Electrochemical genosensor based on graphene oxide modified iron oxide–chitosan hybrid nanocomposite for pathogen detection

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  • Accepted Manuscript

    Title: Electrochemical genosensor based on graphene oxidemodified iron oxide-chitosan hybrid nanocomposite forpathogen detection

    Author: Ida Tiwari Monali Singh Chandra Mouli PandeyGajjala Sumana

    PII: S0925-4005(14)01125-3DOI: http://dx.doi.org/doi:10.1016/j.snb.2014.09.056Reference: SNB 17432

    To appear in: Sensors and Actuators B

    Received date: 20-6-2014Revised date: 2-9-2014Accepted date: 12-9-2014

    Please cite this article as: I. Tiwari, M. Singh, C.M. Pandey, G. Sumana,Electrochemical genosensor based on graphene oxide modified iron oxide-chitosanhybrid nanocomposite for pathogen detection, Sensors and Actuators B: Chemical(2014), http://dx.doi.org/10.1016/j.snb.2014.09.056

    This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.


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    Electrochemical genosensor based on graphene oxide modified iron oxide-chitosan hybrid nanocomposite for pathogen detection

    Ida Tiwari*a

    , Monali Singha,1

    , Chandra Mouli Pandeya, b,1

    , Gajjala Sumanab

    aDepartment of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi-221005,

    IndiabBiomedical Instrumentation Section, CSIR- National Physical Laboratory, New Delhi-110012,



    In this report, a nucleic acid sensor has been fabricated via covalent immobilization of

    Escherichia coli O157:H7 (E. coli) specific probe oligonucleotide sequence, onto graphene

    oxide modified iron oxide-chitosan hybrid nanocomposite (GIOCh) film. The size of the

    GIOCh, as determined by dynamic light scattering and TEM, varies from 350 to 300 nm, while

    the zeta potential for the composite remains near 60 mV at pH 4.0. These prepared GIOCh are

    electrophoretically deposited onto indium tin oxide (ITO) coated glass substrate, used as

    cathode, while parallel platinum plate is used as counter electrode. The pDNA immobilized

    onto GIOCh/ITO electrode has been characterized using scanning electron microscopy; contact

    angle and Fourier transform infrared spectroscopy. Further, the electrochemical response

    studies have been carried out using electrochemical impedance spectroscopy which reveals that

    this nucleic acid sensor exhibits a linear response to complementary DNA in the concentration

    range of 10-6

    to 10-14

    M with a detection limit of 11014

    M. Under optimal conditions, this

    biosensor is found to retain about 90 % of the initial activity after 6 cycles of use.

    Keywords: Graphene oxide, Iron oxide, Chitosan, Electrochemical impedance spectroscopy,

    Escherichia coli.

    *Corresponding author: idatiwari_2001@rediffmail.com1 These authors contributed equally.

  • Page 2 of 25






    1. Introduction1

    2Rapid and accurate detection of nucleic acids have received an unbeaten attention 3

    in the field of biomolecular sensing owing to its important role in future genetic-disease 4

    diagnosis [1]. The hybridization between a DNA probe and its complementary target 5

    plays a pivotal role in the success of drug discovery, pathogen detection and forensic 6

    identifications [2]. Based on the changes in electrochemical signals generated on DNA 7

    hybridization, various electrochemical DNA biosensors have been constructed [3]. These 8

    DNA biosensors are simple to use; can give reliable, clear, fast and inexpensive results 9

    directly at the site of interest. The problems in clinical field (pathogen detection, genetic 10

    diagnosis), can be solved using electrochemical biosensors. However, the electrochemical 11

    signals generated due to DNA bases are very weak on conventional electrodes. To 12

    overcome this problem, various nanomaterials, due to their unique structural, electronic, 13

    magnetic, optical and catalytic properties [8], such as metal nanoparticles [4-6], carbon 14

    nanotubes [6], graphene [7] have been utilized to modify the electrode surface, which 15

    could promote the electrochemical response of DNA and improve the sensitivity of 16

    DNA-based biosensors. Because of their high surface-to-volume ratio and excellent 17

    biocompatibility, nanomaterials can enhance the sensing surface area that greatly 18

    increases the amount of immobilized DNA and also when DNA mixed with 19

    nanomaterials its biological activity is maintained. In this context, nanostructured metal 20

    oxides not only possess high surface area, nontoxicity, good biocompatibility and 21

    chemical stability, but also show fast electron communication, which make the materials 22

    to function as biomimetic membrane [9], make them ideal as immobilization matrices 23

    [10] and as transduction platform [11, 12]. Further, among the metal oxide materials, iron 24

    oxide [13] has risen as a new star in the field of electrochemical sensors for their distinct 25

    catalysis capabilities, unique magnetic properties, large surface area, biocompatibility, 26

    stability in the ambient environment and effectiveness to mediate the electrochemical 27

    redox process of the analyte [14,15].2829

    In recent years graphene, a one-atom-thick planar sheet of sp2-bonded carbon 30

    atoms densely packed in a honeycomb crystal lattice [16], has grabbed appreciable 31

  • Page 3 of 25






    attention to be used as a next generation electronic material, due to its exceptional 1

    properties including high current density, ballistic transport, chemical inertness, high 2

    thermal conductivity, optical transmittance and super hydrophobicity at nanometer scale 3

    [17]. The high surface area of electrically conductive graphene sheets can give rise to 4

    high densities of attached analyte molecules thus facilitating high sensitivity and device 5

    miniaturization [18]. Further, the facile electron transfer between graphene and redox 6

    species opens up opportunities for sensing strategies based on direct electron transfer 7

    rather than mediation [19]. Zhou et al. [20] reported the electrochemical oxidation signals 8

    of four free bases of DNA (guanine (G), adenine (A), thymine (T), and cytosine (C)) at 9

    the chemically reduced graphene oxide modified electrode. Bo et al. constructed a DNA 10

    biosensor at the oxidized graphene and polyaniline nanowire modified GCE [21]. 11

    Zainudin et al. [22] fabricated a biosensor by immobilizing DNA onto graphene 12

    nanosheets by surface functionalization of graphene with 1-pyrenebutyric acid (PyBA), 13

    followed by carbodiimide linkage chemistry. A novel electrochemical DNA biosensor 14

    based on graphene-three dimensional nanostructure gold nanocomposite modified glassy 15

    carbon electrode (G-3D Au/GCE) was fabricated for detection of survivin gene which 16

    was correlated with osteosarcoma was fabricated by Liu et al. [23]. Shi et al. fabricated a17

    sensitive electrochemical DNA biosensor based on gold nanomaterial and graphene 18

    amplified signal [24]. But very few reports are available for detection of E. coli. The iron 19

    oxide nanoparticles and graphene hybrid appears to be more promising [25, 26] than 20

    graphene due to enhanced electrical property. 21

    Chitosan, a natural-biopolymer with unique structure features, possesses the 22

    primary amine at the C-2 position of the glucosamine residues and is soluble in aqueous23

    acidic media at pH< 6.5. It is fascinating over past few decades due to its unique24

    properties such as biodegradability, biocompatibility, physiological inertness and high 25

    mechanical strength [27]. It is commonly used to disperse graphene and nanomaterials 26

    for fabricating biosensors due to its excellent capability for film formation, nontoxicity, 27

    biocompatibility, mechanical strength, and good water permeability. GO nanosheets 28

    dispersed in a matrix like chitosan have been reported to enhance electrochemical 29

    performance, ease of immobilization, biocompatibility and favorable environment for 30

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    fabrication of DNA biosensors. Singh et al. [28] fabricated Graphene oxide-chitosan 1

    nanocomposite based electrochemical DNA biosensor for detection of typhoid.2

    Consequently, Fe3O4/GO/chitosan composites [29] combines the respective advantages, 3

    such as large surface area, good electronic conductivity, good stability, good 4

    biocompability and excellent catalysis, which can be used to fabricate a biosensor.5

    Hence, in this work graphene oxide modified iron oxide-chitosan hybrid 6

    nanocomposite was synthesized in simple steps at room temperature and 7

    electrophoretically deposited onto ITO coated glass substrate. Further, a nucleic acid 8

    sensor was fabricated by immobilizing probe sequence specific to E. coli. 9

    Electrochemical transduction of the hybridization reaction with complementary target 10

    sequence, single-base mismatch, and noncomplementary sequence has been carried out to 11

    investigate response characteristics of this nucleic acid sensor. To the best of our 12

    knowledge, we are reporting first time application of graphene oxide, iron oxide-chitosan 13

    hybrid nanocomposite for the enhancement of the electrochemical signal of the DNA 14

    indicator for rapid and sensitive detection of E. coli.1516

    2. Experimental Section1718

    2.1. Material and Chemicals1920

    Graphite powder, Iron(III) acetylacetonate, 1,2-hexadecanediol, oleic acid, 21

    oleylamine, glutaraldehyde, PLGA (Mw 40 00075 000), 1-ethyl-(dimethylaminopropyl) 22

    carbodiimide (EDC), N-hydroxysuccinimide (NHS), polyvinyl alcohol (PVA), and 23

    chitosan (MW 15 00020 000) were of analytical grade and purchased from Sigma-24

    Aldrich. The probe sequence (17 mer.) specific to E. coli has been identified from the 16s 25

    rRNA coding region of the E. coli genome, complementary, non-complementary and 26

    one-base mismatch target sequences have been procured from Sigma Aldrich, 27

    Milwaukee, USA. 28

    Probe I: probe DNA (pDNA): Amine-5-GGT CCG CTT GCT CTC GC-3 29

    Probe II: Complementary DNA (cDNA): 5-GCG AGA GCA AGC GGA CC-30

    3 31

  • Page 5 of 25






    Probe III: Non-complementary DNA: 5-CTA GTC GTA TAG TAG GC-1

    3 Probe IV: One-base mismatch DNA: 5-GCG AGA 2


    The solution of oligonucleotide are prepared in TrisEDTA buffer (1M TrisHCl, 4

    0.5M EDTA) of pH 8.0 and stored at -20 oC prior to use.56

    2.2. Synthesis of reduced graphene oxide78

    Graphite oxide was prepared by chemical oxidation and exfoliation of natural 9

    graphite according to a modified Hummers method [19]. In a typical preparation process, 10

    concentrated H2SO4 (69 mL) was added to a mixture of graphite flakes (3.0 g) and 11

    NaNO3 (1.5 g, 0.5 wt equiv.), and the mixture was cooled. Freshly prepared KMnO4 (9.0 12

    g, 3 wt equiv.) was added slowly in portions to keep the reaction temperature below 20 13

    C. Further, the solution was heated (35 C) and stirred for 30 min, followed by the slow 14

    addition of water (100 ml). External heating was introduced to maintain the reaction 15

    temperature at 98 C for 15 min, followed by cooling the1617

    solution using a water bath for 10 min. For purification, the mixture was washed and 18

    centrifuged with 5% HCl and deionized water several times to obtain the graphene oxide.1920

    2.3. Synthesis of iron oxide nanoparticle and graphene oxide composite2122

    Iron oxide nanoparticles (IONPs) were synthesized by the reported procedure with slight 23

    modifications [30]. Briefly, Iron(III) acetylacetonate (4 mmol), 1,2-hexadecanediol (10 24

    mmol), and phenyl ether (20 mL) were mixed at constant stirring at 200 oC for 2 h 25

    mechanically under inert nitrogen atmosphere. The obtained black coloured mixture was 26

    cooled to room temperature (25 oC) and purified using repeated washing with pure 27

    ethanol followed by separation via centrifugation. For the preparation of IONP/GO 28

    composite 0.10 g GO was ultrasonicated in 100 mL of Milli-Q water to form a 29

    homogeneous suspension. After that the solution was transferred to a three-neck flask and 30

    purged with N2. The prepared IONPs were put into the GO suspension and the mixture 31

    was heated to 80 oC and stirred constantly under N2 atmosphere. To adjust the pH of the 32

    solution 5 mL of ammonia solution (30%) was added and the mixture was stirred and 33

    kept at 80 oC for 30 min. Finally, 1.0 g trisodium citrate was added to the prepared 34

  • Page 6 of 25






    solution and the temperature was maintained at 80 oC, which result in the formation of 1

    black precipitate (Fig. 1). 23

    2.4. Preparation of graphene-iron oxide-chitosan hybrid nanobiocomposite45

    For the preparation of GIOCh, 0.05g of chitosan was added to 10 ml of acetic acid 6

    solution in water and stirred for 1 hour. Further, 1 mg of IONPs/GO composite was 7

    added in the chitosan solution. Then 0.5 ml (0.5% w/v) EDC was added in this solution 8

    followed by ultrasonication of the solution for 30 minutes (Fig.1) [31].910

    2.5. Electrophoretic deposition of GIOCh nanocomposite onto ITO electrode1112

    The synthesized nanocomposite were cathodically deposited using electrophoretic 13

    deposition technique (EPD) [32,33] onto a pre-hydrolyzed ITO electrode using a two-14

    electrode system, with parallel-placed platinum as the counter electrode. Prior to 15

    deposition, the prepared nanocomposite was diluted with ethanol in a 1:3 ratio, of which 16

    12 mL of the aliquot was taken out and subjected to EPD at a DC potential of 10 V for 60 17

    s. The migration of GIOCh colloids towards cathode shows that the nanocomposite has 18

    net positive charge in the solution which is attributed to the presence of Ch that contains 19

    primary amino groups with pKa value of around 4.0,. Below this pH most of the amino 20

    groups in Ch get protonated. When voltage is applied the Ch molecules experience a 21

    higher pH than pKa near the cathode, wherein amino groups of Ch get deprotonated 22

    making it insoluble thus leading to the deposition of the GIOCh nanocomposite onto the 23

    ITO electrode (Fig.1) [30, 34]. 2425

    2.6. Fabrication of nucleic acid-functionalized GIOCh/ITO electrode2627

    To allow coupling between the amine terminal of pDNA and the GIOCh/ITO28

    electrode, 20 L of pDNA was spread onto the modified electrode surface using 29

    glutaraldehyde as a cross-linker (0.1% v/v; 4 h). The prepared 30

    pDNA/Graphene/IONPs/ITO bioelectrodes were utilized for detection of E. coli by 31

    subjecting them to various concentrations of complementary target DNA for 30 min and 32

    the corresponding difference in Rct value was measured by EIS. The different steps 33

    involved in the fabrication of GIOCh/ITO electrode has been shown in the scheme (Fig. 34

    1). Control experiments have also been carried o...


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