Biosensors and Bioelectronics 63 (2015) 212–217

Contents lists available at ScienceDirect

Biosensors and Bioelectronics

http://d0956-56

n CorrE-m1 Th

journal homepage: www.elsevier.com/locate/bios

Non-enzymatic electrochemical detection of cholesterol using β-cyclodextrin functionalized graphene

Nidhi Agnihotri 1, Ankan Dutta Chowdhury 1, Amitabha De n

Chemical Sciences Division, Saha Institute of Nuclear Physics, 1/AF, Bidhannagar, Kolkata, West Bengal 700064, India

a r t i c l e i n f o

Article history:Received 9 May 2014Received in revised form17 July 2014Accepted 18 July 2014Available online 23 July 2014

Keywords:Graphene-β-CyclodextrinCholesterol sensingCyclic VoltammeteryRedox indicatorDifferential Pulse VoltammeteryInclusion complex

x.doi.org/10.1016/j.bios.2014.07.03763/& Elsevier B.V. All rights reserved.

esponding author.ail address: amitabha.de@saha.ac.in (A. De).ese authors contributed equally.

a b s t r a c t

A non-enzymatic approach towards cholesterol detection is presented here, exploiting the electro-chemical non-enzymatic route of sensing which has a distinct advantage over other conventionalenzymatic processes. Chemically converted Graphene modified with β-CD, being hydrophilic, electro-active and high surface area material, provides a platform for the electrochemical detection of cholesterolusing Methylene Blue as redox indicator. Methylene Blue (MB) forms an inclusion complex with Grp-β-CD and emerges as a cholesterol sensing matrix. MB molecule is replaced by cholesterol molecule andmoves out in the buffer solution, hence, detected electrochemically using Differential Pulse Voltammetric(DPV) technique. The sensing matrix is characterised using FT-IR and Raman spectroscopy. TransmissionElectron Microscopy is carried out to study the morphology of functionalized graphene sheets.

& Elsevier B.V. All rights reserved.

1. Introduction

Graphene has become the most exciting nano-structuredcarbon allotrope over the last few years, gaining its popularity invarious fields of material research including energy storage andconversion (Pumera, 2009), electromechanical resonators (Bunchet al., 2007), ultrafast electronic devices (Novoselov et al., 2004)etc. Applications of graphene have recently been extended even tobiological research viz., in bioelectronics (Choi et al., 2013), drugdelivery (Stoller et al., 2008; Liu et al., 2008; Ang et al., 2008),molecular resolution sensors (Schedin et al., 2007; Novoselovet al., 2006; Zhang et al., 2005) because of its large specific surfacearea, extraordinary electrical and thermal conductivities(Novoselov et al., 2004; Kanghyun et al., 2008), high mechanicalstiffness (Lee et al., 2008), good biocompatibility (Chen et al.,2008), and lower manufacturing cost (Segal, 2009). As the highelectrical and thermal conductivities of graphene originate fromthe extended long-range π-conjugation, it helps in enhancing itsusage in electrochemical approach for biosensors (Zhu et al., 2010)which is one of the most emerging fields in recent biologicalscience.

Cholesterol and its esters are membrane constituents widelyfound in biological systems which serve a unique purpose ofmodulating membrane fluidity, elasticity, and permeability

(Ikonen, 2008). It makes the cell walls rigid and strong, protectingitself from the foreign bodies. In the human serum, 80% ofcholesterol exists in the ester form. The normal level of totalcholesterol in healthy human serum is �200 mg/dL (Motonakaand Faulkner, 1993) where higher levels of cholesterol lead to life-threatening coronary heart diseases, cerebral thrombosis andatherosclerosis (Raines and Ross, 1995). Therefore, cholesterollevel in the serum is one of the most important parameters indiagnostics and prevention of heart diseases. Over various analy-tical methodologies, the electrochemical approach for biosensinghas gained momentum in past few decades, due to their highsensitivity and fast response time. The electrochemical biosensingof cholesterol has been performed by the enzymatic reaction ofcholesterol with cholesterol oxidase, where the concentration ofeither H2O2 generated or oxygen consumed during the enzymaticreaction is being monitored (Karube et al., 1982). Detectionselectivity in most of these methods relies on the use of cholester-ol selective enzymes which are expensive and prone to denatura-tion. As an alternative for simple and cost effective methods, theoptical sensors are highly appreciable whereas an electrochemicalnon-enzymatic sensing process has an ample scope for bettersensitivity.

It is well known that β-cyclodextrin (β-CD) is a cyclic oligosac-charide consisting of 7 β (1–4)-glucopyranose units. The internalcavity is lined with C(3)H and C(5)H hydrogen and ether-likeoxygen that provide a hydrophobic environment, whereas theexternal faces of the cyclodextrin molecule are hydrophilic. Due toits ability to encapsulate hydrophobic compounds, this internal

N. Agnihotri et al. / Biosensors and Bioelectronics 63 (2015) 212–217 213

cavity of β-CD allows hydrophobic cholesterol molecules to besoluble in aqueous solutions (Pitha et al., 1988; López et al., 2011).β-CDs have a high affinity for sterols as compared to other lipidsin vitro (Irie et al., 1992; Ohtani et al., 1989), which make thesecompounds quite effective in modifying cholesterol metabolism.Mondal and Jana (2012) have recently carried out fluorescentdetection of cholesterol using same Graphene-β-CD hybrid system,where the optical detection of cholesterol was carried out usingRhodamin 6G (R6G) dye as a fluorophore. But, to achieve higherdetection sensitivity along with lower detection limit and repro-ducibility, electrochemical sensor delivers more authentic resultsthan optical.

Herein, we have presented a non-enzymatic electrochemicalapproach for cholesterol sensing using Graphene-β-Cyclodextrin(Grp-β-CD) hybrid system as the sensing matrix. Grp-β-CDsolution was synthesized in situ following the route proposed byGuo et al. where treatment of graphene oxide (GO) with β-CD inpresence of ammonia and NaOH was carried out. β-CD is presumedto get covalently attached over GO sheets during the reactionforming GO-β-CD. This complex was then reduced using hydra-zine, forming Grp-β-CD. Methylene Blue (MB), a redox indicator,when added into the Grp-β-CD solution, forms a host–guestcomplex with β-CD (Zhang et al., 2003; Zhao et al., 1999). Onaddition of cholesterol in the solution, the cholesterol moleculewill replace the MB molecule in the cavity due to its higher affinitytowards β-CD, offering better detection sensitivity range viaselective host–guest interaction and graphene sheet networkhelps in rapid transfer of the electrochemical signal (as displayedin Scheme 1). As MB is a well known redox probe and hence can beeasily detected using Differential Pulse Voltammetery (DPV) tech-nique. Although there are ample reports in the literatures on thenon enzymetic electrochemical or optical biosensing proceduresfor detection of different analytes, to the best of our knowledgethis is for the first time, a completely non-enzymatic sensing withhigh sensitivity and low detection limit is being reported, using anelectrochemical DPV metric method with the targeted analyte,cholesterol.

Scheme 1. Demonstrating the mechanism of cholester

2. Materials and method

2.1. Materials

Graphite powder (o20 μm), β-cyclodextrin, cholesterol, ammo-nia solution (25%), hydrazine monohydrate (98%), Methylene Blue(MB) and dialysis membrane (MWCO 12000) were purchased fromSigma Aldrich and used as received. Deionised water from aMillipore Milli-Q ultra purification system having resistivity greaterthan 18.2 MΩ was used for synthesis.

PBS Buffer with pH 7.4 was prepared using NaCl, KCl, Na2HPO4

�2H2O, KH2PO4. All the chemicals were procured from SigmaAldrich and used as received. Interference against cholesterolsensing was studied using various interfering species: NaCl, KCl,MgCl2, Glucose, Glycine, Tyrosine, Tryptophan, Ascorbic acid,Sodium Dodecyl Sulfate (SDS), few hydrophilic drugs like Lido-caine, Chloropramine, Quinine, Quindine and Piroxicam.

2.2. Synthesis of β-cyclodextrin functionalized graphene (Grp-β-CD)from graphene oxide

Grp-β-CD was synthesized following the method reportedearlier (Guo et al., 2010) with some minor modification. Aqueoussolution of GO (5 mg/mL) was prepared by the modified Hummer'smethod (Marcano et al., 2010). In a separate vial, 400 mg of β-cyclodextrin was dissolved in 10 mL water and mixed with 200 μLNH4OH and 1 mL GO solution. The whole solution wascontinuously stirred for an hour, followed by addition of 20 μLhydrazine monohydrate solutions (0.04 M). Next, the solution washeated to 80–90 °C for an hour with constant stirring. The stableblack solution was then dialysed against distilled water withcellulose membrane for overnight. Finally, the solution was dilutedwith 0.1 M Phosphate Buffered Saline (PBS) of pH 7.4 and used asstock solution for the cholesterol sensing.

For the physical characterization of the sensing material,Transmission Electron Microscopy (TEM), FT-IR and Raman spec-troscopy were carried out. The TEM images were taken using atransmission electron microscope (FEI model Tecnei G2 20 S with200 kV accelerating voltage and resolution range of 50 nm to1 mm). FT-IR (Perkin Elmer model Spectrum 100) was done to

ol sensing, using Grp-β-CD as the working matrix.

N. Agnihotri et al. / Biosensors and Bioelectronics 63 (2015) 212–217214

identify the nature of bonding occurring between β-CD andGraphene sheets, whereas Raman spectroscopy was performed(Horiba Raman triple spectrometer T64000 with Ar ion Laser andsample imaging using Olympus 50� microscope) to characterisethe nature of the graphene sheets, under study.

2.3. Preparation of cholesterol solution

As cholesterol has very low solubility in water, 1 mM stocksolution of cholesterol was prepared by dissolving 4 mg of cho-lesterol in 10 mL ethanol. The prepared solution was used furtherfor the electrochemical detection. The cholesterol solution getssolvated in PBS buffer, during its sequential additions in theelectrochemical cell.

2.4. Preparation and functioning of the sensing probe

For the electrochemical studies, a Potentiostat/Galvanostatfrom Princeton Applied Research (Model 263A) having PS Litesoftware were used, connected with a single compartment cellwith three electrodes (where counter as well as working electrodeis Pt wire and an Ag/AgCl electrode acts as a reference electroderespectively). Initially, 3 mL Grp-β-CD solution (solvated in PBS,maintained at pH of 7.4) was taken in the electrochemical cell.

Fig. 1. (a) Color change in Graphene Oxide (GO), chemically converted graphene (CCGsheet with the spotted area showing the agglomeration of the attached β-CD, (c) FTIRshowing the 2D and G bands of Grp-β-CD in comparison with the bare CCG. (For interprweb version of this article.)

2 mL, 10 mMMethylene Blue solution was then added and kept fora while. As suggested earlier, MB molecules are expected to moveinside the cavity of the β-CD molecules, forming an inclusioncomplex of Grp-β-CD-MB (the sensing probe). The sensing probewas then centrifuged, separated and washed thoroughly to re-move the MB traces remaining on the surface. The residual probewas transferred again to the electrochemical cell with 3 mL freshPBS solution, assuming it as a blank solution. The CV and DPVmeasurements were carried out before and just after mixing withsequential addition of stock cholesterol aliquots. CV plot was takenat a scan rate of 10 mV/s, within the potential window of �0.6 to0.6 V. DPV scan was taken with pulse height of 50 mV and pulsewidth of 70 ms. Stock solution of cholesterol were added to it, insuch a manner that its final concentration in the system wouldincreased from 0 to 100 mM. Due to its higher binding affinitytowards β-CD, cholesterol molecules are expected to replace theMB molecules, forming Grp-β-CD-cholesterol complex and theextent of MB molecule moving out of the graphene–CD system,would be detected by DPV and CV measurements after eachaddition. Initially the sensing probe was characterized using CyclicVoltammetric (CV) measurements, to ensure the potential windowrequired for the DPV.

) and Graphene-β-Cyclodextrin hybrid (Grp-β-CD), (b) TEM image of the graphenespectra showing the signature of β-CD in the composite, and (d) Raman spectraetation of the references to color in this figure legend, the reader is referred to the

Fig. 2. (a) Cyclic voltammograms of the Grp-β-CD sensing probe in the PBS (at pH7.4) for the additions of analyte, cholesterol. (b) The peak variations occurring, aftercholesterol additions in the range of �0.3 to 0.3 V.

N. Agnihotri et al. / Biosensors and Bioelectronics 63 (2015) 212–217 215

3. Results and discussions

During the synthesis of Grp-β-CD, initially when graphite istreated in the presence of strong oxidizing agent, KMnO4 andmixed acid (H2SO4/H3PO4), polar oxygen functionalities developedon the graphitic sheets lead to the formation of GO. And, theseoxygen functionalities (mostly epoxy, hydroxyl, carbonyl andcarboxyl groups) render hydrophilic nature, thus making GOsoluble in water (Zhu et al., 2010, 2008). During reduction withhydrazine, chemically converted Graphene (CCG) sheets agglom-erate due to strong π–π stacking interactions with less number ofhydrophilic functional groups on the GO sheets (Zhu et al., 2008).During reduction of GO in the presence of β-CD, covalent interac-tions occur between hydroxyl groups of β-CD and oxygen func-tionalities of GO, hence forming water soluble Graphene-β-CDsystem (Fig. 1a) (Guo et al., 2010; Xu et al., 2010; Konkena andVasudevan, 2012).

3.1. Transmission Electron Microscopy

The images obtained for Grp-β-CD sheets under TEM, showingfew agglomerations in the graphene sheet, possibly for β-CD whichis covalently attached on its surface (Fig. 1b). In the TEM image, thepresence of agglomerated β-CD is well established over the sheetlike structure of graphene.

3.2. FT-IR studies

The characteristic FT-IR spectral signal of β-CD can be observedin the spectra of Grp-β-CD (shown in Fig. 1c), justifying thecovalent interactions occurring between β-CD and CCG and nophysical adsorption of β-CD over graphene sheets. The character-istic peaks of 3370 cm�1 due to stretching vibration of O–H,2925 cm�1 due to C–H stretching vibration, C¼O stretchingvibrations gives a pick at 1720 cm�1 bending stretching of C–Hfrom CH2 and CH3 gives a peak at 1420 cm�1, 1157 cm�1 is due tocoupled stretching vibrations of C–O, C–C and C–O–H, and938 cm�1 peak represents skeletal vibration involving α-1.4 link-age in Grp-β-CD. For CCG, the characteristic peaks are mainly at�3370 cm�1 due to stretching vibration of O–H (a small peak dueto the presence of O–H groups in the graphene sheets afterdeoxygenation of GO), and 1060 cm�1 is due to stretching vibra-tions from C–O, from the carboxyl groups remaining after hydra-zine reduction. The spectrum of our sensing matrix, Grp-β-CDshowed the mixed characteristic peaks of β-CD and CCG, indicatingthe successful covalent attachment of these two (Guo et al., 2010).

3.3. Raman spectroscopy

The Raman spectra (Fig. 1d), for the CCG and Grp-β-CD systemshowed D and G peaks at �1460 and 1700 cm�1 respectively,significant for the pristine graphitic nature in the CCG as well as inGrp-β-CD, even after β-CD was incorporated within the graphenesheets. The two spectra do not show any major changes in thepeak positions or intensities establishing the fact that graphenedidn't lose its graphitic structure after binding with β-CD.

3.4. Electrochemical studies for the sensing matrix

Cyclic Voltammetric (CV) studies were carried out as a char-acterization of the sensing probe for cholesterol sensing. CV plotswere taken at a scan rate of 10 mV/s, in the potential window of�0.6 to þ0.6 V, for the Grp-β-CD solution in PBS, before and afterdipping into MB (Fig. 2a). Cyclic Voltammograms showed that MBquite efficiently displayed changes in the peak current valuebetween �0.1 and þ0.1 V (as shown in Fig. 2b). Further addition

of cholesterol in the system also exhibited significant changes inthe peak current. Hence, CV results confirmed that MB is giving apeak current around �0.1 V potential (Zhang et al., 2003). There-fore, we chose the working window for DPV studies in the range of�0.4 to þ0.4 V. Comprehensively, the trends in the peak positionand intensity obtained in cyclic voltammograms, helped us in thebetter understanding of electroactivity of our sensing probe alongwith the selection of the potential window for further DPVmeasurements.

Progress of MB replacement by cholesterol is manifested in amonotonic increase of the electrochemical signal of MB in DPVwith increase of target cholesterol concentration. It can be seenthat initially, when the cholesterol concentration was zero, therewas no MB peak in the 0 to þ0.4 V regions even when the MB ispresent in the Grp-β-CD (Fig. 3). This indicates that the MB isbound in the cavity of the β-CD therefore, not able to give anyredox signal into the electrode. Though there is some probabilityof MB, which is hydrophilic in nature, to get adsorbed on thegraphene sheets, these will not make any difference to the CV andDPV results, as MB remains intact on the surface of graphene

Fig. 3. DPV of the Grp-β-CD sensing probe in the PBS at pH 7.4 and after addition ofanalyte, cholesterol, starting concentration from 1 to 100 mM. Calibration plot ofanalyte concentration dependent peak current value is given with its fitted curve inthe inset.

Fig. 4. Comparative interference studies using different species in the developedcholesterol detection method, using DPV and keeping all the parameters constant.The cholesterol concentration is 30 mM against the concentration of all othersubstances, which is kept at 2 mM.

N. Agnihotri et al. / Biosensors and Bioelectronics 63 (2015) 212–217216

sheets in the beginning as well as at the end of the experiment. Acontrol experiment carried out to exclude the possibility ofdesorption of MB from the graphene surface during cholesteroldetection proved that only the MB molecule coming out from theCD cavity is responsible for the DPV signal (Fig. S4). After additionof the cholesterol, it readily moved inside the cavity of the β-CD,replacing the MB into the solution and hence, a characteristic peakwas obtained at þ0.05 V. Cholesterol can only go inside a hydro-phobic pocket (that is of β-CD) and would replace MB molecule, ascholesterol has higher affinity towards CD as compared to MB. Themeasured binding constant between cholesterol and β-CD-G was�1.6 M�1 which is similar to the earlier observed value with freeβ-CD (Breslow and Zhang, 1996). The sensing signal of Grp-β-CDsystem increased periodically with increasing concentration ofcholesterol, up to 100 mM, as presented in the calibration curve(Fig. 3 inset). Above 100 mM, the signal reached its saturation.Detection limit was less than 1 mM which was quite low andsatisfactory with respect to other recently reported articles. Table 1illustrates few of the recent literatures on cholesterol biosensors,through both enzymatic and non-enzymatic sensing routes, estab-lishing the edge of the present work. The detection limit andsensitivity of the present sensing matrix is comparatively betterthan the reported ones, with a decent sensing time. The peakpotential of MB showed a little shift towards the positive potential.Being a pH dependent redox indicator, the peak potential of MBshifted from 0.05 to 0.1 V with decreasing pH, as the cholesterolsolution used in DPV was soluble in acidic buffer. Therefore, in the

Table 1Comparison of the present work with other recent literatures, using various immobiliza

Immobilization matrix Method ofimmobilization

Detection range(mM)

D

Tetraethylorthosilicate ChOx/HRP Covalent 2–12 ATetraethylorthosilicate ChEt/ChOx Covalent 0.184–12 PDithiobissuccinimidyl ChEt/ChOx Covalent 0.77–6.14 DGC/CS–SiO2–MECNTs ChOx Entrapment 0.004–0.7 CCHIT–SiO2–MWCNT/ITO ChET/

CHOxCovalent 0.15–7.68 D

ChOx/nano-ZnO/ITO Sol-gel entrapment 0.278–22.2 C

Grp/β-CD/Rhodamine 6G Entrapment 0.005–0.03 FGrp/β-CD/Methylene Blue Entrapment 0.001–0.10 D

higher concentration range, the MB peak in DPV shifted slightlytowards right. The cyclic stability of the sensing matrix was alsotested, before and after the addition of 10 mM cholesterol solution,by performing 100 cycles in cyclic voltammetry at a scan rate of100 mV/s, in the potential window of �0.6 to þ0.6 V. It is evidentfrom obtained voltammograms that the sensing matrix was quitestable towards its cyclability (Figs. S1 and S2).

3.5. Interaction of the interfering species

As we know, human blood serum contains many more biocomponents like salts, amino acids, carbohydrates, lipids etc.,those can interfere with cholesterol detection and hamper theselectivity of the biosensor. Therefore, we studied a wide range ofresponse in DPV for the species which can interfere with thedetection process of cholesterol in the blood serum. It is quiteevident from Fig. 4 that salts like NaCl, KCl and MgCl2; carbohy-drates and amino acids like glucose, glycine etc. showed negligibleinterference even at the concentration of 2 mM, except trypto-phan. Anionic surfactant like Sodium Dodecyl Sulfate (SDS)showed minute interference in the system, compared to choles-terol detected for only 30 mM concentration. Similarly, few hydro-phobic drugs (lidocaine, chloropramine, quinine, quinidine andpiroxicam) were checked for their interference with cholesterol.Results obtained showed very little interference for 2 mM drugconcentrations against 30 mM cholesterol concentration.

tion matrix and techniques for cholesterol sensing.

etection Process Sensitivity SensingTime (s)

References

mperometric – – Kumar et al. (2006)hotometric 8.3�10�7 Abs/mM 180 Singh et al. (2007)PV – – Salinas et al. (2006)V 1.55 mA/mM 13 Tan et al. (2005)PV 3.8 mA/mM 10 Solanki et al. (2009a,

2009b)V 0.059 A/mg dl�1

cm�210 Solanki et al. (2009a,

2009b)luorescence Micromolar range – Mondal and Jana (2012)PV 0.01 lA/lM � 20 Present Work

N. Agnihotri et al. / Biosensors and Bioelectronics 63 (2015) 212–217 217

4. Conclusion

In conclusion, we have developed an electrochemical methodof cholesterol detection using β-CD functionalized graphene. Thedetection limit of cholesterol is achieved as low as 1 mM which isquite impressive compared to recent literatures. Salient features ofthe present study include: (1) Graphene's increased solubility afterβ-CD functionalization, due to the covalent interactions occurringbetween the hydrophilic surfaces of the two; (2) The sensor caneasily detect cholesterol using DPV technique, where cholesterolmolecule is replacing MB molecule and forming the inclusioncomplex within the hydrophobic core of Grp-β-CD. Also, thedeveloped detection method is important as it does not use anyenzyme or antibody for detection and still, detects cholesterolefficiently in the micro molar concentration range with outstand-ing selectivity over the common interfering species.

Acknowledgement

The authors would like to thank MMDDA (Grant no. SIN-5.04-0200) and BARD (Grant no. SIN-5.04-0103) for funding. ADCacknowledges CSIR, India for financial support. We also thankMr. Pulak Roy, SINP for providing TEM facility, Dr. Subodh KumarDe, IACS, Kolkata for Raman studies and Mr. Sourav Chowdhury,IICB Kolkata, for providing drug samples.

Appendix A. Supplementary Information

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.bios.2014.07.037.

References

Ang, P.K., Chen, W., Wee, A.T.S., Kian, P.L., 2008. J. Am. Chem. Soc. 130, 14392–14393.Breslow, R., Zhang, B., 1996. J. Am. Chem. Soc. 118, 8495–8496.Bunch, J.S., Van Der Zande, A.M., Verbridge, S.S., 2007. Science 315, 490–493.Chen, H., Muller, M.B., Gilmore, K.J., Wallace, G.G., Li, D., 2008. Adv. Mater. 20,

3557–3561.

Choi, J., Wang, M.C., Cha, R.Y.S., Park, W., Nam, S. W., I.I., 2013. Biomed. Eng. Lett. 3,201–208.

Guo, Y., Guo, S., Ren, J., Zhai, Y., Dong, S., Wang, E., 2010. ACS Nano 7, 4001–4011.Ikonen, E., 2008. Nat. Rev. Mol. Cell Biol. 9, 125–138.Irie, T., Fukunaga, K., Pitha, J., 1992. J. Pharm. Sci. 81, 521–523.Kanghyun, K., P.Hyung, J., Woo, B.C., Kook, J.K., Gyu, T.K., Wan, S.Y., 2008. Nano Lett.

8, 3092–3096.Karube, I., Hara, K., Matsuoka, H., Suzaki, S., 1982. Anal. Chim. Acta 139, 127–132.Kumar, A., Pandey, R.R., Brantley, B., 2006. Talanta 69, 700–705.Konkena, B., Vasudevan, S., 2012. Langmuir 28, 12432–12437.Lee, C., Wei, X., Kysar, J.W., Hone, J., 2008. Science 321, 385–388.Liu, Z., Robinson, J.T., Sun, X., Dai, H., 2008. J. Am. Chem. Soc. 130, 10876–10877.López, C.A., de Vries, A.H., Marrink, S.J., 2011. PLoS Comput. Biol. 7, e1002020.Marcano, D.C., Kosynkin, D.V., Berlin, J.M., Sinitskii, A., Sun, Z., Slesarev, A., Alemany,

L.B., Lu, W., Tour, J.M., 2010. ACS Nano 4, 4806–4814.Mondal, A., Jana, N.R., 2012. Chem. Commun. 48, 7316–7318.Motonaka, J., Faulkner, L.R., 1993. Anal. Chem. 65, 3258–3261.Novoselov, K.S., Geim, A.K., Morozov, S.V., 2004. Science 306, 666–669.Novoselov, K.S., McCann, E., Morozov, S.V., 2006. Nat. Phys. 2, 177–180.Ohtani, Y., Irie, T., Uekama, K., Fukunaga, K., Pitha, J., 1989. Eur. J. Biochem. 186, 17–

22.Pitha, J., Irie, T., Sklar, P.B., Nye, J.S., 1988. Life Sci. 43, 493–502.Pumera, M., 2009. Chem. Rec. 9, 211–223.Raines, W.E., Ross, R., 1995. J. Nutr. 125, 624S–630S.Salinas, E., Rivero, V., Torriero, A.A.J., Benuzzi, D., Sanz, M.I., Raba, J., 2006. Talanta

69, 700–705.Schedin, F., Geim, A.K., Morozov, S.V., 2007. Nat. Mater. 6, 652–655.Segal, M., 2009. Nat. Nanotechnol. 4, 612.Singh, S., Singhal, R., Malhotra, B.D., 2007. Anal. Chim. Acta 582, 335–343.Solanki, P.R., Kaushik, A., Ansari, A.A., Tiwari, A., Malhotra, B.D., 2009a. Sens.

Actuators B 137, 727–735.Solanki, P.R., Kaushik, A., Ansari, A.A., Malhotra, B.D., 2009b. Appl. Phys. Lett. 94,

143901.Stoller, M.D., Park, S., Yanwu, Z., An, J., Ruoff, R.S., 2008. Nano Lett. 8, 3498–3502.Tan, X., Li, M., Cai, P., Luo, L., Zou, X., 2005. Anal. Biochem. 337, 111–120.Xu, C., Wang, X., Wang, J., Hu, H., Wan, L., 2010. Chem. Phys. Lett. 498, 162–167.Zhang, G., Shuang, S., Dong, C., Pan, J., 2003. Spectrochim. Acta A: Mol. Biomol.

Spectrosc. 59, 2935–2941.Zhang, Y., Tan, Y.W., Stormer, H.L., Kim, P., 2005. Nature 438, 201–204.Zhao, G.-C., Zhu, J.-J., Zhang, J.-J., Chen, H.-Y., 1999. Anal. Chim. Acta 394, 337–344.Zhu, L., Zhao, R., Wang, K., Xiang, H., Shang, Z., Sun, W., 2008. Sensors 8, 5649–5660.Zhu, Y., Murali, S., Cai, W., Li, X., Suk, J.W., Potts, J.R., Ruoff, R.S., 2010. Adv. Mater. 22,

3906–3924.