Journal of Electroanalytical Chemistry 686 (2012) 69–72

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Journal of Electroanalytical Chemistry

journal homepage: www.elsevier .com/locate / je lechem

Chitosan-modified graphene electrodes for DNA mutation analysis

Subbiah Alwarappan a,b, Kyle Cissell c, Suraj Dixit a, Chen-Zhong Li d,⇑, Shyam Mohapatra a,⇑a USF Nanomedicine Research Center and Division of Translational Medicine, Department of Internal Medicine, USF Morsani College of Medicine, University of South Florida,Tampa, FL 33612, United Statesb Nanomaterials Research and Education Center, University of South Florida, Tampa, FL 33620-5350, United Statesc TransGenex Nanobiotech Inc., Nebraska Business Park, Tampa, FL 33613, United Statesd Nanobiosensors/Bioelectronics Laboratory, Department of Biomedical Engineering, Florida International University, Miami, FL 33174, United States

a r t i c l e i n f o

Article history:Received 29 July 2012Received in revised form 19 September 2012Accepted 20 September 2012Available online 28 September 2012

Keywords:GrapheneDNAChitosanMutation

1572-6657/$ - see front matter � 2012 Elsevier B.V. Ahttp://dx.doi.org/10.1016/j.jelechem.2012.09.026

⇑ Corresponding authors. Addresses: NanobiosensoDepartment of Biomedical Engineering, Florida Intern33174, United States (C.-Z. Li), USF Nanomedicine ResTranslational Medicine, Department of Internal MediMedicine, University of South Florida, Tampa, FL 33patra).

E-mail addresses: licz@fiu.edu (C.-Z. Li), smohapat

a b s t r a c t

Graphene has remarkable electrochemical properties that make it an ideal material for constructing bio-sensors. Herein, we report on a chitosan-modified graphene platform for the electrochemical detection ofchanges in DNA sequences. For this purpose, graphene synthesized chemically and characterized byRaman spectroscopy and Transmission electron microscopy, was covalently modified with positivelycharged chitosan to facilitate the immobilization of a single-stranded DNA ‘capture’ oligonucleotide.The covalent attachment of chitosan to graphene was confirmed by FT-IR spectroscopy and then the cap-ture DNA was immobilized on to the chitosan modified graphene electrode. Then, the target DNA (com-plementary or mismatched ‘mutant’ DNA) was applied to the electrode and cyclic voltammetry wasperformed. The results of the voltammetric experiments indicate that the chitosan modified grapheneelectrodes immobilized with ssDNA+complementary DNA exhibit a significantly higher magnitude ofredox peak current than the chitosan modified graphene electrodes immobilized with the non-comple-mentary mutant DNAs. Together, these results demonstrate that the chitosan-graphene platform pro-vides a rapid, stable and sensitive detection of mismatched DNA and has the potential to be used forpoint-of-care diagnostic tests for specific DNA mutations associated with disease conditions.

� 2012 Elsevier B.V. All rights reserved.

1. Introduction

Graphene [1], one of the exotic new nanomaterials of the 21stcentury, is a two-dimensional sheet comprised solely of sp2 carbonatoms arranged in a chicken-wire like framework [2–5]. Graphenepossesses outstanding electronic [6–8], mechanical [9–11], ther-mal [12–14], optical [15,16] and charge transport [17,18] proper-ties that make it an attractive choice for biosensors [19–23], fuelcells [24,25], batteries [26,27], ultracapacitors [28,29], electrome-chanical resonators [30,31] and field effect transistors [32,33]. Al-tered DNA sequences in specific genes are characteristic of manydiseases from cancer to asthma and their identification is impor-tant for correct diagnosis and treatment. While DNA mutationsor single-nucleotide polymorphisms can be detected by directsequencing or PCR, these methods require costly instruments,

ll rights reserved.

rs/Bioelectronics Laboratory,ational University, Miami, FLearch Center and Division ofcine, USF Morsani College of612, United States (S. Moha-

@usf.edu (S. Mohapatra).

extensive training and relatively long run times. A rapid, inexpen-sive, specific, and sensitive device for detecting mismatched DNAsthat could be used in the field or during a clinic visit would proveinvaluable to physicians, especially in resource-limited areas. In or-der to develop such a device, we modified graphene electrodeswith biocompatible chitosan. Single-stranded DNA (ssDNA) oligo-nucleotides antisense to the target DNA were then electrostaticallybonded to chitosan as capture molecules. Chitosan is a natural bio-polymer that contains primary amine groups at the C-2 position ofthe glucosamine moiety and is soluble in buffers at pH < 6.5[34,40]. In solution, chitosan acquires a positive charge due tothe formation of �NHþ3 groups and these interact electrostaticallywith the negatively charged phosphate backbone of the ssDNA toform a strong and stable bond that holds the molecules in positionfor hybridization with other ssDNAs.

In this work, we prepare a chitosan modified graphene blendand then immobilize them onto a glassy carbon electrode. Thechitosan modified graphene electrode thus obtained was thenmodified with the capture ssDNA. A buffer solution containingthe target DNA or mismatched DNA was applied to the electrodeand cyclic voltammetry was performed. The chitosan-modifiedgraphene electrodes acted as a stable and sensitive platform forthe rapid and specific electrochemical detection of DNA and

70 S. Alwarappan et al. / Journal of Electroanalytical Chemistry 686 (2012) 69–72

allowed us to rapidly and easily distinguish complementary fromnoncomplementary DNA.

2. Materials

Hydrazine hydrate, graphite, potassium hexacyanoferrate (III),potassium hexacyanoferrate (IV), sodium phosphate (dibasic salt),sodium chloride, trisodium citrate, and bovine serum albuminwere all purchased from Sigma–Aldrich (St. Louis, MO) and usedwithout any further purification. Chitosan (33 kDa) was a gift fromTransgenex (Tampa, FL). Glassy carbon electrodes, Ag/AgCl (3.0 MKCl) reference electrodes and platinum wire counter electrodeswere all purchased from CH Instruments (Austin,Texas).

2.1. DNA sequences

� 50-GACTCTGGTAACTAGAGATC-30 (capture DNA).� 50-GATCTCTAGTTACCAGAGTC-30 (complementary DNA).� 50-CCATATCACCTAGAACTTTA-30 (mismatch DNA).

These DNA oligonucleotides were synthesized by IntegratedDNA Technologies Inc. (Coralville, IA).

3. Methods

3.1. Graphene synthesis

Graphene was synthesized according to the method reportedearlier [19]. Initially graphene oxide (GO) was synthesized by fol-lowing Hummers method [35]. GO was mixed with de-ionizedwater and sonicated until it becomes clear. The clear solutionwas treated with hydrazine hydrate and heated to 100 �C in anoil-bath for 24 h. This resulted in the formation of a black precipi-tate. This precipitate was filtered and washed several times withde-ionized water. The precipitate was finally dried in nitrogenatmosphere for about 6 h to get pure graphene.

3.2. Raman Spectroscopy

Raman measurements were conducted at room temperaturewith the help of a Reinshaw spectrometer in the back scatteringconfiguration that operates with a 514 nm Ar+ laser at 50 mW.Raman spectra were collected for each sample after a 15-min expo-sure time using a high throughput holorographic imaging spectro-graph that contains volume transmission grating, holorographicnotch filter and a �70 �C Peltier cooled CCD with 4 cm�1

resolution.

3.3. Transmission electron microscopy (TEM)

TEM measurements were made using TECHNAI F20. The elec-tron source is a Schotty Field emitter operating at 0.7 eV. TECHNAIF20 employed in this work has a point resolution of 0.24 nm, lineresolution of 0.102 nm and information limit of 0.14 nm.

3.4. Synthesis of chitosan-modified graphene

Graphene as obtained above (5 mg/100 mL) was sonicated(Model: Branson 2510) in de-ionized water for an hour at 25 �Cto yield a homogeneous suspension. To this suspension, chitosan(5 mg) was added and vortexed for one hour at 25 �C to yield achitosan-modified graphene (CMG) suspension. Following this,5 lL of CMG suspension was pipetted onto the surface of a glassycarbon electrode to serve as the electrochemical platform for the

electroanalysis. The as prepared CMG platform is stable between4 �C and 50 �C.

3.5. Fourier transform infra-red spectroscopy

FTIR characterization of graphene and chitosan modified graph-ene nanosheets were performed using a Nicolet IR-100 spectrom-eter. 10 ll (of the sample was dropped onto a disposablepolyethylene IR card and the solution was dried under vacuumprior taking the measurements.

3.6. Capture DNA immobilization procedure

About 30 pg of capture DNA in 0.1 M phosphate buffered saline(PBS) was pipetted onto the CMG electrode and allowed to remainat room temperature for 15 min. The positive charge on the CMGwill exert an electrostatic interaction onto the negatively chargedphosphate backbone to hold the capture DNA in place. The elec-trodes were rinsed twice with PBS to remove excess DNA. Theamount of capture DNA (30 pg) that needs to be immobilized toachieve a steady and observable signal was optimized by testinga range of DNA concentrations.

3.7. Hybridization of target DNA with capture DNA

The hybridization was performed by pipetting an aliquot of thetarget DNA in 5 � SSC (pH = 6.1) onto the electrode coated with thecapture DNA and allowing it to remain at room temperature for30 min. The electrodes were then rinsed twice with SSC to removenonspecifically bound target DNA.

3.8. Cyclic voltammetry

The electrochemical readings were performed using a CHI-630Aelectrochemical analyzer (CH Instruments, Inc.). The working, ref-erence and counter electrode(s) were CMG, Ag/AgCl (3.0 M KCl)and platinum wire respectively. In all experiments, the surfaceareas of the CMG modified glassy carbon electrodes, Ag/AgCl andPt-counter electrodes were identical. All the voltammetric experi-ments were performed in a 5 mL vial containing the redox probe0.1 mM K4[Fe(CN)6]3�/4� in 1.0 M KCl. Before performing voltam-metry, the vial containing the redox probe was purged 2 min withnitrogen to remove oxygen.

4. Results and discussion

Initially, graphene was synthesized as detailed in the methodssection. Next, the surface of graphene nanosheets was character-ized by Raman spectroscopy and transmission electron micros-copy. The Raman spectrum of graphene at 514 nm shows a Gpeak at �1590 cm�1 and a 2D peak at �2700 cm�1 (Fig. 1A). TheG peak is attributed to the doubly degenerate zone center (E2g

mode) [36,37] and the 2D peak is due to the presence of a pair ofphonons with opposite momentum in the highest optical branchnear the K [38,39,41]. The number of layers of graphene as deter-mined by the high resolution electron micrograph was found tobe 7 and the d value (inter-layer spacing) was calculated to be0.35 nm (Fig. 1B). Following this, graphene was subjected to FT-IR analysis. The FT-IR spectrum of graphene (Fig. 2) exhibited theIR peaks at 2920 cm�1 and 2849 cm�1 (sp2 mC–H stretching),1731 cm�1 and 1626 cm�1 (mC=O), 1481 cm�1 (skeletal vibrationsof graphene sheets), 1461 cm�1 (carboxyl mC–O stretching), 1177cm�1 (alkoxy mC–O stretching at the edges of graphene) and1051 cm�1 (mC–O stretching). Then, the graphene surface was cova-lently modified with chitosan to make the surface more positive

Wave number (cm-1)

Inte

nsity

(a.u

)

250

200

150

100

50

3000250020001500

10 nm

(A) (B)

Fig. 1. Raman spectrogram (A) and transmission electron micrograph (B) of graphene.

Fig. 2. FT-IR spectra of graphene and chitosan-modified graphene.

(a)

(b)

(c)

(a)

(b)

(c)

Fig. 3. Illustration depicting the DNA orientation on a CMG electrode and thecorresponding voltammetric response: (a) ssDNA, (b) ssDNA plus complementaryDNA, and (c) ssDNA plus mismatch DNA.

0.40.20.0-0.2-0.4

E/V vs Ag/AgCl

I/ µA

5µA(b)

(a)

(c)

Fig. 4. Cyclic voltammogram of the CMG electrode with (a) ssDNA alone, (b) ssDNAplus complementary DNA, and (c) ssDNA plus mismatch DNA.

S. Alwarappan et al. / Journal of Electroanalytical Chemistry 686 (2012) 69–72 71

and electrostatically trap the capture ssDNA onto the CMG plat-form. Chitosan modified graphene exhibited all the FT-IR bandsas discussed above with the addition of a band at 1635 cm�1

(mCONH) indicating the covalent modification of graphene withchitosan (Fig. 2).

In order to determine whether the graphene electrode couldidentify a mutant DNA, electrodes were loaded with captureDNA then cyclic voltammetry was performed in 0.1 mMK4[Fe(CN)6]3�/4�/1.0 M KCl with capture ssDNA alone, ssDNA pluscomplementary DNA and ssDNA plus non-complementary DNA.The introduction of ssDNA on to the CMG surface is expected tosterically hinder the diffusion of the [Fe(CN)6]3�/4� ions towardsthe electrode surface. The steric hindrance experienced by the[Fe(CN)6]3�/4� ions is mainly due to the way in which the ssDNAorients itself on the electrode surface (Fig. 3a). Furthermore, thenegatively charged [Fe(CN)6]3�/4� ions are expected to experiencean electrostatic repulsion from the negatively charged phosphategroups on the capture ssDNA. As a result of this combinedhindrance, the diffusion of the [Fe(CN)6]3�/4� towards the electrodesurface was restricted compared to an electrode with no DNA (datanot shown). Next, we applied the complementary target DNA tothe ssDNA-loaded CMG platform. The complementary DNA hybrid-ized perfectly with the capture DNA and a double-stranded struc-ture was formed by hydrogen bonding between the bases. Uponforming the double-stranded helix, the dsDNA is expected to orientitself perpendicular to the electrode platform as shown in scheme(Fig. 3b). This perpendicular orientation causes less steric hin-

drance to the negatively charged [Fe(CN)6]3�/4� ions diffusing to-wards the electrode surface. Also, the formation of hydrogenbonds will weaken the negative charge on the phosphate backbonethereby minimizing the electrostatic repulsion between the dsDNAand the [Fe(CN)6]3�/4� ions. This explanation was confirmed fromthe voltammogram obtained in this work using dsDNA modifiedCMG electrode which showed an increase in the magnitude ofthe voltammetric peak current (Fig. 4a and b). Though there is asignificant increase in the peak current value after hybridization,the DEp value (60 ± 3.5 mV, N = 7) remained the same before andafter hybridization.

In the next set of experiments, we applied a non-complemen-tary target DNA onto the ssDNA-modified CMG platform and cyclicvoltammetry was performed in 0.1 mM K4[Fe(CN)6]3�/4�/1.0 M KCl

72 S. Alwarappan et al. / Journal of Electroanalytical Chemistry 686 (2012) 69–72

(Fig. 4c). The significant decrease in the magnitude of the peak cur-rent seen here is attributed to the major steric and repulsive effectoffered to the [Fe(CN)6]3�/4� ions jointly by the capture and targetDNAs. The non-complementary target DNA does not hybridize tothe capture ssDNA but it is expected to bind electrostatically tothe surface of the CMG electrode itself and cause a severe blockageto the [Fe(CN)6]3�/4� ions diffusing towards the electrode surface(Fig. 3c). However, there was no significant change in the DEp va-lue upon comparison of the electrode with capture DNA vs captureDNA+complementary DNA. The magnitude of the voltammetricpeak current varies in the following order with a negligiblevariation in their DEp value: i(complementary DNA) > i(capture DNA alone) >i(noncomplementary DNA), suggesting the usefulness of this approach fordetecting DNA sequence alterations.

5. Conclusions

In this report, we have demonstrated the ability of the CMGplatform to electrochemically distinguish complementary DNAfrom noncomplementary DNA providing a simple, sensitive, andstable platform for detecting specific mutations. Our work iscontinuing with the construction of a prototype CMG electrode ar-ray for the identification of single nucleotide polymorphisms asso-ciated with various human diseases.

Acknowledgments

This work is supported by the following grants 1RO1CA152005-01 (National Cancer Institute), N00014-09-1-1008 (Office of NavalResearch) awarded to Prof. Shyam Mohapatra and W81XWH-10-1-0732 (TATRC) and Kauffman Professor Award to Dr CZ Li. We thankDr. Gary Hellerman, Nanomedicine Research Center, USF MorsaniCollege of Medicine, University of South Florida, Tampa, FL forhis critique to enhance the quality of the manuscript. We wouldalso like to thank Dr. Yusuf Emirov, Nanomaterials Research andEducation Center (NREC) at USF for helping us during TEM imaging.Authors thank Prof. Kumar at the College of Engineering, Universityof South Florida for kindly allowing us to use the electrochemicalwork station available in his lab to perform our experiments

References

[1] K.S. Novoselov, A.K. Geim, S.V. Morozov, D. Jiang, Y. Zhang, S.V. Dubonos, I.V.Grigorieva, A.A. Firsov, Science 306 (2004) 666.

[2] A.K. Geim, P. Kim, Sci. Am. 298 (2008) 90.[3] A.K. Geim, Science 324 (2009) 1530.[4] V.I. Falko, A.K. Geim, Eur. Phys. J. Special Topics 148 (2007) 1.

[5] F. Schedin, A.K. Geim, S.V. Morozov, E.W. Hill, P. Blake, M.I. Katsnelson, K.S.Novoselov, Nature Mater. 6 (2007) 652.

[6] A.H. Castro Neto, F. Guinea, N.M.R. Peres, K.S. Novoselov, A.K. Geim, Rev. Mod.Phys. 81 (2009) 109.

[7] N.M.R. Peres, F. Guinea, A.H. Castro Neto, Phys. Rev. B 73 (2006) 125411.[8] K.S. Novoselov, S.V. Morozov, T.M.G. Mohinddin, L.A. Ponomarenko, D.C. Elias,

R. Yang, R.I.I. Barbolina, P. Blake, T.J. Booth, D. Jiang, J. Giesbers, E.W. Hill, A.K.Geim, Phys. Stat. Solidi B – Basic Solid State Physics 244 (2007) 4106.

[9] T.J. Booth, P. Blake, R.R. Nair, D. Jiang, E.W. Hill, U. Bangert, A. Bleoloch, M. Gass,K.S. Novoselov, M.I. Katnelson, A.K. Geim, Nano Lett. 8 (2008) 2442.

[10] J.C. Meyer, A.K. Geim, M.I. Katnelson, K.S. Novoselov, D. Obergfell, S. Roth, C.Girit, A. Zettl, Solid. Stat. Commun. 143 (2007) 101.

[11] S. Scharfenberg, D.Z. Rocklin, C. Chialvo, R.L. Weaver, P.M. Goldbart, N. Mason,Appl. Phys. Lett. 98 (2011) 091908.

[12] F. Clement, F. Blaise, M. Orlita, M. Potemskim, R.R. Nair, A.K. Geim, ACS Nano 4(2010) 1889.

[13] A.A. Balandin, Nat. Mater. 10 (2011) 569.[14] A.A. Balandin, S. Ghosh, W. Bao, I. Callzo, D. Teweldebrhan, F. Mlao, C.H. Lau,

Nano Lett. 8 (2008) 902.[15] K.S. Kim, Y. Zhao, H. Jang, S.Y. Lee, J.M. Kim, J.H. Ahn, P. Kim, J.Y. Choi, B.H.

Hong, Nature 457 (2009) 706.[16] S. Bae, H. Kim, Y. Lee, X.F. Xu, J.S. Park, Y. Zheng, J. Balakrishnan, T. Lei, H.R. Kim,

Y.I. Song, Y.J. Kim, K.S. Kim, B. Ozyilmaz, J.H. Ahn, B.H. Hong, S. Iijima, Nat.Nanotechnol. 5 (2010) 575.

[17] S. Cho, M.S. Fuhrer, Phys. Rev. B 77 (2008) 08140. R.[18] M.Y. Han, J.C. Brant, P. Kim, Phys. Rev. Lett. 104 (2010) 056801.[19] S. Alwarappan, A. Erdem, C. Liu, C.-Z. Li, J. Phys. Chem. C 113 (2009) 8853.[20] S. Alwarappan, C. Liu, A. Kumar, C.-Z. Li, J. Phys. Chem. C 114 (2010) 12920.[21] Z. Tang, H. Wu, J.R. Cort, D.W. Buchko, Y. Zhang, Y. Shao, I.A. Aksay, J. liu, Y. Lin,

Small 6 (2010) 1205–1209.[22] L. Zhu, L. Luo, Z. Wang, Biosens. Bioelectron. 35 (2012) 507–511.[23] F. Li, Y. Huang, Q. yang, Z. Zhong, D. Li, L. Wang, S. Song, C. Fan, Nanoscale 2

(2010) 1021–1026.[24] C. Liu, S. Alwarappan, Z. Chen, X. Kong, C.-Z. Li, Biosens. Bioelectron. 25 (2010)

1829.[25] B. Seger, P.V. Kamat, J. Phy, Chem. C 113 (2009) 7990.[26] Z.S. Wu, W.C. Ren, L. Wen, L.B. Gao, J.P. Zhao, Z.P. Chen, G.M. Zhou, F. Li, H.M.

Cheng, ACS Nano 4 (2010) 3187.[27] P.S. Min, E.-J. Yoo, H. Itaru, Nano Lett. 9 (2009) 72.[28] M.D. Stoller, S.J. Park, Y.W. Zhu, J.H. An, R.S. Ruoff, Nano Lett. 8 (2008) 3498.[29] C. Liu, Z. Yu, D. Neff, A. Zhamu, B.Z. Jang, Nano Lett. 10 (2010) 4863.[30] J.S. Bunch, A.M. Van der Zande, S.S. Verbridge, I.W. Frank, D.M. Tanenbaum,

J.M. Parpia, H.G. Craighead, P.L. McFuen, Science 315 (2007) 490.[31] C. Chen, S. Rosenblatt, K.I. Bolotin, W. Kalb, P. Kim, I. Kymissis, H.L. Stormer,

T.F. Heinz, J. Hone, Nat. Nanotechnol. 4 (2009) 861.[32] Y.M. Lin, K.A. Jenkins, A. Valdes-Garcia, J.P. Small, D.B. Farmer, P. Avouris, Nano

Lett. 9 (2009) 422.[33] F. Schwierz, Nat. Nanotechnol. 5 (2010) 487.[34] H. Hengames, M. Barikani, Monatsh. Chem. 140 (2009) 1403.[35] W.S. Hummers, R.E. Offeman, J. Am. Chem. Soc. 80 (1958) 1339.[36] F. Tuinstra, J.J. Koenig, Chem. Phys. 53 (1970) 1126.[37] A.C. Ferrari, J.C. Meyer, V. Scardaci, C. Casiraghi, M. Lazzeri, F. Mauri, S.

Piscanec, D. Jiang, K.S. Novoselov, S. Roth, A.K. Geim, Phys. Rev. Lett. 97 (2006)187401.

[38] A.C. Ferrari, A.C. Robertson, J. Phys. Rev. B 6 (1) (2000) 14095.[39] S. Piscanec, M. Lazzeri, F. Mauri, A.C. Ferrari, J. Robertson, Phys. Rev. Lett. 93

(2004) 1855503.[40] P. Shradha, C.Z. Li, Biosens. and Bioelectron. 26 (2010) 1743.[41] S. Alwarappan, P. Shradha, A. Durygin, C.Z. Li, J. Nanosci. Nanotechnol. 9 (2009)

2991.