedcorrespondbands at 28(curve b). In(curve c), tband at 59THiGR nanreported lit</p><p>3.2. Electro</p><p>In orderstructed suchange of eFig. 4. It wacircle domawas modicle domainTHi as an ewith GNPs </p><p>Fig. 4. EIS of BSA/anti-CEA/5 mmol/L K3Fepectrometry was employed to explore the interactionsR and THi. Fig. 3 showed the typical UVvis spectraHiGR (b) and pure THi solution (c). The spectrum of</p><p> an absorption band at about 266 nm (curve a), whiched to the chemically reduced GO [40]. Two absorption0 and 599 nm were observed at THiGR nanocomposite</p><p> comparison with those obtained at pure THi solutionhe slightly red shift and broadened for the absorption9 nm provided further evidence for the formation ofocomposite. This phenomenon was consistent with theerature [31].</p><p>chemical characteristics of the immunosensor</p><p> to conrm that the sensing interface has been con-ccessfully, we utilized EIS to monitor the impedanceach modication process. The results were shown ins observed that the bare GCE showed a small well semi-in at higher frequencies (curve a). After the electrodeed with GNPTHiGR nanocomposites, the semicir-</p><p> became smaller in curve b. The reason may be thatxcellent electron transfer mediator and GR togetherin the GNPTHiGR nanocomposites possessing goodbare GCE (a), GNPTHiGR/GCE (b), anti-CEA/GNPTHiGR/GCE (c),GNPTHiGR/GCE (d) and CEA/BSA/anti-CEA/GNPTHiGR/GCE (e) in(CN)6/K4Fe(CN)6 (1:1) solution containing 0.1 mol/L KCl.</p></li><li><p>F.-Y. Kong et al. / Talanta 85 (2011) 2620 2625 2623</p><p>Fig. 5. The different modied electrodes in 0.1 mol/L PBS: (a) bare GCE,(b) GNPTHiGR/GCE, (c) anti-CEA/GNPTHiGR/GCE, and (d) BSA/anti-CEA/GNPTHiGR/GCE, all potentials were given vs. SCE and the scan ratewas 50 mV/s.</p><p>conductive properties are favorable for the electron transfer. Afterthe GNPTHiGR/GCE was incubated with anti-CEA, a large semi-circle domain was found in curve c. Subsequently, when BSA wasadsorbed on the surface, the electron-transfer resistance obvi-ously increBSA/anti-CEtance was protein memunicationthe conductransfer [41</p><p>On the omonitor eawere shownpeak in PBThen a pairwith GNPTtwo peaks wnanocompoof the redowas immerobserved (cwith the re</p><p>Moreove6.0) at diffshown in Fcurrents inction, both </p><p>Fig. 6. Cyclic vinner to outer(pH 6.0) undecurrents on th</p><p>ffects of (a) pH of detection solution, (b) incubation temperature, and (c)on time on the immunosensor.</p><p>tional to the potential scan rates in the range of 20400 mV/sn in the inset), suggesting a surface conned redox processased (curve d). At last, after CEA was coupled onto theA/GNPTHiGR/GCE, an increase of interfacial resis-</p><p>obtained (curve e). The reason may be that the outermbrane on the electrode acts as the electron com-</p><p> and mass-transfer blocking layer, further insulatingtive support and counteracting the interfacial electron].ther hand, cyclic voltammetry (CV) was employed toch immobilization step and the corresponding results</p><p> in Fig. 5. It can be seen that the bare GCE had no redoxS solution (curve a) for the absence of redox probes.</p><p> of well-dened redox peak was appeared in curve bHiGR nanocomposites modied GCE. Therefore, theseere from the redox reaction of THi in the GNPTHiGRsites. However, the loading of anti-CEA led to a decreasex peak currents (curve c). After the modied electrodesed in BSA, a further decrease of the current can beurve d). These measurements were in good agreementsults obtained from EIS.r, The CVs of the resulting immunosensor in PBS (pH</p><p>erent scan rates were also studied. The results wereig. 6. It can be observed that the couple of redox peakreased with the increase of potential scan rate. In addi-the anodic and cathodic peak currents were directly</p><p>Fig. 7. Eincubati</p><p>propor(show[42].oltammograms of the modied electrode at different scan rates (from): 20, 50, 100, 150, 200, 250, 300 and 400 mV/s in 5 mL 0.1 mol/L PBSr room temperature. The inset shows the dependence of redox peake potential sweep rates.</p><p>3.3. Optimi</p><p>The pH trochemicacause proteelectrochemser...</p></li></ul>