Microstructural and electrochemical impedance characterization of bio-functionalized ultrafine ZnS nanocrystals–reduced graphene oxide hybrid for immunosensor applications

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<ul><li><p>dfcp</p><p>K</p><p>ci</p><p>y</p><p>electrochemically reduced GO sheets through a cross linker, 1-pyrenemethylamine hydrochloride, by</p><p>Introduction</p><p>Nanomaterials, including carbreceived great attention due toapplications in many areas, incltronics and photonics. Grapheneof sp2-bonded carbon atoms wistructure1 and unique physicochigh surface area, excellent emechanical strength, biocompation and mass production, whicsubject for electro-chemical app</p><p>Received 18th May 2013Accepted 16th August 2013</p><p>DOI: 10.1039/c3nr02575f</p><p>www.rsc.org/nanoscale</p><p>Nanoscale</p><p>PAPER</p><p>Publ</p><p>ished</p><p> on </p><p>22 A</p><p>ugus</p><p>t 201</p><p>3. D</p><p>ownl</p><p>oade</p><p>d by</p><p> Uni</p><p>vers</p><p>ity o</p><p>f Virg</p><p>inia</p><p> on </p><p>17/1</p><p>1/20</p><p>13 1</p><p>8:17</p><p>:16.</p><p> electric devices5 transistors,6 andGraphene is considered to b</p><p>aCSIR-National Physical Laboratory, Dr K.</p><p>India. E-mail: rajesh_csir@yahoo.combDepartment of Applied Chemistry, Delhi</p><p>Delhi, 110042, India</p><p>10494 | Nanoscale, 2013, 5, 104941carbodiimide reaction and have been characterized by scanning electron microscopy, transmission</p><p>electron microscopy and energy dispersive X-ray spectroscopy. The transmission electron microscopic</p><p>characterization of the ZnSRGO hybrid shows the uniform distribution of ultra-ne nanoparticles of</p><p>ZnS in nano-sheets of GO throughout the material. The protein antibody, Ab-cMb, was covalently linked</p><p>to ZnSRGO nanocomposite hybrid for the fabrication of the bioelectrode. A detailed electrochemical</p><p>immunosensing study has been carried out on the bioelectrode towards the detection of target Ag-</p><p>cMb. The optimal tted equivalent circuit model that matches the impedance response has been</p><p>studied to delineate the biocompatibility, sensitivity and selectivity of the bioelectrode. The bioelectrode</p><p>exhibited a linear electrochemical impedance response to Ag-cMb in a range of 10 ng to 1 mg mL1 in</p><p>PBS (pH 7.4) with a sensitivity of 177.56 U cm2 per decade. The combined synergistic eects of the high</p><p>surface-to-volume ratio of ZnS(MPA) nanocrystals and conducting RGO has provided a dominant charge</p><p>transfer characteristic (Ret) at the lower frequency region of </p></li><li><p>impedance spectroscopy (EIS) represents a powerful method for</p><p>Paper Nanoscale</p><p>Publ</p><p>ished</p><p> on </p><p>22 A</p><p>ugus</p><p>t 201</p><p>3. D</p><p>ownl</p><p>oade</p><p>d by</p><p> Uni</p><p>vers</p><p>ity o</p><p>f Virg</p><p>inia</p><p> on </p><p>17/1</p><p>1/20</p><p>13 1</p><p>8:17</p><p>:16.</p><p> View Article Onlinesheets, the electrochemical reduction method is found to be apromising route for preparing reduced graphene oxide (RGO)modied electrode surface because it is simple, fast, inexpen-sive and more ecient than other methods such as chemicaland thermal reduction, with no additional element such as Nincorporated into the obtained RGO lm.11 Recent applicationsof RGO includes its use as a kind of novel electrochemicalsensing material in biological systems, such as detection ofDNA,4 proteins and pathogens,12 design of cell/bacterial nano-devices13 and drug delivery carriers.14</p><p>To obtain enhanced mechanical, thermal and electro-chemical properties, the RGO surface has been functionalizedwith dierent materials such as polymers, inorganic metal/semiconducting nano materials and biomaterials.1517 Theability to retain the native structure of RGO while enabling thebioactivity of the functionalizing moiety through a surfaceconned process, as well as eective direct electron transferreaction properties, means that RGO is a suitable material forconstruction of electrochemical substrates. Wang et al. recentlyprepared a glucose oxidase biosensor based on graphene andCdS nanocrystals.18 For green chemistry, ZnS is very suitableas a good substitute of CdS due to its nontoxicity to the humanbody and low cost. Jian Du et al. have done a comparison studyof the electrochemical and photo electrochemical behaviors ofthree biosensors, based on the use of Au, CdS, and ZnS nano-particlesglucose oxidase (GOD) systems and they found thatbiosensors based on ZnS NPs are more sensitive and much lesstoxic to humans and the environment than CdS NPs.19 Recentapplication of ZnS in the eld of biosensor includes theformation of single-walled carbon nanotube based chemir-esistive label-free DNA sensors.20 The purpose of using water-soluble ZnS nanoparticles coated with carboxyl capping agentsis that it can allow greater anity of binding or interaction withthe bio-target molecules, and the biocompatible nature of theZnSRGO nanocomposite lm can provide a favorable micro-environment to retain the activity of the immobilized proteins.</p><p>According to the WHO, acute myocardial infarction (AMI) isthe result of a sudden occlusion that decreases blood ow to aportion of the myocardium, causing cell death. Symptoms ofAMI include chest pain, pressure, shortness of breath, and/ornausea but these symptoms may also be seen with non-heart-related conditions. So it is very crucial that physicians areprovided with additional information in a short space of time,enabling them to carry out quick and accurate diagnoses.Cardiac biomarker tests are intended to help detect AMI and toevaluate its severity as soon as possible so that appropriatetherapy can be initiated. Cardiac biomarkers are substancesthat are released into the blood when there is damage to theheart muscle. Typical cardiac markers used for diagnosis of AMIare cardiac myoglobin, creatine kinase-MB and cardiac tropo-nins I and T. Although not cardiac-specic, myoglobin (cMb) isone of the very earliest known markers that increase aer acutemyocardial infarction, and its rapid screening under acutephysiological conditions is fundamental. Due to its small size(17.8 kDa), facilitating its quick release into circulation (as earlyas 13 h upon symptom onset), as well as its high sensitivity andhigh predictive value, cMb is considered as a valuable earlyThis journal is The Royal Society of Chemistry 2013the investigation of bulk and interfacial electrical properties ofany kind of solid or liquid material which is connected to, orpart of, an appropriate electrochemical transducer providing asensitive, non-destructive, and rapid electrochemical sensingmethod for the characterization of the electrical properties ofbiological interfaces. EIS measures the response (current and itsphase) of an electrochemical system to an applied oscillatingpotential as a function of the frequency. It is an eectivemethod to detect antigenantibody complex formation, biotinavidin complexation and oligonucleotideDNA interaction,27 ascompared with other methods such as radiochemical, colori-metric and chemiluminescent methods.</p><p>This work demonstrates a facile strategy to synthesize a ZnSRGO nanocomposite consisting of 3-mercaptopropionic acid(MPA) capped ZnS nanocrystals, ZnS(MPA), anchored on RGOsheets through a linker and deposited onto silane modiedindium-tin-oxide (ITO) glass plate for the fabrication of a bio-electrode. In this work, we have utilized large surface ZnS (MPA)nanocrystals, where the surrounding carboxyl functional groupsprovided a high loading of protein antibody, Ab-cMb, moleculesthrough strong carbodiimide linkage. The composition,morphology and the microstructure of the as-obtainedZnS(MPA)RGO nanocomposite was characterized usingvarious instrumental techniques such as SEM, TEM and elec-trochemical techniques. The impedimetric sensing perfor-mance of the bioelectrode with ZnSRGO nanocompositetowards the quantitative detection of target protein antigen, Ag-cMb, in phosphate buer solution (PBS; pH 7.4) was studiedand compared with that of native RGO sheets without ZnSnanoparticles, to highlight the contribution of the ZnS nano-particles in the overall enhanced immunosensing performance.</p><p>ExperimentalChemicals and reagents</p><p>Ab-cMb (Cat4M2 MAb 4E2) &amp; Ag-cMb (Cat 8M50) were obtainedfrom Hytest (Turku, Finland). Mouse immunoglobulin-G (Ag-IgG) (Cat IGP3) was obtained from GENEI, Bangalore, India.3-Aminopropyltriethoxysilane (APTES) was purchased fromMerck chemicals (Germany). N-(3-Dimethylaminopropyl)-N0-ethyl carbodiimide hydrochloride (EDC) and N-hydroxy succi-nimide 98% (NHS), zinc nitrate hexahydrate (Zn(NO3)2$6H2O),sodium sulde nonahydrate (Na2S$9H2O), 1-pyrenemethyl-amine hydrochloride (PyMe-NH2), 1-pyrene butanoic acidscreening test for AMI.21 The cut-o concentrations of cardiacmyoglobin may vary from 50 ng mL1 (Behring Diagnosticsmethod, Nanogen cardiac STATus panel) and 56 ng mL1</p><p>(Stratus CS STAT, for female) to 170200 ngmL1 (triage cardiacpanel22) with majority of researchers holding the cut-o toabout 100 ng mL1.23 Several conventional methods have beenemployed to detect and quantify Mb. These include enzymelinked immunosorbent assay (ELISA),24 and chromatographic25</p><p>or spectrophotometric methods.26 These methods, however,lack the required specicity and/or involve several steps, aretime consuming and require very expensive reagents.</p><p>As one of the electrochemical technologies, electrochemicalNanoscale, 2013, 5, 1049410503 | 10495</p></li><li><p>APTES from the surface of the substrate and dried under N2</p><p>succinimidyl ester (PyBtNHS) without undergoing any func-tionalization with ZnS nanocrystals to fabricate Ab-cMb/RGO/APTES/ITO-glass for comparative study. The stepwiseconstruction of the prototype assembly is represented inScheme 1.</p><p>Results and discussionsContact angle measurement</p><p>Contact angle measurements based on the sessile drop methodwere used to determine the hydrophobic/hydrophilic characterof the surface. The image of the drop deposited on the modiedITO electrode surface was recorded by a video camera and animage-analysis system calculates the contact-angle (q) from theshape of the drop. Measurements were repeated with four drops</p><p>Nanoscale Paper</p><p>Publ</p><p>ished</p><p> on </p><p>22 A</p><p>ugus</p><p>t 201</p><p>3. D</p><p>ownl</p><p>oade</p><p>d by</p><p> Uni</p><p>vers</p><p>ity o</p><p>f Virg</p><p>inia</p><p> on </p><p>17/1</p><p>1/20</p><p>13 1</p><p>8:17</p><p>:16.</p><p> View Article OnlineHowever, this reduction peak at 1.1 V disappeared in thenext two consequent scans and is therefore irreversible,gas ow. The APTES modied ITO glass plates were thenimmersed in the GO solution (0.3 mg mL1) for a period of 1h followed by washing with distilled water and dried underN2 to form the GO/APTES/ITO-glass electrodes. Negativelycharged GO akes were deposited on the positively chargedamino modied APTES/ITO-glass plates due to electrostaticinteractions and were not removed even aer repeatedwashing. The GO/APTES/ITO-glass electrodes were then elec-trochemically reduced by cyclic voltammetry (CV) in 0.5 MKCl solution saturated with N2 gas from 0.7 to 1.1 V for 3CV cycles, at a scan rate of 50 mV s1 (Fig. 1) to reducedgraphene oxide (RGO). The large reduction current at 1.1 Vcorresponds to the reduction of the surface oxygen groupsonly and not water since the reduction of water to hydrogenoccurs at more negative potential (e.g., 1.5 V) as shown inthe scheme.</p><p>GO + aH+ + be/ RGO + cH2Osuccinimidyl ester and 3-mercapto propionic acid (MPA) wereobtained from Sigma-Aldrich Corp. All other chemicals were ofanalytical grade and used without further purication.</p><p>Apparatus</p><p>Contact angles were recorded on Drop Shape Analysis System;model DSA10MK2 from Kruss GmbH, Germany. High resolu-tion transmission electron microscopy (HR-TEM) was doneusing an FEI model: Tecnai G2 F30 and JEOLmodel: JEM 2100F.Scanning electronmicroscopy (SEM) images were obtained witha LEO 440 PC; UK based digital scanning electron micrograph.Cyclic voltammetry and EIS measurements were done on aPGSTAT302N, AUTOLAB instrument from Eco Chemie, TheNetherlands. All measurements were carried out in a conven-tional three-electrode cell conguration consisting of theproposed modied electrode as working electrode, Ag/AgCl as areference electrode and a platinum wire as a counter electrode.Electrochemical impedance spectroscopy was conducted inPBS (pH 7.4, 0.1 M KCl) solution containing 2 mM [Fe(CN)6]</p><p>3/[Fe(CN)6]</p><p>4 in the frequency range from 1 Hz to 100 kHz at anAC voltage of 0.05 V.</p><p>Preparation of biofunctionalized of Ab-cMb(BSA)/ZnS(MPA)RGO/APTES/ITO-glass bioelectrode</p><p>The ITO coated glass plates (10 U ,1) were cleaned bysequential ultrasonic cleaning in dextran, acetone, ethanol,isopropyl alcohol and DI for 10 min each and dried invacuum. Then, the cleaned ITO glass plates were exposed tooxygen plasma for 5 minutes in a plasma chamber. The ITOglass plates were immersed in 2% APTES solution preparedin ethanol for 1.5 h, under the ambient conditions, to form aself assembled monolayer (SAM) of APTES. These glass plateswere then rinsed with ethanol in order to remove non-bonded10496 | Nanoscale, 2013, 5, 1049410503indicating the reduction of surface oxygenated species in therst cycle only with the formation of RGO. The remaining twoCV cycles correspond to the electrochemical behavior of theresulting RGO/APTES/ITO.</p><p>The RGO/APTES/ITO-glass electrodes were then immersed in6 mM solution of 1-pyrenemethylamine hydrochloride (PyMe-NH2) in DMF, for 2 h, at room temperature, and thereaerwashed extensively with DMF and dried under N2 gas ow. TheZnS(MPA) nanocrystals were synthesized in aqueous solution,at room temperature, by a method reported earlier.20 1-Pyr-enemethylamine functionalized RGO/APTES/ITO-glass elec-trodes were treated with a 1 mg mL1 aqueous solution ofZnS(MPA) nanocrystals containing 0.1 M EDC and 0.05 M NHSfor 2 h and were rinsed thoroughly with double distilled water toobtain the ZnS(MPA) functionalized RGO/APTES/ITO-glasselectrodes. Ab-cMb was then covalently immobilized onZnS(MPA)-RGO/APTES/ITO-glass electrodes by immersing themin PBS buer (pH 7.4) containing 100 mg mL1 Ab-cMb over-night at 4 C, followed by washing with PBS and drying with N2gas ow. The protein antibody immobilized electrodes werefurther immersed in 1% BSA (W/V) solution to block thenonspecic binding sites and the remaining unbound freecarboxyl groups as well, followed by washing with PBS to removeany physically adsorbed antibodies and nally dried under N2ow and stored at 4 C. Ab-cMb was covalently immobilizeddirectly over the RGO sheets through 1-pyrene butanoic acid</p><p>Fig. 1 Electrochemical reduction of GO/APTES/ITO-glass surface in a deaeratedsolution of 0.5 M KCl, at a scan rate of 50 mV s1.This journal is The Royal Society of Chemistry 2013</p></li><li><p>on ZnS (MPA) nanocrystals. However, upon immobilizationof hydrophobic protein antibody, Ab-cMb, molecules onZnS(MPA)RGO/APTES/ITO-glass the contact angle signicantlyincreased to 97.24 1(Fig. 2f) indicating the formation of thebioelectrode.</p><p>Microstructural characteristics</p><p>Fig. 3 shows the SEM images RGO/APTES/ITO-glass andZnS(MPA)RGO/APTES/ITO-glass. The isolated akes of GO asobserved in Fig. 3a of SEM image demonstrate that many RGOsheets have been uniformly dispersed throughout the structure</p><p>Paper Nanoscale</p><p>Publ</p><p>ished</p><p> on </p><p>22 A</p><p>ugus</p><p>t 201</p><p>3. D</p><p>ownl</p><p>oade</p><p>d by</p><p> Uni</p><p>vers</p><p>ity o</p><p>f Virg</p><p>inia</p><p> on </p><p>17/1</p><p>1/20</p><p>13 1</p><p>8:17</p><p>:16.</p><p> View Article OnlineScheme 1 Schematic representation of the stepwise fabrication of thebioelectrode.of water as a test liquid probe at dierent regions of themodied surface and are shown in Fig. 2.</p><p>The obtained contact angle value for bare ITO-glass wasfound to be 40.15 2 (Fig. 2a) analogous to a hydrophilicsurface with hydroxyl groups present on it. A signicantincrease in contact an...</p></li></ul>

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