Electrochemical Detection of Cardiac Biomarkers Utilizing …cssharma/assets/pdf/34.pdf · 2018. 1. 5. · DOI: 10.1002/elan.201501163 Electrochemical Detection of Cardiac Biomarkers

Seediscussions,stats,andauthorprofilesforthispublicationat:https://www.researchgate.net/publication/305818637

ElectrochemicalDetectionofCardiacBiomarkersUtilizingElectrospunMultiwalledCarbonNanotubesEmbedded...

ArticleinElectroanalysis·August2016

DOI:10.1002/elan.201501163

CITATIONS

0

READS

43

4authors,including:

Someoftheauthorsofthispublicationarealsoworkingontheserelatedprojects:

DesignFabricationandCharacterizationofMEMSBio-SensorforDetectionofCholeraandDiarrhea

Viewproject

Lowtemperature,LowpressureCu-Cufinepitchthermo-compressionbondingfor3DICintegration

applicationsViewproject

DurgaPrakashM

KLUniversity

11PUBLICATIONS2CITATIONS

SEEPROFILE

ShivgovindSingh

IndianInstituteofTechnologyHyderabad

110PUBLICATIONS415CITATIONS

SEEPROFILE

ChandraShekharSharma

IndianInstituteofTechnologyHyderabad

49PUBLICATIONS300CITATIONS

SEEPROFILE

AllcontentfollowingthispagewasuploadedbyDurgaPrakashMon31August2016.

Theuserhasrequestedenhancementofthedownloadedfile.Allin-textreferencesunderlinedinblueareaddedtotheoriginaldocumentandarelinkedtopublicationsonResearchGate,lettingyouaccessandreadthemimmediately.

DOI: 10.1002/elan.201501163

Electrochemical Detection of Cardiac Biomarkers UtilizingElectrospun Multiwalled Carbon Nanotubes EmbeddedSU-8 NanofibersM. Durga Prakash,*[a] S. G. Singh,[a] C. S. Sharma,[b] and V. Siva Rama Krishna[a]

1 Introduction

The focus of the present decade is towards achievingsmart and intelligent health care, which is possible onlywith the development of reliable, low cost, robust, selec-tive and sensitive biosensors. For a large set of diseasessuch as cancer, genetic disorders, vascular diseases etc.,ultrasensitivity is paramount as the concentration of tar-geted analytes is in the range of few nano-gram/ml to fewpico-gram/ml [1–3]. Apart from being ultrasensitive, thetransduction mechanism should be extremely selective.One of the robust ways to achieve high selectivity is touse immunoassay techniques. These techniques rely onhighly specific molecular recognition between antigensand antibodies and are integral part of all the main ana-lytical methods in clinical diagnostics. Techniques such asEnzyme-linked immunosorbent assay (ELISA), chemilu-minescence immunoassay, radio immunoassay, Fluoroim-munoassay are being routinely deployed in the clinics foridentifying various biomarkers [4–7]. However the equip-ment associated with these techniques are costly, bulky,time consuming and need skilled manpower to operatethem. Electrochemical immunoassay, a viable alternative,offers several advantages such as high sensitivity, fastanalysis, simple pre-treatment, small analyte volume,simple instrumentation, ease of miniaturization, to namea few [8–10]. In this technique, immunoassay reactionsoccurring at the electrode/electrolyte interface inducechanges in the electrochemical kinetics which can be ana-lysed using standard electroanalytical techniques.

Immobilization of antibodies onto the electrode surfaceis one of the critical steps in the development of an elec-trochemical immunosensor. An effective and simple im-

mobilization method enhancing the amount of antibodiesonto the electrode surface is of a great value addition asit not only results in high sensitivity but also detection oftargeted antigen over a wide range of concentrations. Thesensitivity and selectivity can be boosted manifold by de-veloping novel materials which perform the dual role ofimmobilization as well as transduction.

Nanohybrid materials are frontrunners for these appli-cations. These synthetic materials comprise

organic and inorganic components that are linked to-gether by non-covalent or covalent bonds at nanoscale.They can be synthesized in such a way that the inherentadvantages of individual components are utilized ina complementary manner resulting in a material havingadvantages of the all the constituents. Furthermore, theadvantages of nano-regime such as high surface tovolume ratio, availability of large amount of active sites,ease of surface functionalization, increased interactionbetween the materials and biomolecules owing to bothhaving similar dimensions etc., make them ideal candi-dates for developing ultrasensitive nanobiosensors [11].

[a] M. D. Prakash, S. G. Singh, V. S. R. KrishnaDepartment of Electrical EngineeringIndian Institute of TechnologyHyderabadIndia*e-mail: [email protected]

[b] C. S. SharmaDepartment of Chemical EngineeringIndian Institute of TechnologyHyderabadIndia

Abstract : In this paper we demonstrate synthesis andcharacterization of MWCNTs embedded SU-8 electro-spun nanofibers and their application towards ultrasensi-tive detection of cardiac biomarkers using Electrochemi-cal Impedance spectroscopy (EIS). The composite nano-fibers have excellent electrical and transduction proper-ties owing to the presence of MWCNTs in addition toease of functionalization and biocompatibility, which canbe attributed to the presence of SU-8. Thus the synthe-sized nanofibers are ideal candidates for sensitive biosen-

sor applications. As a proof concept, the detection of car-diac biomarkers, Myoglobin (Myo), cardiac Troponin I(cTn I) and Creatine Kinase MB (CK-MB) is demonstrat-ed. The synthesized nanofibers were functionalized withthe antibodies of the biomarkers and the detection wascarried using Electrochemical Impedance Spectroscopy,an excellent technique for understanding the adsorptionkinetics. A minimum detection limit of nano-gram/ml isdemonstrated using this nanobiosensor platform.

Keywords: Electrospinning · Nanofiber · Electrochemical Impedance Spectroscopy · MWCNT · SU-8 · Cardiac Biomarkers

www.electroanalysis.wiley-vch.de � 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2016, 28, 1 – 8 &1&

These are not the final page numbers! ��

Full Paper

Biocompatibility is one of the critical requirements fordeveloping biomedical diagnostic devices. Several bio-compatible polymers such as polystyrene (PS), poly-carbonate (PC), Poly(ethylene glycol) (PEG), Poly-(acrylic acid) (PAA), Polydimethylsiloxane (PDMS),epoxy-based negative photoresist (SU-8), chitosan [12–15] have been explored for developing miniaturized bio-sensors. One such biocompatible material whose utiliza-tion in the fabrication of biomedical devices increaseddrastically over the past few years SU-8. It is an epoxybased negative photoresist. Apart from its regular utiliza-tion in microfabrication and biocompatibility, the ease offunctionalization comes in handy for developing highlyselective biosensors. Several well developed, straight for-ward protocols for functionalization of different groupsonto SU-8 have been reported [16]. For developing nano-biosensors, SU-8 is preferable as synthesis of SU-8 basednanofibers using electrospinning technique is well ex-plored for different applications [17–20]. Electrostaticspinning or electrospinning is a very simple, robust tech-nique to create nanofibers through uniaxial stretching ofelectrically charged jet of viscoelastic polymer solution.The diameters of the fibers obtained are in range of10 mm to 10 nm [21]. In order to utilize SU-8 nanofibersfor electrochemical/electrical applications, it is essentialto increase the conductivity of the fibers as they are insu-lating in nature. Multiwall carbon nanotubes (MWCNTs)find wide range of applications in developing sensors in-cluding electrochemical sensors because of their excellentelectrical, mechanical properties. Though they have excel-lent transduction properties, surface functionalization ofMWCNTs is an issue for developing robust biosensors.Well established protocols can functionalize MWCNTs

easily, however, minimal percentage of functional groupsand their randomness may result in repeatability issue.Thus the primary focus of this work is to demonstrate theapplicability of MWCNTs embedded SU-8 hybrid nano-fibers for ultrasensitive biosensing applications as bothmaterials complement each other by overcoming their re-spective shortcomings viz., minimal functional groups forMWCNTs and lack of conductivity for SU-8. By embed-ding MWCNTs into SU-8, highly conductive nanofibersthat are biocompatible, amenable for functionalization,were synthesized. As a proof of concept, we demonstrateelectrochemical detection of three cardiac biomarkers:Myoglobin (Myo), cardiac Troponin I (cTn I) and Crea-tine Kinase MB (CK-MB) using the synthesized nanofib-ers. The schematic shown in the Figure 1 illustrates de-tailed protocol of achieving the same. The rest of the sec-tions focus on explaining these steps in detail.

2 Experimental

2.1 Materials

Standard multi-walled carbon nanotubes (MWCNTs)with diameter range of 5–20 nm was purchased from Re-inste Nano Ventures Pvt. Ltd (New Delhi, India). SU-8(2015) was purchased from MicroChem Crop (USA). My-oglobin from equine skeletal muscle, Troponin I fromhuman heart, Creatine Kinase MB fraction from humanheart and Indium Tin Oxide (ITO) Coated Substrateswere purchased from Sigma-Aldrich (USA). Monoclonalanti-Myoglobin antibody, monoclonal anti-cardiac Tropo-nin I antibody, monoclonal anti-Creatine Kinase MB anti-body, cross-linker molecules N-Hydroxysuccinimide

Fig. 1. Schematics of stepwise fabrication of bioelectrode: (A) fabrication procedure for MWCNT/SU-8 composite and (B) the im-munosensor.

www.electroanalysis.wiley-vch.de � 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2016, 28, 1 – 8 &2&

These are not the final page numbers! ��

Full Paper

sodium salt (NHS) and 1-ethyl-3-(3-dimethylamino pro-pyl)�carbodiimide (EDC) were purchased from Abcambiochemical (UK). Chloroform (CHCl3), Toluene, potassi-um ferrocyanide (K4Fe(CN)6), potassium ferricyanide(K3Fe(CN)6) were purchased from MERCK, India. Phos-phate buffer solution (PBS) were purchased from Sigma-Aldrich (USA) and all other reagents used were analyti-cal grade. All solutions were prepared with deionizedwater of 18 MW-cm purified from a Milli-Q purificationsystem.

2.2 Apparatus

Synthesis of MWCNTs embedded SU-8 nanofibers wascarried out using Electrospinning set up (E-Spin NanoPvt. Ltd, India). For structural and physical characteriza-tion of these synthesized nanofibers X-Ray Diffractionanalysis (XRD) (X Pert PRO,USA), Raman Spectrosco-py (Senterra, Bruker, UK), Scanning Electron Microsco-py (SEM)(Quanta 200, FEI, Frankfurt am Main, Germa-ny; SUPRA 40 VP, Gemini, Carl Zeiss, Oberkochen, Ger-many), Transmission Electron Microscopy (TEM) (Phi-lips, CM200) were carried out. All the electrochemical ex-periments were carried out using CH660EElectrochemical Workstation (CH Instruments, USA).

2.3 Synthesis of MWCNTs Embedded SU-8 Nanofibers

Electrospinning is a simple, robust, low cost technique toproduce nanofibers at large scale. This technique involvesapplication of a very high electric field is applied betweena syringe containing a polymer solution and a cathodewhich typically is grounded. Sub-micron fibers jet out ofthe syringe at a critical field when electrostatic forcesovercome surface tension forces and are collected ontocathode which also serves as the collector of fibers. Themorphology of fibers can be precisely controlled by opti-mizing the electrospinning parameters. In this work,MWCNTs embedded SU-8 is used as polymer solution.SU-8 is an epoxy based negative photoresist marketed byMicrochem, USA and is available in different viscosities.We have used SU-8 2015 as it meets the desired viscosityrequirements. Prior to spinning, desired weight percent-age of MWCNTs were dispersed in an organic solventand then mixed with SU-8 2015 and probe sonicated foran hour. The weight percentage of MWCNTs dictates theconductivity of the composite as SU-8 is insulating innature. It is necessary to have very high conductivity inorder to use this as an electrode material for carryingelectrochemical impedance spectroscopy. In our previouswork, we have proven that 11% w/w dispersion providesbest conductivity and it is not possible to increase theconcentration of MWCNT dispersion beyond 13% as itclogs the syringe thereby precluding the synthesis of uni-form nanofibers. Hence 11 % w/w MWCNTs dispersionwas used in this experimentation [22]. The solution wasimmediately loaded into a 26-gauge needle (internal di-ameter of 0.26 mm) and electrospinning process was car-

ried out with a clean ITO substrate as collector foralmost 30 min. The resultant mat of nanofiber was ex-tracted from ITO substrate and was used for further ex-perimentation.

2.4 Functionalization of GCE with MWCNTs EmbeddedSU-8 Nanofibers and Monoclonal Antibodies

GCE was polished with 0.05 m m alumina and washedthoroughly with DI water. MWCNTs/SU-8 dispersion wasprepared by dispersing 1 mg of synthesized MWCNTs/SU-8 nanofibers in 1 ml of toluene and sonicated for30 min. The dispersion was then drop cast onto the pol-ished electrode and was allowed to dry for 2 hours. De-pending on the target analyte, the respective antibodywas chemically attached onto this modified electrodeusing the following protocol.

A mixture of EDC/NHS along with monoclonal anti-bodies of cardiac biomarkers was sonicated for 20 min.This mixture was then drop cast onto modified electrodeand was allowed to dry for 2 hours. The electrode wasrinsed thoroughly with DI water to remove any physisor-bed monoclonal antibodies. In the subsequent sections,“modified GCE” refers to MWCNTs/SU-8 nanocompo-site coated GCE and “antibody modified GCE” refers toMWCNTs/SU-8 nanocomposite modified GCE function-alized with monoclonal antibodies.

2.5 Functionalization of GCE with MWCNTs EmbeddedSU-8 Nanofibers and Monoclonal Antibodies

Electrochemical studies were performed using an electro-chemical analyser (CH660E, CH Instruments, USA) em-ploying a three-electrode system comprising a 0.5 mmplatinum wire as the counter-electrode, Ag/AgCl (saturat-ed, 0.1 M KCl) as the reference electrode and GlassyCarbon Electrode (GCE) as the working electrode withthe dimension of 3 mm diameter. All the electrodes werepurchased from CH instruments Inc, USA. EIS measure-ments were carried out in 0.1 mM phosphate buffer solu-tion (pH 7.0, 5 ml volume) containing 2.5 mM each ofK4Fe(CN)6 and K3Fe(CN)6. Antibody modified GCE wasused as working electrode. A potential of +0.2 V was ap-plied between the working electrode and Ag/AgCl refer-ence electrode. This is the formal potential of the redoxcouple. The impedance offered by the modified workingelectrode for the electron transfer was measured betweenthe working and platinum counter electrode. The fre-quency ranges used for the measurement was from25 mHz to 1 MHz with a sine wave amplitude of 5 mV.Cyclic Voltammetry measurements were carried out spe-cifically for myoglobin detection and fresh pH 7 phos-phate buffer was used as electrolyte in those experiments.

www.electroanalysis.wiley-vch.de � 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2016, 28, 1 – 8 &3&

These are not the final page numbers! ��

Full Paper

3 Results and Discussion

3.1 HRTEM Analysis

To understand the internal structure of the MWCNT/SU-8 nanocomposite, TEM analysis was carried out. Nano-composite extracted from ITO substrate dispersed intoethanol and then drop coated onto the TEM grid. Thedispersion process resulted in tiny nanoparticles of thesynthesized nanofibers. The high resolution images of thesame is shown in Figure 2. The Figure 2 (a) shows thattubular-shaped MWCNTs are well dispersed in SU-8polymer. The length range of the MWCNTs is severaltens of micrometers, and they have an external diameterof approximately 10–50 nm. The presence of MWCNTs isconfirmed with the help of selected area electron diffrac-tion (SAED) pattern which shows graphitic (002) and(004) reflections which are key signature features ofMWCNTs (Figure 2 (b)). The high-resolution image ofthe individual MWCNTs shows outer diameters of 34 nm.The Figure 2 (c) shows higher magnification of Figure 2(a). From the atomic scale image of the MWCNTs/SU-8(Figure 2 (d)), it can be concluded that MWCNTs in thenanofiber retains its crystalline nature, with an interlayerspacing of 3.41 � (d002 for MWCNT, shown in the sub-figure inset). These results clearly reveal a successful for-mation of the MWCNT embedded SU-8 nanofibers.

3.2 Detection of Cardiac Biomarkers

The interaction of cardiac biomarkers with monoclonalantibody functionalized, MWCNTs/SU-8 nanocomposite

is shown in Figure 1 (b). When antibody modified GCE isused as working electrode the corresponding antigen getsimmobilized onto the electrode surface through immuno-assay reaction. The charge transfer resistance (Rct) ofstandard redox couple [Fe(CN)6]

3�/[Fe(CN)6]4� depends

on the electrode surface. Different electrodes have differ-ent charge transfer resistance and it depends on the sur-face condition of the electrode.

Any adsorption on the surface of an electrode acts asan inhibition to the electron transfer process thereby in-creasing the charge transfer resistance. This principle canbe used to study the adsorption behaviour at an electrodeinterface and is applied to detect cardiac biomarkers.

3.2.1 Myoglobin

a) Electrochemical Impedance Spectroscopic Study

The Nyquist plots for different concentrations of Myoglo-bin on anti-myoglobin modified GCE are shown inFigure 3. The X-axis represents the real part of the impe-dance and the Y-axis represents the imaginary part of theimpedance. The charge transfer resistance (Rct) is foundto increase with increase in the concentration of Myoglo-bin. This confirms that there is an adsorption on the elec-trode surface which is inhibiting the electron transfer rateof the redox couple [Fe(CN)6]

3�/[Fe(CN)6]4� thereby in-

creasing the charge transfer resistance. The higher is theconcentration of Myoglobin, the more is the extent of ad-sorption leading to higher charge transfer resistance. Thecontrol test for the same is carried out by repeating thesame experiment with modified GCE. In this caseMWCNTs/SU-8 nanocomposite was coated on GCE andthey were not functionalized with anti-myoglobin. In suchcase, myoglobin cannot get adsorbed onto the electrodesurface owing to absence of its corresponding antibodies.

Fig. 2. HRTEM images: (a) MWCNTs embedded SU-8 nano-composite; (b) an individual MWCNT (inset: the SEAD patternof the MWCNT); (c) illustrates a good dispersion of MWCNTsin SU-8 (d) an atomic-scale of a MWCNT/SU-8.

Fig. 3. EIS Nyquist plots obtained for anti-myoglobin immobi-lized, MWCNTs/SU-8 nanocomposite modified GCE before andafter the addition of Myoglobin; inset image (a) Variation ofstandardized charge transfer resistance with respect to concentra-tion of myoglobin.

www.electroanalysis.wiley-vch.de � 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2016, 28, 1 – 8 &4&

These are not the final page numbers! ��

Full Paper

No change in the charge transfer resistance should occurin this case. From the resultant Nyquist plot shown inFigure 4, it can be clearly inferred that there is no changein charge transfer resistance when the electrode surfacewas not functionalized with antibodies.

b) Cyclic Voltammetry Study

After the EIS experiments were completed, the workingelectrode was rinsed with DI water to remove any physi-sorbed myoglobin. Fresh phosphate buffer solution wastaken and cyclic voltammetry measurements were carriedout with this working electrode, with Pt and Ag/AgCl ascounter and reference electrodes respectively. These ex-periments were carried out to validate the chemisorptionof Myoglobin onto the modified GCE electrode andstrengthen the claim that the change in charge transfer re-sistance is due to the chemisorption of myoglobin, notphysisorption. Since myoglobin is a redox active speciescontaining (Fe2+), adsorbed species of the same ontoa working electrode should show redox behaviour even ina pure buffer solution. Furthermore, the peak currentshould be proportional to the scan rate as in the case ofany adsorbed redox species [23]. Figure 5 shows cyclicvoltammograms of the same at different scan rates. Myo-globin is showing an irreversible behaviour with a cathodicpeak at �0.35 V. This is because of the reduction of Fe3+

to Fe2+. The peak current Vs scan rate is linear (insetFigure 5), indicating the process is an adsorption processas opposed to a diffusion based mass transport processwherein the peak current is proportional to the squareroot of the scan rate [23]. Ideally, any redox active couplelike Fe2+/Fe3+ would should both anodic and cathodicpeaks at the same voltage [23]. Any shift in the peaks orappearance can be attributed to the nature of the elec-trode/adsorbed species interface. The electron tunnelingdistance is the key in this case. Lower the electron tunnel-

ing distance, the system would be closer to the ideal be-havior. As the electron tunneling distance increases, thekinetics decreases resulting in a quasireversible or irrever-sible behavior. In this case, the irreversible behaviour inthis adsorbed state is expected because the heme groupin myoglobin is embedded deep inside the protein. Theelectron tunnelling distance from the electrode surface islarge owing to the presence of the nanocomposite andmonoclonal antibody. This results in reduction in kineticsthus making the process irreversible. Cyclic Voltammetryresults indicate affirmatively that Myoglobin was ad-sorbed onto the electrode surface and was detected suc-cessfully using EIS.

Rct is calculated from Nyquist plots by fitting the curvewith parameters in the modified Randles circuit shown inFigure 6 (a). This circuit is modified to effectively capturemass transport and kinetic processes that occur in thismeasurements. A typical Randles circuit comprises ofa double layer capacitor Cdl in parallel with a series com-bination of charge transfer resistance Rct and Warburgimpedance W. This parallel combination is in series withthe solution resistance Rs [23]. However Randles circuitcannot accurately model all the behaviours especiallywhen there is a non-uniformity of the electrode and thereis a change in the environment near the electrode/electro-lyte interface. Frequency dispersion is a predominanteffort and is taken care of by using a constant phase ele-ment [24]. In the proposed circuit a parallel constantphase element (Q) is connected in parallel with solutionresistance Rs to capture the effects of frequency disper-sion on the solution resistance. An additional capacitoralong with a constant phase element is added parallel tothe double layer capacitor Cdl. This captures the effect ofsurface roughness, non-uniform surface modification andvariations in the double layer capacitance due to adsorp-tion. Rct and W represent the charge transfer resistanceand the Warburg impedance respectively. Randles circuit

Fig. 4. EIS Nyquist plots obtained for non-immobilized withMWCNTs/SU-8 nanocomposite modified GCE after the additionof Myoglobin. Fig. 5. Cyclic Voltammogram of Myglobin adsorbed, modified

GCE electrode in fresh buffer solution.

www.electroanalysis.wiley-vch.de � 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2016, 28, 1 – 8 &5&

These are not the final page numbers! ��

Full Paper

with these modifications incorporated accurately fits inthe model with an error of <2% as opposed to the origi-nal Randles circuit wherein the error is >10%. Figure 6(b). The values of Rct with Myoglobin were standardizedto the reference value of Rct without Myoglobin and aredenoted by Rnorm. The variation of Rnorm with respectto the logrithmic concentration of Myoglobin is shown inFigure 3 (a) and it is linear with a correlation coefficientof 0.999 with a CV of less than 5 %. The linearity range inthis case is from 1 ng/ml to 50 ng/ml. The limit of detec-tion achieved was 0.1 ng/ml. This is calculated using stan-dard sigmoidal binding curve analysis [25].

3.2.2 Troponin I and Creatine Kinase-MB

The procedure adapted for detecting cardiac Troponin Iand Creatine Kinase-MB is same but for the antibodythat was functionalized to the modified GCE. Their re-spective monoclonal antibodies were functionalized ontothe surface to carry out the detection. Figure 7 andFigure 8 shows the EIS plots for cTn I and CK-MB re-spectively. The inset shows the linearity range for thesebiomarkers. These markers are not electroactive. Howev-er since the antibody-antigen reactions are highly specific,the selectivity is not a concern. In these cases also, the co-efficient of variation across experiments is less than 5%.The limit of detection for cTn I and CK-MB as calculatedusing standard sigmoidal analysis were found to be 0.1 ng/ml and 1 ng/mL respectively. In the case of CTn I the lin-earity range was found to 0.1 ng/mL–10 ng/mL whereas in

CK-MB, we observed a wider range of linearity from10 ng/mL–10 mg/mL.

4 Conclusions

Synthesis and characterization of MWCNTs embeddedSU-8 electrospun nanofibers and their application to-wards ultrasensitive detection of cardiac biomarkers usingElectrochemical Impedance spectroscopy (EIS) is demon-strated. The synthesized nanohybrid composite combinedexcellent electrical and transduction properties ofMWCNTs and ease of functionalization and biocompati-

Fig. 6. (a). Modifies Randles electrical equivalent circuit and(b) Experimental and simulated (modified Randles circuit andoriginal Randles circuit) fit impedance data obtained for Myomodified GCE.

Fig. 7. EIS Nyquist plots obtained for anti-cardiac troponin Iimmobilized, MWCNTs/SU-8 nanocomposite modified GCEbefore and after the addition of cardiac troponin I; inset image(a) Variation of standardized charge transfer resistance with re-spect to concentration of cardiac troponin I.

Fig. 8. EIS Nyquist plots obtained for anti- Creatine Kinase-MB immobilized, MWCNTs/SU-8 nanocomposite modified GCEbefore and after the addition of Creatine Kinase-MB; insetimage (a) Variation of standardized charge transfer resistancewith respect to concentration of Creatine Kinase-MB.

www.electroanalysis.wiley-vch.de � 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2016, 28, 1 – 8 &6&

These are not the final page numbers! ��

Full Paper

bility of SU-8. Electrochemical detection of cardiac bio-markers, Myoglobin (Myo), cardiac Troponin I (cTn I)and Creatine Kinase MB (CK-MB) is demonstrated. Theproposed system is label-free. This reduces the complexitymanifold as labelling is a complex process. Furthermoredeveloping three electrode system using screen printedprocess is very well established and the entire process isamenable for point of care applications. A minimum de-tection limit of nano-gram/ml is demonstrated using thisnanobiosensor platform for all the biomarkers witha wide range of linearity.

References

[1] X. Chen, X. Jia, J. Han, J. Ma, Z. Ma, Biosensors and Bio-electronics, 2013, 50, 356 –361.

[2] B. V. Chikkaveeraiah, A. A. Bhirde, N. Y. Morgan, H. S.Eden, X. Chen, ACS nano, 2012, 6, 6546 –6561.

[3] A. Bhimji, A. A. Zaragoza, L. S. Live, S. O. Kelley, Analyti-cal chemistry, 2013, 85, 6813–6819.

[4] A. Voller, A. Bartlett, D. Bidwell, Journal of Clinical Path-ology, 1978, 31, 507 –520.

[5] W. Chen, W. Jie, Z. Chen, X. Jie, J. Huang-Xian, ChineseJournal of Analytical Chemistry, 2012, 40, 3–10.

[6] D. Skelley, L. Brown, P. Besch, Clinical chemistry, 1973, 19,146–186.

[7] A. Qureshi, Y. Gurbuz, J. H. Niazi, Sensors and ActuatorsB: Chemical, 2012, 171, 62 –76.

[8] D. Grieshaber, R. MacKenzie, J. Voeroes, E. Reimhult, Sen-sors, 2008, 8, 1400–1458.

[9] J. Wang, Analyst, 2005, 130, 421 –426.[10] B. J. Sanghavi, O. S. Wolfbeis, T. Hirsch, N. S. Swami, Micro-

chimica Acta, 2015, 182, 1–41.

[11] L.-H. Liu, R. M�tivier, S. Wang, H. Wang, Journal of Nano-materials, 2012, 28, 325–347.

[12] A. Alrifaiy, O. A. Lindahl, K. Ramser, Polymers, 2012, 4,1349–1398.

[13] L. Shen, Journal of functional biomaterials, 2011, 2, 355 –372.

[14] B. Lakard, G. Herlem, M. De Labachelerie, W. Daniau, G.Martin, J.-C. Jeannot, L. Robert, B. Fahys, Biosensors andBioelectronics, 2004, 19, 595–606.

[15] A. Deepu, V. Sai, S. Mukherji, Journal of Materials Science:Materials in Medicine, 2009, 20, 25–28.

[16] G. Blagoi, S. Keller, A. Johansson, A. Boisen, M. Dufva,Applied Surface Science, 2008, 255, 2896 –2902.

[17] C. S. Sharma, A. Sharma, M. Madou, Langmuir, 2010, 6,2218–2222.

[18] C. S. Sharma, R. Vasita, D. K. Upadhyay, A. Sharma, D. S.Katti, R. Venkataraghavan, Industrial & EngineeringChemistry Research, 2010, 49, 2731 –2739.

[19] S. Huang, Y. Ding, Y. Liu, L. Su, R. Filosa, Y. Lei, Electroa-nalysis, 2011, 23, 1912 –1920.

[20] C. S. Sharma, H. Katepalli, A. Sharma, M. Madou, Carbon,2011, 49, 1727 –1732.

[21] N. Bhardwaj, S. C. Kundu, Biotechnology advances, 2010,28, 325–347.

[22] P. M. Durga, K. V. Siva Rama, A. Dutta, C. Sharma, S. G.Singh, SENSORS, 2014 IEEE, 2014, pp. 2093–2096.

[23] A. J. Bard, L. R. Faulkner, Electrochemical methods: funda-mentals and applications, Wiley New York, 1980, vol. 2.

[24] P. C�rdoba-Torres, T. J. Mesquita, R. P. Nogueira, The Jour-nal of Physical Chemistry C, 2015, 119, 4136 –4147.

[25] M.J. Warwick, Immunoassay: A practical guide, Taylor &Franics Ltd., London, UK, pp. 160.

Received: December 19, 2015Accepted: July 13, 2016

Published online: && &&, 0000

www.electroanalysis.wiley-vch.de � 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2016, 28, 1 – 8 &7&

These are not the final page numbers! ��

Full Paper

FULL PAPERS

M. D. Prakash,* S. G. Singh,C. S. Sharma, V. S. R. Krishna

&& –&&

Electrochemical Detection ofCardiac Biomarkers UtilizingElectrospun Multiwalled CarbonNanotubes Embedded SU-8Nanofibers

www.electroanalysis.wiley-vch.de � 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2016, 28, 1 – 8 &8&

These are not the final page numbers! ��

Full Paper

View publication statsView publication stats