physicochemical characteristics of reduced graphene oxide based pt-nanoparticles-conducting polymer...

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Research Article Received: 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 reduced graphene oxide based Pt-nanoparticles- conducting polymer nanocomposite film for immunosensor applications Nidhi Puri, a,b Sujeet K. Mishra, a Asad Niazi, b Avanish K. Srivastava a and Rajesh a* Abstract 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 on PtNP(PPy-PPa)-RGO nanocomposite through pendant carboxyl groups of the polymer chain using carbodiimide coupling reaction 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 bioelectrode was investigated, as an impedimetric immunosensor, for the quantitative detection of the target human cardiac myoglobin antigen (Ag-cMb) in phosphate buffer silane (pH 7.4). RESULTS: The EIS studies of the bioelectrode towards Ag-cMb showed a dominant charge transfer resistance (R et ) characteristic in the frequency region 50 to 100 Hz. The EIS of the bioelectrode showed an effective immunoreaction with Ag-cMb with a significant increase in both R et and the constant phase element parameter (n) from 0.61 to 0.85. The bioelectrode exhibited a linear dynamic range of detection from 10 ng mL 1 to 1 g mL 1 , sensitivity of 107.08 cm 2 per decade and a lowest detection limit of 4.0 ng mL 1 Ag-cMb. CONCLUSION: The polymer nanocomposite with uniformly distributed PtNP in conjunction with conducting RGO support has been demonstrated to be a high performance immunosensor. This has been attributed to the synergistic combination of conducting RGO support and electroactive PtNP embedded conducting functional copolymer for efficient covalent biomolecular immobilization, responsible for high protein loading and fast interfacial electron exchange. © 2014 Society of Chemical Industry Keywords: electrochemistry; characterization; proteins; immobilization INTRODUCTION Myoglobin (cMb), a small sized (17.6 kDa) cardiac protein, is a cardiac marker released into the blood stream very shortly after acute myocardial infarction (AMI). The normal concentration range of cMb in human blood is 30 to 90 ng mL 1 , which increases to 200 ng mL 1 within 1 h of onset of myocardial infarction and reaches a highest value of 900 ng mL 1 during the peak hours. The high sensitivity and predictive value of cMb make it a valuable screening test in the 1 – 3 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 critical time window. Conventional laboratory methods used for the quan- titative detection of cMb include enzyme linked immunosorbent assay (ELISA), 3 chromatography 4 and spectrophotometry. 5 How- ever, these methods suffer from numerous disadvantages such as long reaction time, complex preparatory method, expensive reagents and requirement for trained personnel. Electrochemi- cal impedance spectroscopy (EIS) based immunosensing methods have attracted significant attention in the last few years because of their rapidity, high sensitivity and non-destructive nature. Billah et al. utilized the EIS technique to characterize the sensor sur- face assembly and successfully recognize cMb, immobilized over the self-assembled monolayer on gold electrode. 6 However, this suffers from the disadvantages of using expensive biotin function- alized Mb antibody immobilized at the transducer surface that does not make it a cost effective method. Therefore, there is still a great interest in the development of a high performance biosensor based on advanced electroactive materials for cMb detection. Correspondence to: Rajesh, CSIR-National Physical Laboratory, Dr. K.S. Krish- nan Road, New Delhi-110012, India. E-mail: [email protected] 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, New Delhi-110025, India J Chem Technol Biotechnol (2014) www.soci.org © 2014 Society of Chemical Industry

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Page 1: Physicochemical characteristics of reduced graphene oxide based Pt-nanoparticles-conducting polymer nanocomposite film for immunosensor applications

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*

Abstract

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 bioelectrode wasinvestigated, as an impedimetric immunosensor, for the quantitative detection of the target human cardiac myoglobin antigen(Ag-cMb) in phosphate buffer silane (pH 7.4).

RESULTS: The EIS studies of the bioelectrode towards Ag-cMb showed a dominant charge transfer resistance (Ret) characteristicin the frequency region 50 to 100 Hz. 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 10 ng mL−1 to 1𝛍g mL−1, sensitivity of 107.08 𝛀 cm2 per decade and a lowest detectionlimit of 4.0 ng mL−1 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 ofconducting RGO support and electroactive PtNP embedded conducting functional copolymer for efficient covalent biomolecularimmobilization, 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 afteracute myocardial infarction (AMI). The normal concentration rangeof cMb in human blood is 30 to 90 ng mL−1, which increasesto 200 ng mL−1 within 1 h of onset of myocardial infarction andreaches a highest value of 900 ng mL−1 during the peak hours. Thehigh sensitivity and predictive value of cMb make it a valuablescreening test in the 1–3 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 criticaltime window. Conventional laboratory methods used for the quan-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 immunosensing methods

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 the development of a high performance biosensorbased on advanced electroactive materials for cMb detection.

∗ Correspondence to: Rajesh, CSIR-National Physical Laboratory, Dr. K.S. Krish-nan Road, New Delhi-110012, India. E-mail: [email protected]

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

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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.7 –10 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 on MNP incorporation the conducting polymer showed supe-rior catalytic property compared to one without MNP 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 carbonallotrope 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.16 – 19 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 4 M23) and myoglobin derived from human heart tissue(Ag-cMb; Cat 8 M50) were procured from Hytest (Turku, Finland).Mouse immunoglobulin-G (Ag-IgG) (Cat IGP3) was obtained fromGENEI, Bangalore. Pyrrole, pyrrolepropylic acid, hexachloropla-tinic acid hexahydrate (H2PtCL6·6H2O, 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 Sigma–Aldrich 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 2 mmol L−1 K3[Fe(CN)6] and2 mmol L−1 K4[Fe(CN)6]. CVs were conducted in a potential win-dow of –0.1 V to +0.5 V. The EIS measurements were carried out ata bias voltage of 0.3 V with an A.C. voltage of 0.05 V over a wide fre-quency range from 1 Hz 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 5 min 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 (0.3 mg mL−1) 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 in0.5 mol L−1 degassed (N2 purged) KCl aqueous solution, in a poten-tial window 0.1 V to −1.1 V, at a scan rate of 50 mV s−1, 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 0.1 mol L−1 pyrrole (Py), 0.03 mol L−1

pyrrolepropylic acid (Pa), 0.1 mol L−1 p-TSA and 0.2 mg mL−1 PtNP,at a fixed current density of 1 mA cm-2 with a total induced chargedensity 100 mC cm-2. This was taken as an optimum inducedcharge density, since any charge density <100 mC cm-2 resulted inthe formation of an unstable polymer composite film with unevensurface features, whereas a thick film with comparatively lesselectroactive features was obtained at higher charge density.

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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 μg mL−1 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 30 min 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 AND DISCUSSIONHRTEM and EDAX characterization of PtNP(PPy-PPa)-RGO filmA 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, PtM𝛼 and 2.12 keV, PtM𝛽 ), C (0.278 keV, CK𝛼), N (0.392 keV,NK𝛼), O (0.525 keV, OK𝛼) and Cu (0.930 keV, CuL𝛼) as delineated in theinset (iii) of Fig. 1(a).

In order to further investigate the morphology of PtNP withinthe 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 to Miller 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-polymer-RGO composite system under study. Asmall amplitude ac signal over a wide frequency range in the EISwas used to measure the charge transport properties and com-plex electrical impedance (Z) to characterize the electrode surfacemodifications during fabrication of the bioelectrode. Figure 2(a)shows the Nyquist plot (real part Z’ vs imaginary part Z′′) of thebioelectrode at various surface modification steps and the insetdepicts the corresponding Randles equivalent circuit. The Nyquistplot of the impedance spectrum is analyzed by mapping theexperimental data to the computer simulated spectra from theRandles equivalent circuit, which is an electronic network consist-ing of an ohmic resistance of the electrolyte solution, Rs, in a seriescombination with a R||C network. This R||C network is divided into two parallel branches: one is the Faradaic-current branch andanother is a non Faradaic-branch. The Faradaic-current branchis the series combination of (i) the charge transfer resistance,Ret, resulting from the interfacial electron-transfer rate between

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Figure 1. (a) HRTEM micrograph showing the co-existing RGO, copolymer (PPy-PPa) and PtNP; insets: (i) bare Pt-nanoparticles, (ii) honey-comb structureof graphene, and (iii) EDS of composite material with X-axis: 0 to 4.2 keV and Y-axis: intensity in arb. unit; (b) HRTEM micrographs showing atomic scalefringes of bare PtNP; and (c) diffuse contrast on PtNP due to presence of copolymer.

the redox probe in solution and the electrode support; and (ii)the Warburg impedance, WR, resulting from ionic diffusionfrom the bulk solution to the electrode surface interface. Thenon-Faradaic branch consists of the double layer capacitance (Cdl)or constant phase element (CPE), wherein the CPE indicates thedegree of surface inhomogeneity, roughness or fractional geom-etry and electrode porosity. The impedance, (Z), of the systemassociated with CPE can be expressed by:

Z(CPE) = Q−10 (j𝜔)−n (1)

where j is√

(–1), 𝜔 is an angular frequency, n and Q0 are CPEparameters which are independent of frequency. Z(CPE) corre-sponds to a pure resistance (1/ Q0) , when n= 0; pure capaci-tance (Q0 =C) when n= 1; pure inductance (Q0 = L), when n= –1;Warburg impedance (WR), when n= 1/2. In the present system,CPE is used instead of Cdl in the Randles equivalent circuit forEIS measurements because of the inhomogeneous surface ofthe polymer nanocomposite film. This is supported by the factthat the value of ‘n’ was found to be significantly <1 (0.53),which is indicative of pseudo-capacitive behavior of the elec-trode surface with inhomogeneous features. The chi-squaredfunction (𝜒2), which is the square of the standard deviationbetween the original data and calculated spectrum, was foundto be 0.013 for the bioelectrode, where the fitting lines nearlymatch Bode and Nyquist curves, indicating the best fit equivalentcircuit.

The EIS spectrum of RGO/APTES/ITO-glass electrode showed anRet value of 119.2 Ω cm2 with CPE parameters Q0 = 31.96 μF cm-2

and n= 0.74. A large fall in the Ret value to 2.3 Ω cm2 with acorresponding increase in Q0 to 38.83 μF cm-2 was observedwith the electrochemical deposition of PtNP(PPy-PPa) film overRGO/APTES/ITO-glass electrode (Table 1). This may be ascribed tothe positive charge induced on the PPy-PPa polymer nanocom-posite film, at the bias voltage of 0.3 V used in AC impedancemeasurements, resulting in easy charge transport due to its strongaffinity towards the negatively charged redox moiety. Contrary tothe above, an increase in Ret value (29.6Ω cm2) was obtained uponbiomolecular immobilization of the PtNP(PPy-PPa)-RGO with anti-body Ab-Mb. This may be attributed to the insulating nature ofthe biomolecular layer formed over the polymer nanocompositefilm resulting in slow interfacial electron exchange between the

electrode surface and electrolyte, confirming the formation of thebioelectrode.

In order to understand the frequency dependent impedancecharacteristics of the bioelectrode, the bode plot (Fig. 2(b)) of thelogarithm of the absolute impedance, lZl and the phase shift, wasstudied over a wide frequency range of 1 Hz to 100 kHz. In thehigh frequency region 10 kHz to 100 kHz, insignificant changeswere observed in both the impedance and phase shift, showinga purely resistive behavior due to the presence of solution resis-tance Rs. At the low frequency region <10 Hz, the RGO modi-fied electrode exhibited a Ret dominant characteristics, whereasin the mid-frequency region from ∼10 Hz to 1 kHz, it mostly cor-responded to capacitive behavior. On further modification withPtNP(PPy-PPa) film, the bode plots showed Ret characteristics ina comparatively high frequency region of 0.2 to 2.0 kHz withdiffusive characteristics dominating in the low frequency region<10 Hz, a fact supported by the low value of n= 0.53. This trans-formation of Ret to a diffusive characteristic in the low frequencyregion showing an almost linear curve (log|Z| vs log f ) is attributedto fast electron exchange at the solution electrolyte interface ofthe PtNP(PPy-PPa)-RGO. After biomolecular immobilization of theabove polymer nanocomposite film, two phase angles at 30∘ and24∘ were found at high and low frequency, respectively. Theseare attributed to a kinetic and a diffusive process at high andlow frequencies, respectively.25 It is noteworthy that while thediffusive characteristic behavior is still prominent in the low fre-quency region <10 Hz, the Ret characteristics are observed at fre-quencies down to 50 to 100 Hz. This shift in Ret characteristics ofthe bioelectrode towards a lower frequency compared with barepolymer-nanocomposite electrode may be attributed to the morecompact and homogenous bioelectrode surface, as is evident by asignificant increase in the value of n from 0.53 to 0.61.

To observe a diffusion controlled process at the bioelectrodeCV scans were taken at different scan rates (25–125 mV s−1) ina potential window –0.1 V to 0.5 V in PBS (pH 7.4, 0.1 mol L−1

KCl) solution, containing a mixture of 2 mmol L−1 K3[Fe(CN)6] and2 mmol L−1 K4[Fe(CN)6]. A curve plotted of cathodic peak cur-rent Ipc vs 𝜈1/2 (Fig. 3) shows a linear relationship, indicating asemi-infinite linear diffusion controlled process at the bioelectrodesurface.26 The above linear relationship can be expressed as:

Ipc

(𝜈1∕2

)= b𝜈1∕2 + c (2)

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Table 1. EIS characteristic parameters at various stages of surface modifications of the electrode

CPE

Electrodes Ret (Ω cm2) Q0 (μF cm−2) n WR (Ω cm2) 𝜒2 (10−2)

RGO/APTES/ITO-glass 119.2 31.96 0.74 1.25× 10-3 1.8

PtNP(PPy-PPa)-RGO/ APTES/ITO-glass 2.3 38.83 0.53 1.22× 10-3 4.4

Ab-Mb/PtNP(PPy-PPa)- RGO/APTES/ITO-glass 29.6 14.21 0.61 0.20× 10-3 1.3

Figure 2. (a) Nyquist plot for RGO/APTES/ITO-glass;PtNP(PPy-PPa)-RGO/APTES/ITO-glass; and Ab-Mb/PtNP(PPy-PPa)-RGO/APTES/ITO-glass in PBS (pH 7.4, 0.1 mol L−1 KCl) containing 2 mmol L−1

[Fe(CN)6]3-/4-. Inset: Randles equivalent circuit; and (b) the correspondingbode plots.

where slope b= 5.029± 0.02 μA (mV s−1)-1/2; c= –11.14 μA with acorrelation coefficient of 0.993. This diffusion controlled process ofthe bioelectrode makes it suitable for biosensing applications.

EIS detection of protein antigen, Ag-cMbEIS response studies of the bioelectrode were carried out forthe quantitative detection of Ag-cMb in PBS (pH 7.4, 0.1 mol L−1

Figure 3. CV of bioelectrode as a function of scan rate in PBS (pH 7.4,0.1 mol L−1 KCl) containing 2 mmol L−1 [Fe(CN)6]3-/4- at scan rates 25 to125 mV s−1. Inset: plot of cathodic peak current Ipc vs v1/2.

KCl). EIS measurements of the bioelectrode were made eachtime after its incubation with 10 μL sample solution contain-ing different concentration of Ag-cMb for 10 min, followed bywashing and drying under N2 gas flow. With the observation ofno significant changes in EIS of the bioelectrode after 10 minof sample incubation, it was taken as an optimum incubationtime for the completion of immunoreaction. Figure 4 shows theNyquist and the corresponding Bode plot of the bioelectrode onimmunoreaction with different concentrations of target Ag-cMb.The diameter of the semicircle in the Nyquist plot of the bioelec-trode was found to increase gradually on successive incubationswith increasing sample concentration of Ag-cMb. This is attributedto the antigen–antibody complexes formed, due to the specificimmunoreaction interactions at the bioelectrode surface, produc-ing a kinetic barrier to interfacial electron transport, which givesrise to an overall increase in the impedance change from the base-line response at the electrode/solution interface (Table 2). Theseresults are supported by the corresponding Bode plot (Fig. 4(b))showing a trend of increasing phase angle from 30∘ to 60∘ inthe mid-frequency region 100 Hz to 2.5 kHz on immunoreaction,indicating a pseudo-capacitive behavior with significant changesin CPE. A very small change in the phase angle was observed onsubsequent immunoreaction with increasing concentration ofAg-cMb, in the low frequency region <100 Hz, where V/t is nearly

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Figure 4. (a) Faradaic impedance spectra of the bioelectrode in PBS (pH 7.4;0.1 mol L−1 KCl) solution containing 2 mmol L−1 [Fe(CN)6]3-/4- before andafter incubation with different concentration of Ag-cMb; (b) the corre-sponding Bode plot.

Table 2. EIS parameters of the bioelectrode on immunoreaction withdifferent concentration of Ag-cMb in PBS (pH 7.4)

CPE

Immunoreactionwith [Ag-cMb]

Ret

(Ω cm2)Q0

(μF cm-2) nWR

(Ω cm2)𝜒2

(10-2)

Control 29.6 14.21 0.61 0.20× 10-3 1.30.001 μg mL−1 89.2 13.18 0.68 0.17× 10-3 1.50.01 μg mL−1 129.2 11.78 0.77 0.17× 10-3 1.10.05 μg mL−1 171.0 9.35 0.80 0.17× 10 -3 9.40.10 μg mL−1 220.0 7.12 0.83 0.16× 10-3 5.60.50 μg mL−1 280.0 6.27 0.84 0.16× 10-3 5.71.00 μg mL−1 354.2 5.70 0.85 0.16× 10-3 6.8

in phase with I/t, representing the charge transfer characteristics ofthe bioelectrode along with the diffusion controlled process. How-ever, it is interesting to note that the diffusive characteristic of thebioelectrode observed at <10 Hz was significantly changed to Ret

behavior with a major shift in the smallest phase angle at low fre-quency on subsequent immunoreaction with Ag-cMb. This domi-nant Ret behavior in the low frequency region indicated good bio-compatibility and immunosensing capability of the bioelectrode.

Figure 5. Calibration curve of the bioelectrode for Ag-cMb and IgG concen-trations; the error bars represent the standard deviation from three sepa-rate experiments.

Figure 5 shows a calibration curve of the bioelectrode depictingchange in charge transfer resistance (ΔRet = (Ret)after immunoreaction

– (Ret)control) on immunoreaction, exhibiting a linear correlationwith the logarithm of the target Ag-cMb over the concentrationrange 10 ng mL−1 to 1000 ng mL−1, with a correlation coefficientof 0.921, according to ΔRet(Ω cm2)= 291.29+ 107.08 [Ag-cMb].The sensitivity of the bioelectrode (slope of the calibration curve)was found to be 107.08 Ω cm2 per decade. This sensitivity isabout 4 times higher than that of the pristine PPy-PPa copoly-mer film based bioelectrode.27 The lowest detection limit ofthe bioelectrode was found to be 4.0 ng mL−1 based on threetimes the signal to noise ratio. The analytical performance ofthe bioelectrode has been found to be better in terms of sensi-tivity, linear dynamic range and detection limit than previouslyreported biosensors27 – 31 (Table 3). This better performance ofthe bioelectrode may be attributed to (i) high loading and effi-cient covalent binding ability of conducting copolymer throughits pendant carboxyl groups, and (ii) the synergistic combina-tion of highly conducting RGO, PtNP and conducting PPy-PPain the nanocomposite. The highly electro-active RGO supportprovided a conducting and highly accessible surface area forthe electrochemical deposition of PtNP(PPy-PPa), wherein PtNPinduces a further increase in both the conductivity and inho-mogeneity in the polymer film for enhanced diffusion of ionsin the low AC frequency region. This synergistic effect of RGOand PtNP offered enhanced ionic and electronic transport in thenanocomposite film, responsible for enhanced immunoreaction atthe electrode/electrolyte interface, providing a better biosensingperformance.

The selectivity of the bioelectrode was investigated by record-ing ΔRet from EIS, on incubation with a 10 μL sample solution con-taining a non-specific protein antigen, Ag-IgG, over the concentra-tion range 1.0 ng mL−1 to 1000 ng mL−1 (Fig. 5). The ΔRet responsefor Ag-IgG was found to be negligible compared with values forAg-cMb over the same concentration range, indicating that thebioelectrode has high selectivity for the target Ag-cMb.

The reproducibility of the bioelectrode was investigated in termsof relative standard deviation (RSD) by taking response measure-ments with three different bioelectrodes prepared in the samemanner and was found to be in the acceptable range 3.8 to11.4% over the concentration range 1.0 ng mL−1 to 1000 ng mL−1

Ag-cMb. The stability of the bioelectrode was examined after 30days storage in a refrigerator at 4 ∘C. The bioelectrode retained

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Physicochemical characteristics of RGO based PtNP-CP composite www.soci.org

Table 3. Comparative analytical performance of the bioelectrode with existing electrochemical systems for cMb detection

Sensing technique Transducing matrix Detection range Limit of detection Sensitivity Ref.

Flow injection/amperometry

MWNTs/GCE 0.1 to3 μmol L−1(∼1.78to ∼5.34 μg mL−1)

20 nmol L−1 (∼0.35 μg mL−1) - [28]

Amperometry SPE 85 to 925 ng mL−1 - - [29]Cyclic voltammetry Ti-NT/GCE 50 nmol L−1 to

6 μmol L−1 (∼0.89to ∼106.8 μg mL−1)

1 μg mL−1 18 μA mg−1 mL [30]

Fluorescence APTES/PS 20 to 230 ng mL−1 16 ng mL−1 - [31]Impedimetric PPy-PPa/ITO 10 to 1000 ng mL−1 - 27.7 Ω cm2 [27]Impedimetric PtNP(PPy-PPa)-

RGO/APTES/ITO10 to 1000 ng mL−1 ∼4.0 ng mL−1 107 Ω cm2 Present work

about 89% of its initial sensitivity for 100 ng mL−1 Ag-cMb detec-tion, indicating good bioelectrode stability for potential applica-tions.

CONCLUSIONWe have reported a bioelectrode based on Pt nanoparticles-polymer-RGO nanocomposite, for the quantitative detection ofhuman cardiac myoglobin, Ag-cMb. The bioelectrode comprisesconducting copolymer film having evenly distributed PtNP of aver-age particle size 5 nm, electrochemically deposited on RGO sup-port. The RGO provided a conducting and highly accessible surfacearea for the electrochemical deposition of PtNP(PPy-PPa), offer-ing enhanced ionic and electronic transport in the nanocompos-ite film. Biomolecular immobilization was accomplished throughstrong covalent binding of Ag-cMb to pendant carboxyl groups ofthe PPy-PPa copolymer film using a carbodiimide coupling reac-tion. The bioelectrode performance was evaluated in terms of itssensitivity, limit of detection, dynamic range, reproducibility, andspecificity for Ag-cMb. The bioelectrode exhibited a linear dynamicrange of Ag-cMb detection from 10 ng to 1 μg mL−1 and a sen-sitivity of 107.08 Ω cm2 per decade with good reproducibility.These results demonstrate that the synergistic effects of PtNP, con-ducting polymer and RGO in nanocomposite result in enhancedsensing performance of the bioelectrode and suggest its poten-tial applications in the detection of Ag-cMb for further study in theclinical environment.

ACKNOWLEDGEMENTSWe are grateful to Professor R.C. Budhani, Director, National Phys-ical Laboratory, New Delhi, India, for providing research facilities.Authors, Nidhi Puri and Sujeet K. Mishra are grateful to CSIR forproviding Senior Research Fellowships.

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