Physicochemical characteristics of reduced graphene oxide based Pt-nanoparticles-conducting polymer nanocomposite film for immunosensor applications
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Research ArticleReceived: 12 May 2014 Revised: 1 July 2014 Accepted article published: 8 July 2014 Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/jctb.4480
Physicochemical characteristics of reducedgraphene oxide based Pt-nanoparticles-conducting polymer nanocomposite film forimmunosensor applicationsNidhi Puri,a,b Sujeet K. Mishra,a Asad Niazi,b Avanish K. Srivastavaa andRajesha*
BACKGROUND: This study describes, electrochemically prepared Pt-nanoparticles (PtNP) embedded poly(pyrrole-co-pyrrolepropylic acid) (PPy-PPa) copolymer film over reduced graphene oxide (RGO) sheets deposited on a silane modi-fied indium-tin-oxide coated glass plate. The cardiac myoglobin protein antibody (Ab-Mb) was covalently immobilized onPtNP(PPy-PPa)-RGO nanocomposite through pendant carboxyl groups of the polymer chain using carbodiimide couplingreaction to construct a bioelectrode. The bioelectrode was characterized by high resolution transmission electron microscopy,energy dispersive spectra and electrochemical impedance spectroscopy (EIS). The sensing performance of the bioelectrodewasinvestigated, as an impedimetric immunosensor, for the quantitative detection of the target human cardiacmyoglobin antigen(Ag-cMb) in phosphate buffer silane (pH7.4).
RESULTS: The EIS studies of the bioelectrode towards Ag-cMb showed a dominant charge transfer resistance (Ret) characteristicin the frequency region 50 to 100Hz. The EIS of the bioelectrode showed an effective immunoreaction with Ag-cMb with asignificant increase in both Ret and the constant phase element parameter (n) from 0.61 to 0.85. The bioelectrode exhibited alinear dynamic range of detection from 10ngmL1 to 1gmL1, sensitivity of 107.08 cm2 per decade and a lowest detectionlimit of 4.0 ngmL1 Ag-cMb.
CONCLUSION: The polymer nanocomposite with uniformly distributed PtNP in conjunction with conducting RGO support hasbeen demonstrated to be a high performance immunosensor. This has been attributed to the synergistic combination ofconductingRGOsupportandelectroactivePtNPembeddedconducting functional copolymer forefficientcovalentbiomolecularimmobilization, responsible for high protein loading and fast interfacial electron exchange. 2014 Society of Chemical Industry
Keywords: electrochemistry; characterization; proteins; immobilization
INTRODUCTIONMyoglobin (cMb), a small sized (17.6 kDa) cardiac protein, is acardiac marker released into the blood stream very shortly afteracutemyocardial infarction (AMI). The normal concentration rangeof cMb in human blood is 30 to 90 ngmL1, which increasesto 200 ngmL1 within 1 h of onset of myocardial infarction andreaches a highest value of 900 ngmL1 during the peak hours. Thehigh sensitivity and predictive value of cMb make it a valuablescreening test in the 13 h immediately following an AMI.1,2 Thus,there is a need of immense importance to devise a rapid investiga-tive technique, which can be conducted within the initial criticaltimewindow.Conventional laboratorymethodsused for thequan-titative detection of cMb include enzyme linked immunosorbentassay (ELISA),3 chromatography4 and spectrophotometry.5 How-ever, these methods suffer from numerous disadvantages suchas long reaction time, complex preparatory method, expensivereagents and requirement for trained personnel. Electrochemi-cal impedance spectroscopy (EIS) based immunosensingmethods
have attracted significant attention in the last few years becauseof their rapidity, high sensitivity and non-destructive nature. Billahet al. utilized the EIS technique to characterize the sensor sur-face assembly and successfully recognize cMb, immobilized overthe self-assembled monolayer on gold electrode.6 However, thissuffers from the disadvantages of using expensive biotin function-alized Mb antibody immobilized at the transducer surface thatdoes not make it a cost effective method. Therefore, there is still agreat interest in thedevelopment of a highperformancebiosensorbased on advanced electroactive materials for cMb detection.
Correspondence to: Rajesh, CSIR-National Physical Laboratory, Dr. K.S. Krish-nan Road, NewDelhi-110012, India. E-mail: firstname.lastname@example.org
a CSIR-National Physical Laboratory, Dr. K.S. Krishnan Road, New Delhi-110012,India
b Department of Physics, Faculty of Natural Sciences, Jamia Millia Islamia, NewDelhi-110025, India
J Chem Technol Biotechnol (2014) www.soci.org 2014 Society of Chemical Industry
www.soci.org N Puri et al.
Over the past decade, nanomaterials have attracted tremen-dous interest in different fields of basic and applied scientificresearch, because of the size (surface/volume) effects on theirproperties such as electrical conductivity, magnetism, electrocat-alytic activity, optical response and biosensitivity.710 Compositematerials based on noble metal nanoparticles of Au, Ag, Pd andPt are potential candidates in the development of electrochemicalbiosensors11,12 owing to their biocompatibility, high surface area,excellent conductivity as well as catalytic activity. These materialshave an additional advantage of enhanced diffusion due to con-vergent rather than linear diffusion at slow scan rates.13 Metalnanoparticle (MNP) composites with conducting polymers havebeen studied extensively in the past due to their unique propertiesinherited from the individual components.14 It has been reportedthat onMNP incorporation the conducting polymer showed supe-rior catalytic property compared to onewithoutMNP owing to theenlarged active catalytic surface area of the dispersed MNP andthe good conductivity of the polymer.15 In addition to the above,the controlled thickness of the conducting polymer prepared bythe electrochemical method could effectively construct a repro-ducible biosensor.Graphene, a two-dimensional, single atom thick carbon
allotrope has been widely used as an ideal transducing andsupporting base material because of its high 2D electricalconductivity, specific surface area and very fast heteroge-neous electron-transfer rates.1619 However, owing to thepoor dispersibility of graphene in water, graphene oxide (GO),a functionalized form of graphene, which possesses excellenthydrophilicity has been exploited, as a potential material, forbiosensing applications.20,21 While the excellent mechanical sta-bility of GO provides a good conducting support for biomolecularimmobilization (such as DNA and antibodies), the stability of thedirectly immobilized biomolecules is quite poor,22 which can bereadily removed by simple washing.Keeping this in view, we have functionalized the electro-
chemically reduced GO (RGO) with conducting polymer Ptnanoparticles (PtNP) the composite material having pendantcarboxyl functional groups for effective biomolecular immobiliza-tion. For this purpose, an in situ electrochemical polymerizationof pyrrole (Py) and pyrrolepropylic acid (Pa) in the presence ofPtNP was carried out on RGO support, wherein the preparedPtNP-(pyrrole-co-pyrrolepropylic acid) composite [PtNP(PPy-PPa)]offers pendant carboxyl groups of PPa for the covalent immobiliza-tion of biomolecules towards the construction of a bioelectrode.The bioelectrode was characterized for microstructural featuresand electrochemical properties, and exhibited enhanced ionicand electronic transport due to the uniform distribution of elec-troactive PtNP with large surface to volume ratio, within thePPy-PPa film. The electrochemical sensing performance of thebioelectrode was studied for the quantitative detection of cMb inphosphate buffer saline (PBS; pH 7.4).
EXPERIMENTALReagentsMonoclonal mouse anti-human cardiac myoglobin (Ab-Mb;Cat 4M23) and myoglobin derived from human heart tissue(Ag-cMb; Cat 8M50) were procured from Hytest (Turku, Finland).Mouse immunoglobulin-G (Ag-IgG) (Cat IGP3) was obtained fromGENEI, Bangalore. Pyrrole, pyrrolepropylic acid, hexachloropla-tinic acid hexahydrate (H2PtCL66H2O, 37.50% as Pt), sodiumborohydride, polyvinylpyrrolidone (PVP), sodium p-Toluene
sulphonic acid (p-TSA), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) and N-hydroxy succinimide98% (NHS) were obtained from SigmaAldrich chemicals.3-aminopropyltriethoxysilane (APTES) was purchased from Merckchemicals (Germany).
InstrumentationMicrostructural characterization was performed on a high reso-lution transmission electron microscopy (HRTEM model: TecnaiG2 F30 STWIN with field emission gun, operated at 300 kV),FEI Europe BV, Eindhoven, The Netherlands. Electropolymer-ization, cyclic voltammetry (CV) and EIS measurements weretaken on a PGSTAT302N, AUTOLAB instrument from Eco Chemie,The Netherlands. CV and EIS measurements were carried out inPBS (pH 7.4) containing a mixture of 2mmol L1 K3[Fe(CN)6] and2mmol L1 K4[Fe(CN)6]. CVs were conducted in a potential win-dow of 0.1 V to+0.5 V. The EISmeasurements were carried out ata bias voltage of 0.3 Vwith an A.C. voltage of 0.05 V over awide fre-quency range from 1Hz to 100 kHz. The EIS experimental data wascircuit fitted by general purpose electrochemical system (GPES,version 4.9, Eco Chemie) software and corresponding values of EISparameters were obtained. All the electrochemical measurementswere carried out in a three-electrode cell configuration consistingof the proposed bioelectrode, as working electrode, Ag/AgClas reference electrode and platinum wire as counter electrode,throughout the experiment.
Preparation of Ab-Mb/PtNP(PPy-PPa)-RGO/APTES/ITO-glassbioelectrodeThe indium-tin-oxide (ITO) glass plates, after cleaning sequentiallyin water, extran, acetone, ethanol, 2-propanol and double dis-tilled water were exposed to oxygen plasma for 5min and thenimmersed in 2% APTES solution (prepared in ethanol) for 1.5 h,to form a self-assembled monolayer (SAM) of APTES. These silanemodified ITO-glass plates (APTES/ITO-glass) were then washedwith ethanol, dried under N2 and masked with insulating tape toopen a working zone area of 0.25 cm2. These were then immersedin aqueous suspension of GO (0.3mgmL1) for 1 h for the elec-trostatic deposition of negatively charged GO sheets on the pos-itively charged SAM of APTES, followed by washing with distilledwater and drying under vacuum. Subsequently, the surface boundoxygenated functional groups of GO such as alchohol, ketone,epoxides, and esters were electrochemically reduced by CV in0.5mol L1 degassed (N2 purged) KCl aqueous solution, in a poten-tial window 0.1 V to 1.1 V, at a scan rate of 50mV s1, for threeconsecutive CV cycles, to obtain the RGO/APTES/ITO-glass.23
The polyvinylpyrrolidone (PVP) capped PtNP was prepared byreducing hexachloroplatinic acid hexahydrate (H2PtCl6.6H2O)using NaBH4, followed by stabilization of the colloidal solu-tion by adding PVP, using a procedure reported earlier.24 ThePtNP incorporated PPy-PPa polymer nanocomposite film waselectrochemically grown on the RGO/APTES/ITO-glass in asingle step electrochemical polymerization method using adegassed aqueous solution of 0.1mol L1 pyrrole (Py), 0.03mol L1
pyrrolepropylic acid (Pa), 0.1mol L1 p-TSA and 0.2mgmL1 PtNP,at a fixed current density of 1mA cm-2 with a total induced chargedensity 100 mC cm-2. This was taken as an optimum inducedcharge density, since any charge density
Physicochemical characteristics of RGO based PtNP-CP composite www.soci.org
Scheme 1. Schematic representation of the stepwise fabrication of the polymer nanocomposite film based bioelectrode and its immunoreactions withcomplementary Ag-cMb.
The PtNP(PPy-PPa)-RGO nanocomposite film was immobilizedwith myoglobin protein antibody, Ab-Mb, by first activating thecarboxyl groups of the copolymer film by treating it with an aque-ous solution of NHS:EDC (1:5) for 1 h, followed by their cova-lent linkage to the active primary amino groups of Ab-Mb inPBS containing 100 gmL1 Ab-Mb, for a period of 3 h at 4 C.This was then washed three times with PBS and dried under vac-uum and was treated with 1% (w/v in PBS) bovine serum albu-min (BSA), for 30min to passivate the substrate surface throughblocking of the nonspecific binding sites. This was followed bywashing with PBS, drying under vacuum to obtain the desiredAb-Mb/PtNP(PPy-PPa)-RGO/APTES/ITO-glass bioelectrode, whichwas stored at 4 C. The stepwise fabrication of the bioelectrode isrepresented schematically in Scheme 1.
RESULTS ANDDISCUSSIONHRTEMand EDAX characterization of PtNP(PPy-PPa)-RGOfilmA composite microstructure of PtNP(PPy-PPa)-RGO prepared ona Cu grid, by the procedure described above, was investigatedby HRTEM and EDAX spectroscopy. A uniform microstructure wasdiscerned throughout the specimen (Fig. 1(a)). As an illustrativeexample an image of the ultra-fine bare-nanoparticles of Pt isdisplayed as an inset (i) in Fig. 1(a). These nanoparticles repre-sent a narrow size distribution of approximately 4 to 8 nm withirregular and rounded shapes. The nanoparticles, upon deposi-tion on the copolymer with a layer of RGO underneath, show adiffused character at their boundary surfaces (Fig. 1(a)) and there-fore the contrast between individual nanoparticles may not beso sharp as seen in their original condition (inset (i) of Fig. 1(a)).The composite microstructure in Fig. 1(a) clearly reveals that thebase surface of Pt NP is uniform because the copolymer with con-tours of graphene-sheets constitutes a layer on which copolymerwas deposited. It is important to mention that such nano-scalemicrostructural changes were possible only under adequate oper-ating conditions of magnification, contrast, and tilting, etc, whencarrying out electron microscopy measurements. The wrinklesmarked by a set of curly arrows in the micrograph are indica-tive of overlapping sheets of RGO. An interplanar spacing of
about 0.32 nm is observed on a typical honey-comb structureof graphene (inset (ii) of Fig. 1(a)). Energy dispersive X-ray spec-troscopy (EDS) performed on the composite shows the presenceof prominent elemental peaks at different energy levels for Pt(2.050 keV, PtM and 2.12 keV, PtM ), C (0.278 keV, CK), N (0.392 keV,NK), O (0.525 keV,OK) andCu (0.930 keV, CuL) as delineated in theinset (iii) of Fig. 1(a).In order to further investigate the morphology of PtNP within
the copolymer, HRTEM images were taken of bare Pt NP as wellas PtNP(PPy-PPa)-RGO composite. Figure 1(b) shows bare PtNPof size about 5 nm with sharp contrast and clearly distinguish-able inter-atomic planes. The interplanar spacing of 0.23 nm corre-sponds toMiller indices (hkl): 111 of the bulk Pt fcc structure (spacegroup: Fmm 3, and lattice parameter: a= 0.39 nm, ref. JCPDS cardno. 040802). The PtNP in the copolymer (Fig. 1(c)) also displays anatomic scale fringe contrast due to Pt. However, the contrast is dif-fused due to the presence of the copolymer. The glazy appearanceof atomic scale fringes is presumably evolved due to the coexis-tence of graphene and copolymer in conjunction with PtNP.
Electrochemical characterization of the bioelectrodeElectrochemical impedance spectroscopy (EIS) was used to char-acterize the PtNP-polyme...