electrochemical genosensor based on graphene oxide modified iron oxide–chitosan hybrid...

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

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http://dx.doi.org/doi:10.1016/j.snb.2014.09.056

http://dx.doi.org/10.1016/j.snb.2014.09.056

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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,

India

Abstract

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 1×10−14

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: [email protected] These authors contributed equally.

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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

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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 40 000–75 000), 1-ethyl-(dimethylaminopropyl) 22

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

chitosan (MW 15 000–20 000) 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

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

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

GAA AGC GGA CC-3´3

The solution of oligonucleotide are prepared in Tris–EDTA buffer (1M Tris–HCl, 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

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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 μL 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 out by immobilizing pDNA onto 3-35

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glycidoxypropyltrimethoxy silane (GPTS) modified ITO coated glass substrates. BSA 1

was used to block the free sites to reduce the false positive results during the 2

hybridization process.3

2.7. Characterization45

The morphological investigations of GIOCh have been carried out using 6

transmission electron microscopic (TEM, Hitachi Model, H-800) studies. The scanning 7

electron microscopy (SEM) images have been recorded using a JEOLJSM-6700F field-8

emitting scanning electron microscope (FESEM, 15 kV). Fourier transform infra-red (FT-9

IR) spectroscopy measurements have been carried out using Perkin-Elmer spectrometer 10

(Model Spectrum BX) at 25oC. Electrochemical analysis has been conducted on an Auto 11

lab potentiostat/galvanostat (Eco Chemie, Netherlands) using a three-electrode cell using 12

ITO as working electrode, platinum as auxiliary electrode and Ag/AgCl as reference 13

electrode in phosphate buffer (PBS, 100 mM, pH 7.4, 0.9% NaCl) containing 5 mM 14

[Fe(CN)6]3-/4-

.15

3. Results and discussion1617

3.1. Transmission electron microscopic (TEM) studies1819

TEM studies have been carried out to reveal the microstructure of the formed 20

GIOCh. Figure 2, panel (i) shows the TEM image of IONPs which are mono-dispersed 21

and is of spherical shape having particle size from 8 to 12 nm [30]. The EDAX spectra 22

for the IONP show peaks around 0.8, 6.3, and 6.8 keV relating to the binding energies of 23

Fe (Fig.S2 (a)). After the addition of GO and chitosan these nanoparticle get struck to the 24

sheet of the graphene sheet showing crumbled, glossy and flake structure [35] well 25

covered with the chitosan (Fig. 2; panel ii). Many small spheres are attached on the 26

graphene-chitosan composite with a mean diameter 24 nm which implies that graphene 27

provide a large surface for IONPs which are uniformly decorated on the graphene sheets 28

(Fig. 2; panel iii). As, GO is derived from the exfoliation of graphite oxide it consists of 29

abundant oxygenated functional groups, such as hydroxyl and epoxides on the basal 30

plane with carbonyl and carboxyl groups at the edges. These oxygenated functional 31

groups can serve as nucleation sites for IONPs to form IONPs/GO nanocomposites. Due 32

to the presence of hydroxyl and epoxides on the basal plane and the carbonyl and 33

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carboxyl groups at the edges, graphene oxide is negatively charged. The positively 1

charged Fe2+ ions attach to the surface and edges of the GO resulting in the formation of 2

nucleation sites. The above observation is well supported by EDAX analysis which 3

clearly shows the formation of GIOCh nanocomposite (Fig. S2).45

3.2. Zeta potential studies for characterization of particle size67

DLS measurement shows that the average particle size of the iron oxide 8

nanoparticle and the prepared GIOCh nanocomposite is 26 nm (Fig. 3; panel i) and 300 9

nm (Fig. 3; panel ii) respectively with a polydispersity index of ~0.23. Further to 10

investigate the stability of the particle, zeta potential measurements [36] have been 11

conducted and is found to be 60 mV at the pH value of 4.0, indicating the stability of this 12

composite at this pH (Fig. 3; panel iii).1314

3.3. FT- IR studies1516

The formation of GO, IONPs/GO and GIOCh nanocomposite was investigated by 17

FTIR spectroscopy (Fig. S1). The spectrum of GO shows intense bands at 3450 and 126218

cm-1 attributed to stretching of the O-H band of CO-H (Fig. S1; panel (a)). The band at 19

1827 cm-1 is associated with stretching of the C=O bond of carboxyl groups. The FTIR 20

spectrum of IONP/GO differs from that of GO as evidenced by the weakening of the 21

peak at 3450 cm-1. Two additional vibrational bands at around 1620 and 1029 cm-122

appeared, which can be assigned to the formation of either a monodentate complex or a 23

bidentate complex between the carboxyl group and Fe on the surface of the magnetic 24

particles, supporting that the iron oxide nanoparticles are covalently bonded to GO (Fig. 25

S1; panel (b)). Moreover, the peak at 674 cm-1 can be ascribed to lattice absorption of 26

iron oxide, indicating the strong interaction of the nanoparticles with the ester O [30, 37].27

In IR spectrum of chitosan (Fig. S1; panel (c)), a band in the range 3347 cm-1–28

3493 cm-1 is observed which may be assigned to OH vibrations. Peak at 2347 cm-1 is due 29

to the stretching vibrations of aliphatic C–C. The peak observed at 1637 cm-1 may be 30

ascribed to stretching vibrations of Amide I (-NH deformation of – NHCOCH3) [38]. In31

the IR spectrum of composite (Fig. S1; panel (d)) peak due to Fe – O shifts to the 678 32

cm-1.which is due to absorption of Fe-O in the lattice. Peaks due to stretching vibrations 33

of Amide I shift to 1623 cm-1from 1637 cm-1. Peaks due to stretching vibrations of OH 34

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are observed at 3098 cm-1 and 3755 cm-1. Peaks observed at 2375 cm-1 is due to 1

stretching vibrations of aliphatic C–C which is shifted to higher wave number. A band 2

can be seen on 1441 cm-1 and 1558 cm-1 which is due to stretching vibrations of CH.34

3.4. Raman analysis56

Raman analysis was done to characterize disorder in sp2 carbon materials. Figure 7

S3 shows the raman spectra of GO and GIOCh nanocomposite. In Raman spectra of GO 8

(Fig. S3; panel i), two prominent peaks are observed at 1590 cm-1, and 1353 cm-1. The 9

peak at 1590 cm-1 is due to G band which arises from the E2g mode, caused by the 10

stretching motion of the ordered sp2 bonded carbon and the peak at 1353 cm-1 is due to D 11

band which arises from sp3 bonded carbon atom indicating disorder in the structure. Here 12

the ID/IG (intensity ratio of D and G band) ratio was found to be 0.873. In the raman 13

spectrum of the GIOCh nanocomposite (Fig. S3; panel ii), G band and D band observed 14

at 2166 cm-1 and 1100cm-1 respectively, implies that graphene main structure is retained 15

but peaks were shifted. Both the peaks were broadened and ID/IG was found to be 0.80716

in the GIOCh nanocomposite. It can be observed that there is decrease in value of ID/IG17

which implies increase in ordered structure. Probably, it may be due to increased 18

graphitic domains in nanocomposite than that of GO, which may be due to the reduction 19

of graphene oxide during composite formation [39].20

213.5. Scanning electron microscopic (SEM) studies22

23SEM analysis was done to study the surface morphology of GIOCh/ITO and 24

pDNA/GIOCh/ITO electrodes. Figure 4, image (i) shows the SEM image of GIOCh 25

deposited onto ITO electrode, which shows flake-like, glossy and crimple shapes of 26

graphene [40]. It can be observed that graphene is well dispersed on the surface of ITO 27

electrode. A large number of spherical shaped structure can be observed in the SEM 28

image which implies that graphene provide large surface area for the spread of iron oxide 29

nanoparticles. After the immobilization of pDNA onto the GIOCh/ITO electrode shiny 30

and fibrous globules structure were seen which confirm that the DNA molecule is 31

covalently attached to the GIOCh (Fig. 4; image ii).3233

3.6. Contact Angle Studies (CA) for ITO, GIOCh/ITO and pDNA/GIOCh/ITO electrodes3435

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The CA measurements were carried out using the sessile drop method to 1

investigate pDNA/GIOCh/ITO bioelectrode fabrication. CA of ITO was found to be 2

84.6o (data not shown), which decreases to 65.2o after the deposition of GIOCh (Fig.S3, 3

panel i). The decrease in CA may be due to the introduction of graphene oxide having 4

polar groups –COO- which increases the hydrophilicity of the electrode surface. A 5

further decrease (Fig.S3, panel ii) in CA (23.84o) with the immobilization of DNA was 6

observed which may be due to enhancement of hydrophilicity caused by the presence of 7

negatively charged phosphodiester backbone of DNA [38].8

3.7. Electrochemical studies9

3.7.1. Electrochemical characterization10

Electrochemical impedance spectroscopic (EIS) is a powerful and sensitive tool 11

for studying the charge transfer processes occurring at electrode−solution or modified 12

electrode−solution interface [41,42]. The impedance was performed with 10 mV 13

sinusoidal modulation amplitude at an applied bias potential of +0.23 V from 0.1–105 Hz 14

frequency range. Randles equivalent circuit (Figure S4 (b); inset) was used for all the 15

modifications in which Rs represents the resistance of the solution; Rct corresponds to 16

resistance to the charge transfer between the solution and the electrode surface; double 17

layer capacitance (Cdl) was replaced with CPE (due to the interface between the electrode 18

and the electrolytic solution) and a Warburg resistance (W). In the Nyquist plot of the 19

impedance spectra, the semicircle corresponds to the electron transfer resistance process 20

(Rct) which usually depends on the dielectric and insulating features at the 21

electrode−electrolyte interface [43].22

Figure S4 (b) shows the EIS studies of GIOCh/ITO electrode, GPTS/ITO 23

electrode, pDNA/GIOCh/ITO bioelectrode and pDNA/GPTS/ITO bioelectrode. The 24

surface modification of the ITO electrode with GPTS results in the increase in the Rct25

value to 125.4 Ω (Fig.S4 (b), curve (ii)). But, when the same ITO electrode was 26

modified with GIOCh there was a decrease in the Rct value Fig.S4 (b), curve (i)). This 27

clearly indicates that the addition of GO results in the fast electron transfer kinetics in 28

compare to that of the electrode without GO. Further on incubation of both the 29

GIOCh/ITO electrode, GPTS/ITO electrode with pDNA there was an increase in the Rct30

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value which could be ascribed to the repellence of redox probe from approaching 1

electrode surface by negative-charged phosphate skeletons of DNA (Fig.S4 (b)). Similar 2

results were also obtained using cyclic voltammetry (Fig. S4 (a)).34

Cyclic voltammograms of pDNA/GIOCh/ITO electrode were recorded as a 5

function of scan rate (10-300 mV/s) to investigate the various kinetic parameters. It was 6

observed that magnitude of electrochemical response current [anodic (Ipa) and cathodic 7

(Ipc), Fig. 5 inset i] for the bioelectrode varies linearly with the scan rate and follows Eqs. 8

(1) and (2). It can be inferred from the peak separation that the process is not perfectly 9

reversible; however, stable redox peak current and position during repeated scans at a 10

particular scan rate suggests that composite based electrodes exhibit a quasi-reversible 11

process [44]. Moreover, the magnitude of both anodic (Ea) and cathodic peak (Ec) 12

potentials (Fig. 5 inset ii) increases linearly as function of scan rate (Eqs. (3) & (4)) 13

revealing the electron transport form redox moieties to the electrode are very facile.1415

Ipa (A) [pDNA/GIOCh/ITO] = 49.789 (A) + 60.957 A (s/mV) [scan rate (mV/s)]

R = 0.998, SD = 1.208×10-5

(1)

Ipc(A) [pDNA/GIOCh/ITO] = -32.56 (A) – 124.9 A (s/mV) [scan rate (mV/s)]

R = 0.989, SD = 1.8612×10-5

(2)

Ea (V) [pDNA/GIOCh/ITO] = 0.06013 (V) + 0.0712 (V) ٭Log [scan rate]

R = 0.938, SD = 0.0043 (3)

Ec (V) [pDNA/GIOCh/ITO] = - 0.047 (V) +0.191 (V) ٭Log [scan rate]

R = 0.977, SD = 0.0025 (4)16The relatively high currents observed in the voltammetric studies of 17

pDNA/GIOCh/ITOelectrode are proportional to the amount of pDNA immobilized, that 18

is, the maximum surface coverage (Γmax). This parameter can be evaluated from the 19

charge required for20

oxidation of the surface-accumulated substance by use of the equation:

Γmax = Q /nFA (5)21

where Q is the maximum charge measured under the adsorptive voltammetry 22

peak, and A is the electrode geometric area (cm2). Evaluating the charge under the peaks 23

in the voltammograms the average surface concentration of pDNA has been found to be 24

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5.9 x10-8 mol cm-2 which is much higher in comparison to pDNA immobilized onto 1

GPTS/ITO electrode (3.4 x 10-9). This obtained Γmax value was greater in comparison to 2

results obtained by Sharma et al. and Pandey et al [30,34]. This enhancement in the Γmax3

may be due to presence of large number of functional group on the surface of GIOCh 4

which help in covelent binding of the pDNA. Further, on the basis of the linear slope of 5

the anodic peak currents vs. square root of potential sweep rates, and the Randle Sevick 6

equation:78

Ip = (2.99 x 105) α

1/2 n

3/2 ACD

1/2 υ

1/2(6)9

the diffusion coefficient (D) is calculated as 3.23 x 10-7 cm2/s which is found to be 10

greater than that of the pDNA immobilized onto GPTS/ITO electrode (1.43 x 10-8 cm2/s).11

Based on the above calculation it can be inferred that the pDNA/GIOCh/ITO bioelectrode 12

exhibits good electron transfer kinetics which is in consistent with DNA based biosensor 13

reported in literature. [30, 45, 46] This may perhaps be due to the oriented 14

immobilization of the pDNA on the GIOCh/ITO electrode surface which provides an 15

easier path for the transfer of electrons from the redox species to the electrode and vice 16

versa.17

3.7.2. Optimum conditions for E. coli measurement18

The oligonucleotide chain is composed of several nucleoside residues and it is 19

expected that there may be occurrence of free functional groups on the GIOCh/ITO20

surface which lead to the chemisorption of the cDNA, thereby providing false positive 21

signal. To evade the non-specific adsorption of the cDNA, a study for optimization of the 22

pDNA concentration has been executed. As shown in Figure S5 (panel (a)), by increasing 23

the amount of pDNA from 0.05 µM to 1.8 µM, the Rct value get leveled off at 1 µM24

pDNA concentration. Hence 1 µM concentration of pDNA was used throughout the 25

experiment, presuming that at this concentration maximum immobilization is achieved.26

The DNA hybridization response is closely related to variation in temperature and 27

generally elevated temperature may speed up the hybridization efficiency by improving 28

the movement of DNA molecules. On the other hand, when the temperature is higher 29

than the melting temperature there is denaturation of DNA leading to decreasing current 30

signals. Figure S5 (panel (b)) displays the amperometric signals after hybridization with 1 31

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µM cDNA in the temperature range from 20 to 45 oC. It was found that with increase in 1

temperature there was amplification in the hybridization signal which was maximum at 2

∼37 oC, signifying that the cDNA was adequately hybridized with the target pDNA. 3

To understand the hybridization process the analytical performance of the nucleic 4

acid towards recognition of the target oligonucleotides has been optimized. Figure S5 5

(panel (c)) shows the influence of incubation time (10-60 min) on the EIS response of the 6

system in the presence of 1 µM complementary target DNA. The EIS signal is found to 7

increase with increasing the incubation time upto 60 min, and after that it is observed to 8

get retarded showing that the extent of hybridization is accomplished within this duration.9

3.7.3. Response Studies of pDNA/GIOCh/ITObioelectrode10

Using EIS, the sensitivity of the pDNA/GIOCh/ITO bioelectrode was determined 11

by changing the concentration of cDNA from 10-6 M to 10-14 M (Fig. 6; panel i).There 12

was change in Rct value with the variation in cDNA concentration. This signify that the 13

hybridization is occurring at the bioelectrode due to which there is more accumulation of 14

negatively charged phosphate backbones, hence the Rct value increases following the 15

formation of double stranded DNA. The difference between the value of the pDNA 16

immobilized electrode and that after hybridization with cDNA (ΔRct = Rct(cDNA)-17

Rct(pDNA)) has been used as the measurement signal. The analytical signal (ΔRct) shows 18

linear relationship with the logarithmic value of the complementary target DNA 19

concentration ranging from 10-14 to 10-6M (Fig. 6, panel ii) and follows the following 20

equation:21

Rct = 383.79 + 21.485 log cDNA (7)22

with linear regression coefficient of 0.995. The detection limit is calculated to be 1 × 23

10−14 M. The detection limit has been calculated using the equation 3σ/sensitivity where 24

σ is the standard deviation of blank and sensitivity is the slope of the calibration curve25

[5]. Similarly the detection limit calculated for pDNA/GPTS/ITO electrode was found to 26

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be 2.3 x 10-12 M (Fig. S7). The sensing experiment was repeated for 4 times and the 1

imprecision of the data as indicated by the error bars was found to be within 5.3%. It has 2

been observed that this platform had lower detection limit and wider detection range for 3

DNA hybridization than previous DNA based biosensors in the literatures (Table S1and 4

S2).56

3.7.4. Selectivity of the bioelectrode78

The specificity of the pDNA/GIOCh/ITO bioelectrode towards different target 9

DNA sequences (complementary, non-complementary and one base mismatch) and with 10

the culture samples of E. coli, S. Typhimurium, N. Meningitidis and K. pneumonia has 11

been studied using EIS and is shown by bar-diagram (Fig. S8). It was observed that after 12

incubation of the bioelectrode with E. coli culture DNA there was a marked increase in 13

Rct value which was nearly same to that of cDNA, showing the process of hybridization. 14

When the pDNA/GIOCh/ITO bioelectrode is incubated with non-complementary and 15

bacterial DNA samples of other water pathogens, there was a slight or negligible change 16

in the Rct value with respect to that of pDNA suggesting that there is no hybridization 17

taking place. While, exposing the pDNA/GIOCh/ITO bioelectrode with one base mis-18

match DNA sequence there was a slight increase in semicircle diameter in comparison to 19

that of the pDNA. This may be due to the partial hybridization of pDNA. These results 20

reveal the selectivity and specificity of the pDNA/GIOCh/ITO bioelectrode towards 21

different target DNA sequences.2223

3.7.5. Reusability and stability of the bioelectrode2425

The biosensor is stable enough to allow for ready regeneration due to strong 26

binding of pDNA on the GIOCh/ITO electrode surface. For this, the electrode is 27

immersed into a buffer Tris−HCl (10 mM) solution, pH 8.0 and EDTA (1 mM) at 100 °C 28

for 5 min, followed by cooling in the ice bath for about 30 min, which completely 29

removes cDNA via thermal denaturation. The Rct value of the biosensor has been found 30

to decrease after each regeneration process with an average signal loss of about 1.3 % 31

(data not shown). This reduction in signal may be possibly due to surface fouling during 32

the regeneration process. The total loss of hybridization signal after 6 cycles is about 15.4 33

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Ω, and it corresponds to ∼10.37 % loss of the initial value, indicating that the 1

bioelectrode reproducibly detects target DNA over repeated uses (Fig. S6 (i)). To 2

investigate storage stability of the fabricated sensor, five measurements have been3

recorded each week for over 30 days of continuous analysis. The decrease in signal 4

response of the bioelectrode was less than 10% when stored at 4 °C (Fig. S6 (ii)).56

4. Conclusions78

In the present work, we synthesized a novel graphene oxide modified iron oxide-9

chitosan hybrid nanocomposite, which was electrophoretically deposited onto ITO coated 10

glass substrate. It was observed that the incorporation of GO enhances the 11

electrochemical properties of the nanocomposite. Using electrochemical impedance 12

spectroscopic technique, it was observed that the prepared nanobiocomposite enhanced 13

DNA detection and can successfully detect E. coli in the range of 10-6 to 10-14 M. This 14

fabricated biosensor is highly selective, sensitive and retains significant activity (90% of 15

the initial activity) after 6 cycles of use. Thus, the biosensor designed in this report could 16

be an important tool for determining the presence of low concentrations of E. coli DNA 17

in biological assays.1819

Acknowledgements2021

M. S. is thankful to DBT, India. C.M.P. is thankful to CSIR, India, for the award 22

of SRF. We thank Dr. A.M. Biradar (NPL, New Delhi), for interesting discussions. 23

Thanks are due to Dr. Seema Sood (AIIMS, New Delhi) for providing the bacterial 24

culture samples. The financial support received from DBT (Grant No.25

BT/PR1791/MED/32/174/2011), India and DST, (Grant No. DST/TSG/ME/2008/18) 26

India is gratefully acknowledged.27

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Biographies:

Dr. Ida Tiwari did her B.Sc. (Hons.) in Chemistry in 1995 from Banaras Hindu University, Varanasi,

India. She received her M.Sc. in Analytical Chemistry in 1997 from Banaras Hindu University,

Varanasi, India. She obtained her doctrait in 2001 in the field of electroanalytical sensors from

Banaras Hindu University, Varanasi, India. There after she was awarded Research Associateship from

C.S.I.R, New Delhi, India in which she worked on sensors. She joined Analytical Chemistry Division,

Department Of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi, India as Assistant

Professor in 2004 and has 13 years research experience in field of sensors, biosensors, composite

materials, nanotechnology.

Ms. Monali Singh B.Sc. (Hons.) in Chemistry in 2008 from Banaras Hindu University, Varanasi, India.

She received her M.Sc. in Analytical Chemistry in 2010 from Banaras Hindu University, Varanasi,

India. She is working for her doctoral degree under guidance of Dr. Ida Tiwari in Department Of

Chemistry, Faculty of Science, Banaras Hindu University, Varanasi, India. Her research interest

includes sensors, biosensors, fluorescence based sensors, nanotechnology and composites.

Mr. Chandra Mouli Pandey received his M.Sc. degree in Chemistry from the Gorakhpur University,

Uttar Pradesh, India in 2008. He is working for his doctrait from Banaras Hindu University, Varanasi

under guidance of Dr. Ida Tiwari in collaboration with Dr. G. Sumanna’s group in the Biomedical

Instrumentation Section at the National Physical Laboratory, New Delhi, India. His main interest of

research is development of biosensor for health care based on microstructure of nano-materials and

bionanocomposites.

Gajjala Sumana received her Ph.D. in 1998 from Jiwaji University in Chemistry. She is currently

working as a senior scientist with the Biomedical Instrumentation Section at the National Physical

Laboratory, New Delhi, India. She has a research experience of 14 years in controlled drug delivery,

liquid crystal polymers, polymer dispersed liquid crystals and biosensors.

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Figure captions

Figure 1 Schematic illustration for the preparation of GIOCh nanocomposite, EPD of

nanocomposite onto ITO electrode and its application for electrochemical detection of E. coli.

Figure 2 (i) Transmission electron micrograph (TEM) images of (i) IONPs, (ii) GIOCh

nanocomposite and (iii) enlarged TEM image of the GIOCh nanocomposite.

Figure 3 DLS measurements showing the particle size distributions of (i) IONPs, and (ii)

GIOCh nanocomposite (iii) zeta potential analysis of the prepared GIOCh nanocomposite.

Figure 4 SEM images of (i) GIOCh/ITO electrode and (ii) pDNA/GIOCh/ITO bioelectrode

Figure 5 Cyclic voltammetric variation of (i) GIOCh/ITO electrode and (ii)

pDNA/GIOCh/ITO electrode with scan rate in PBS (100 mM, pH 7.4, 0.9% NaCl) solution

containing 5 mM [Fe(CN)6]. Inset figure shows the variation of (i) potential with log of

scan rate and (ii) current with square root of scan rate.

Figure 6 EIS response of pDNA/GIOCh bioelectrode as a function of complementary DNA

concentration (10-14-10-6 M) in PBS solution (pH 7.4) containing 5 mM [Fe(CN)6]3-/4-. Plot of

the ratio of charge transfer resistance after and before hybridization, Rct target/Rct pDNA, of

the pDNA/GIOCh/ITO bioelectrode vs. the logarithm of the target DNA concentrations.

3-/4-

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Figure 1:

Figure 2:

Figure 3:

OH

OH

OH

OH

OH

O

OH

O OH

OOH

OH

O

OH

O

OH

O

OH

OH

OH

OH

OH

OH

O

OH

O OH

OOH

OH

O

OH

O

OH

O

OH

OH

O H

O H

OH

O H

O

O H

O OH

OOH

O HO

O H

O

OH

O

OH

OH

O H

O H

OH

O H

O

O H

O O H

OOH

OH

O

OH

O

O H

O

O HOH

OH

OH

OH

OH

O

OH

O O H

OOH

O H

O

O H

O

O H

O

O H

OH

O H

O H

OH

O H

O

O H

O O H

OOH

OHO

OH

O

O H

O

O H

OH

O H

OH

OH

O H

O

O H

O O H

OOH

O HO

O H

O

O H

O

O H

+-

ITO Pt

OHOHOHOHOHOOHOOHOOHOHOOHOOHOOHOHOHOHOHOHOOHOOHOOHOHOOHOOHOOHOHOHOHOHOHOOHOOHOOHOHOOHOOHOOHOHOHOHOHOHOOHOOHOOHOHOOHOOHOOHOHOHOHOHOHOOHOOHOOHOHOOHOOHOOHOHOHOHOHOHOOHOOHOOHOHOOHOOHOOHOHOHOHOHOHOOHOOHOOHOHOOHOOHOOHOHOHOHOHOHOOHOOHOOHOHOOHOOHOOHOHOHOHOHOHOOHOOHOOHOHOOHOOHOOHOHOHOHOHOHOOHOOHOOHOHOOHOOHOOHOHOHOHOHOHOOHOOHOOHOHOOHOOHOOHOHOHOHOHOHOOHOOHOOHOHOOHOOHOOHOHOHOHOHOHOOHOOHOOHOHOOHOOHOOHOHOHOHOHOHOOHOOHOOHOHOOHOOHOOHOHOHOHOHOHOOHOOHOOHOHOOHOOHOOHOHOHOHOHOHOOHOOHOOHOHOOHOOHOOHOHOHOHOHOHOOHOOHOOHOHOOHOOHOOHOHOHOHOHOHOOHOOHOOHOHOOHOOHOOHOHOHOHOHOHOOHOOHOOHOHOOHOOHOOHOHOHOHOHOHOOHOOHOOHOHOOHOOHOOH

Graphene oxide Graphene oxide/IONP composite GIOCh

ChIONP

Electrophoretic depositionpDNA/GIOCh/ITO electrodeHybridization

Complementary DNA probe DNA

45 50 55 60 65 700

50000

100000

150000

200000

Zeta Potential (mV)

Tota

l Cou

nt

18 20 22 24 26 28 30 32 340

5

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35

Size (d, nm)

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(%)

350 400 450 500 550 6000

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(%)

(i) (ii) (iii)

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Figure 5:

Figure 6:

(i) (ii)

(i) (ii)

1 µm 1 µm

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HIGHLIGHTS

Synthesis of graphene oxide modified iron oxide-chitosan hybrid nanocomposite(GIOCh).

GIOCh was electrophoretically deposited onto ITO coated glass substrate and this platform has been utilized for sensitive detection of Escherichia coli O157:H7.

The fabricated nucleic acid sensor demonstrates high performance with enhanced sensitivity (1×10−14 M) in the concentration range of 10-6 to 10-14 M and high selectivity over other bacterial pathogen.

electrochemical genosensor based on graphene oxide modified iron oxide–chitosan hybrid...

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