Biosensors and Bioelectronics 68 (2015) 726735

Contents lists available at ScienceDirect

Biosensors and Bioelectronics

journal homepage: [Link]/locate/bios

Multifunctional magnetic reduced graphene oxide dendrites: Synth-

esis, characterization and their applications
Ekta Roy a, Santanu Patra a, Deepak Kumar b, Rashmi Madhuri a,n, Prashant K. Sharma c
a
Department of Applied Chemistry, Indian School of Mines, Dhanbad, Jharkhand 826004, India
b
Solid State and Structural Chemistry Unit, Indian Institute of Science, Bangalore 560012 India
c
Functional Nanomaterials Research Laboratory, Department of Applied Physics, Indian School of Mines, Dhanbad, Jharkhand 826004, India

art ic l e i nf o a b s t r a c t

Article history: In this paper, for the rst time, we have reported the novel synthesis of reduced graphene oxide (r-GO)
Received 29 November 2014 dendrite kind of nanomaterial. The proposed r-GO dendrite possesses multifunctional properties in
Received in revised form various elds of sensing and separation. The dendrite was synthesized by chemical reaction in different
29 January 2015
steps. Initially, the r-GO sheet was conjugated with silane group modied magnetic nanoparticle, re-
Accepted 31 January 2015
sulting in nanoparticle decorated r-GO. The above r-GO sheet was further reacted with a new r-GO sheet,
Available online 7 February 2015
resulting in the formation of r-GO dendrite type of structure. Multifunctional behavior of this r-GO
Keywords: dendrite structure was studied by different methods. First, magnetic properties were studied by vibrating
Reduced graphene oxide sample magnetometer (VSM) and it was found that dendrite structure shows good magnetic suscept-
Magnetic nanoparticle
ibility (180.2 emu/g). The proposed r-GO dendrite also shows a very good antibacterial behavior for Es-
Dendrite
cherichia coli and excellent electrochemical behavior towards ferrocyanide probe molecule. Along with
Europium ion
Molecularly imprinted polymer these, it also acts as a substrate for the synthesis of molecularly imprinted polymer for europium metal
ion, a lanthanide. The proposed imprinted sensor shows a very high selectivity and sensitivity for
europium metal ion (limit of detection 0.019 g L  1) in aqueous as well as real samples.
& 2015 Elsevier B.V. All rights reserved.

1. Introduction presence of an enormous -orbital, which permits higher het-

erogeneous electron transfer rates than GO (Martn and Escarpa,
Since the discovery of graphene, a two-dimensional atomic 2014).
sheet entirely made of carbon; immense efforts have been directed Recently, continuous scientic endeavors have been directed
toward the development of reliable methods for obtaining large toward the optimization of r-GO by improving its electronic, me-
quantities of this extraordinary material (Chang et al., 2011). chanical, chemical, and structural properties, such as surface
However, after several year of research, present difculties are in morphology, pore geometry, and functional sites, in order to ad-
obtaining processable graphene sheets in large quantities. One vance various potential applications (Chen et al., 2015). Over the
promising, low-cost, and easily up-scalable alternative is the re- past few years, composites containing r-GOs and inorganic nano-
duction of graphene oxide (GO), a layered material whose con- particles (NPs) have been intensively developed, and found to
stituting graphene layers functionalized with epoxy and hydroxyl exhibit a range of unique and useful properties, which are at-
groups and easily exfoliated in water (Li et al., 2010). The resulting, tracting more and more attention from researchers (Mao et al.,
2011). In these composites, r-GO acts as a high-performance sup-
reduced graphene oxide (r-GO) can be deposited in controllable
port, while other component can be uniformly dispersed on its
density onto a large variety of substrates, thus enabling the pre-
plane, and the charge transfer at the interface of these hybrid
paration of thin conductive lms on solid and exible substrates
materials can show a synergistic effect to induce properties that
(Zeng et al., 2014). Although, both GO and r-GO are widely used in
are different from those of each individual component (Table 1).
electrochemistry for the detection of different target molecules,
Inspired by this, several nanocomposites and/or hybrid materials
r-GO has demonstrated more advantages for electrochemical ap-
of r-GO with metal nanoparticles [Au (Wang et al., 2015), Ag (Qu
plications. The main characteristic of r-GO is the higher content of
et al., 2012), Fe (Metin et al., 2014; Zubir et al., 2014), ZrO2 (Tey-
sp2-carbon compared with GO. The natural consequence is the mourian et al., 2014), and SiO2 (Ge et al., 2014)], quantum dots
(Zhou et al., 2014), polymers (Chen et al., 2015), dendrimer (Jiang
n
Corresponding author. Fax: 91 326 2296563. et al., 2014), chitosan/ionic liquid (Li et al., 2014) and carbon na-
E-mail address: rshmmadhuri@[Link] (R. Madhuri). notubes (Kong, 2013) were developed and explored in various

[Link]
0956-5663/& 2015 Elsevier B.V. All rights reserved.
 E. Roy et al. / Biosensors and Bioelectronics 68 (2015) 726735 727

Table 1
Various applications of reduced graphene oxide with their conjugated nanomaterials.

S.n. Conjugated material Synthesis/Behavior study Application References

1. MNPsa Self-assembly method Schottky diode Metin et al., 2014

2. AgNPsMNPsa Catalytic study Environmental pollutant degradation Qu et al., 2012
3. MNPsa Electrocatalytic activity Electrocatalytic oxidation of NADH and lactate biosensing Teymourian et al., 2013
4. RAFTb agent Platform for MIPc Endocrine disrupting chemicals Li et al., 2010
5. Congored dye Platform for MIPc Sensing of dopamine (DA) Mao et al., 2011
6. Zirconium dioxide Electrocatalytic activity Sensing of AAd, DA and UAe Teymourian et al., 2014
7. Quantum dots Fluorescence and imprinting Detection of paranitrophenol Zhou et al., 2014
8. AuNPs MNPsa Magnetic and imprinting Detection of AFPf and CEAg Wang et al., 2015
9. MNPsa Chemiluminescence and imprinting Determination of epinephrine Qiu et al., 2012
10. MNPsa Adsorption and desorption study Removal of 4-nitrophenol Zeng et al., 2014
11. Dendrimer Support for trypsin immobilization Fast on-plate digestion of proteins Jiang et al., 2014
12. MSNPsh Electrochemical immunosensor Detection of cancer antigen 153 Ge et al., 2014
13. MNPs Computational study Heterogeneous Fenton-like reaction Zubir et al., 2014
14. Chitosan ILi Biodegradable biosorbents Waste water treatment Li et al., 2014
15. MSNPs r-GOj Magnetic, electrocatalytic, antibacterial and For E. Coli inhibition, ferrocynide sensing, Eu 3 imprinting This work
imprinting and detection

a
Magnetic nanoparticle.
b
Reversible addition fragmentation chain transfer.
c
Molecularly imprinting polymer.
d
Ascorbic acid.
e
Uric acid.
f
Alpha feptoprotein.
g
Carcinogenic embryonic antigen.
h
Magnetic silica nanoparticle.
i
Ionic liquid.
j
Reduced graphene oxide.

elds of sensing, separation, drug delivery and many more. Re- was chosen as a template molecule because of their enormous use
cently, Teymourian et al. have reported the simple and one pot in industry, material science, and electronics devices. Due to tre-
synthesis of zirconium dioxide (Teymourian et al., 2014) as well as mendous hike in their use, they entered into our food chain now
iron oxide (Teymourian et al., 2013) decorated-reduced graphene (Richter and Bard, 1996) and the threshold value of Eu 3 reported
oxide composite for simultaneous detection of ascorbic acid (AA), into the literature is o6 mg L  1 (Liang et al., 2001). Furthermore,
dopamine (DA) and uric acid (UA). In addition, some researchers with long-term exposures of their dusts or vapors in the working
have also explored the antibacterial properties of r-GO composites environment, it cause lung embolism including cancer in humans
(Xu et al., 2011). Due to the high surface area r-GO also acts as a (Liang et al., 2001). So, their estimation is very much important in
platform or substrate for molecularly imprinted polymer synth- the aqueous as well as real samples.
esis. Initially, Chang et al. (2011) have reported an MIP for 2,4-
dichlorophenol prepared by ATRP method. However, Li et al.
(2010) have described the rst MIP on functionalized GO using 2. Experimental section
RAFT polymerization technique. Qiu's group reported GO-magne-
tite-MIP composite using RAFT polymerization for chemilumi- 2.1. Materials
nescence sensing epinephrine (Qiu et al., 2012). Zeng et al. (2013)
have prepared surface MIP composites for dopamine recognition 3-Aminopropyltriethoxysilane (APTES), N,N-bisacryl amide,
on the basis of GO/SiO2 as a supporter. In addition, Mao et al. europium nitrate [Eu(NO3)3], ethyl-2-bromo isobutyrate were
(2011) used free radical polymerization (FRP) to prepare congored- supplied by Sigma-Aldrich. Ferric chloride hexahydrate
functionalized r-GO (the complete reduction of GO) for sensing of (FeCl3  6H2O), Ferrous sulfate heptahydrate (FeSO4  7H2O), cop-
dopamine. per chloride (CuCl2), graphite powder, bipyridyl, sodium hydroxide
Herein, we have tried to demonstrate multifunctional behavior (NaOH), ascorbic acid, nitric acid (HNO3), sulfuric acid (H2SO4),
of a new composite of graphene, made up of r-GO and magnetic tetrahydrofuran (THF) and absolute ethanol (99%) were purchased
nanoparticle (MNPs). Herein, we have tried to prepare a graphene from Fluka. All the chemicals used in this work were of analytical
and silane modied Fe3O4 based dendrite type of structure, where reagent grade and used as obtained. All aqueous solutions were
graphene sheet (r-GO), itself used as a core. From the graphene prepared with deionized water obtained from a Milli-Q system.
core, MNPs were attached via chemical bonding and followed by
attachment of another layer of r-GO, resulting in r-GO dendrite 2.2. Instrumentation
type of structure. We have explored various applications of syn-
thesized r-GO dendrite like magnetic behavior, electrocatalytic All electrochemical experiments i.e. cyclic stripping voltam-
activity and antibacterial response towards Escherichia coli. Along metry (CSV), differential pulse stripping voltammetry (DPSV),
with these, herein, r-GO dendrite is used as a platform or substrate square wave stripping voltammetry (SWSV) and electrochemical
for the synthesis of europium (Eu 3) imprinted polymer. For this, impedance spectra (EIS) were performed on CH instrument (USA,
r-GO dendrite was further reacted with acrylamide to produce a model number 660 C), with a conventional three-electrode system
vinyl functionalized monomer. From the r-GO dendrite surface consisting of a modied pencil graphite electrode (PGE) (area
polymerization was initiated by activator generated atom transfer 9.41  10  2 cm2) as a working electrode, a reference electrode (Ag/
radical polymerization technique (ARGET-ATRP), resulting in AgCl/KCl (1 M)) and a platinum wire as auxiliary electrode. Mor-
MIP@r-GO dendrite, specic for europium metal ion. Europium phological images of bare and modied PGE surfaces were
728 E. Roy et al. / Biosensors and Bioelectronics 68 (2015) 726735

recorded by Field emission scanning electron microscope (FE- FeSO4  7H2O (3.0 g, 0.011 mol) were dissolved in 100 mL distilled
SEM), Zeiss, model Super 55. For the binding characterization, IR water (60 C) with vigorous mechanical stirring under N2 atmo-
analysis was carried out on Varian Fourier Transform Infrared sphere. Then, 7.5 mL of NaOH was injected into the reaction
spectrometers (USA). UVvisible study was performed on a Per- mixture resulting in the immediate formation of the black pre-
kin-Elmer Lambda 35 (Singapore) spectrophotometer. The powder cipitate. The reaction was continued for half an hour and the
X-ray diffraction (XRD) study was carried on Bruker D8 Focus mixture was cooled to room temperature. Finally, the Fe3O4 pre-
X-ray diffract meter instrument using a Cu target radiation source. cipitate was ltered, washed thrice with ethyl acetate and dried.
Transmission electron microscopy (TEM) studies were performed
on Tecnai 30 G2 S-Twin electron microscope operated at 300 kV 2.3.2. Surface modication of MNPs with silane
accelerating voltage. All the experiments were performed at room For the surface modication of Fe3O4, rstly, 300.0 mg MNPs
temperature (25 71 C). were dispersed in ethanol and water mixture (600:4 mL) by ul-
trasonication. The above MNPs were then treated with APTES
2.3. Preparation of APTES-coated iron oxide magnetic nanoparticles (120 mL) in an ultrasonic water bath for 30 min followed by me-
(Silane@Fe3O4) chanical stirring for 6 h. The resultant reaction product
(silane@Fe3O4) was separated by centrifugation at 10,000 rpm
2.3.1. Synthesis of magnetic nanoparticle then re-dispersed in ethanol and isolated with magnetic decan-
Fe3O4 magnetic nanoparticles (MNPs) were prepared by co- tation. The washing and separation process was repeated 6 times.
precipitation of Fe2 and Fe3 in the presence of NaOH (Cao et al., The nal product (silane@Fe3O4) was dried at room temperature
2009). In brief, a mixture of FeCl3  6H2O (5.85 g, 0.022 mol) and under vacuum oven.

Scheme 1. The reaction pathway for the preparation of r-GO dendritic monomer.
 E. Roy et al. / Biosensors and Bioelectronics 68 (2015) 726735 729

2.4. Synthesis of r-GO dendrite nanostructure 1 h. To the above solution, 25 mL of 1% (v/v) ethanolic solution of
silane@Fe3O4 was added and the reaction mixture was reuxed at
The r-GO dendrite nanostructure was synthesized in four steps 65 C for 12 h. The mixture was cooled, centrifuged and dried at
that are given below in a sequential manner and shown in room temperature.
Scheme 1:
2.4.4. Step IV: Addition of another sheet of r-GO to
2.4.1. Step I: Synthesis of graphene oxide (GO) r-GO/silane@Fe3O4
Graphene oxide (GO) was prepared from graphite powder by This above-synthesized product was again dispersed in 250 mL
modied Hummer's method (Hummers and Offeman, 1958). The ethanol by sonication (1 h) and mixed with 25 mL dispersed r-GO
graphite powder was oxidized by nitric acid and sulfuric acid in solution. The above reaction mixture was reuxed again at 65 C
order to form carboxyl groups on their surface. Briey, graphite for 12 h. The resulting product (r-GO dendrite) was cooled to room
powder (3.0 g) was oxidized in 60 mL of HNO3 and H2SO4 (1:3, v/ temperature and collected by centrifugation.
v) mixture under reux for 48 h. The pre-oxidized graphite was
separated by centrifugation, washed with dry THF and dried under
vacuum. The pre-oxidized graphite was further oxidized by a 2.5. Antibacterial behavior by disk diffusion method
mixture of 96% H2SO4 (120 mL) and KMnO4 (15.0 g) by reux for
3 h. In the end, the GO was collected and dried under vacuum. The modied disk diffusion method (Xu et al., 2011) was used
to evaluate the antibacterial activity of only silane@Fe3O4, r-GO,
2.4.2. Step II: Acylation of GO and their reduction to r-GO r-GO/silane@Fe3O4, and r-GO dendrite (1.0 mg each dissolved in
The above prepared GO (1.0 g) was rst well dispersed in 20 mL 100 mL water) were used against E. coli (pure strain). This method
of DMF by sonication (45 min) and treated with SOCl2 (60 mL, was performed in Luria-Bertani (LB) medium solid agar Petri dish.
0.82 mol) at 80 C for 48 h. The acyl group-functionalized GO (GO/ Briey, 6 mm lter paper disks was impregnated with 1.0 mL of
COCl) was separated by centrifugation, washed with anhydrous each samples solution and placed on E. coli culture agar plate. Agar
THF, and dried under vacuum. To reduce the acylated GO, GOCOCl plate along with sample containing lter paper was incubated at
(0.1 g) was dispersed in DMF and reuxed with 2 mL hydrazine for 37 C for 24 h and inhibition zone was monitored. After incuba-
24 h. The resultant r-GO was centrifuged, washed with dry THF tion, bacterial growth inhibition was observed by calculating the
several times and dried under vacuum. diameter of bacteria-free area (visualized by blank space around
lter paper).
2.4.3. Step III: Conjugation of r-GO and silane@Fe3O4
For the conjugation of silane@Fe3O4 to the surface of r-GO, rst,
50.0 mg of r-GO was dispersed in 250 mL ethanol by sonication for 2.6. As a platform for synthesis of europium imprinted polymer-

Fig. 1. (A) Schematic representation of europium imprinted polymer@r-GO dendrite synthesis and characterization of various materials by (B) XRD: (1) r-GO, (2)
silane@Fe3O4, (3) r-GO/silane@Fe3O4, (4) r-GO dendrite and (C) FT-IR: (1) silane@Fe3O4, (2) r-GO, (3) r-GO/silane@Fe3O4 and (4) r-GO dendrite.
730 E. Roy et al. / Biosensors and Bioelectronics 68 (2015) 726735

based sensor with the initial concentrations ranging from 1.0 to 50.0 g L  1 and
placed on a dynamic multi-spinner After 10 h of contact, the sa-
To work as a platform for imprinted polymer synthesis, r-GO turated polymers were separated with an external magnet. The
dendrite was modied with the vinyl group in the following free Eu 3 concentration in the aqueous solution was determined
manner (Scheme 1 and Fig. 1A): by the SWSV method. The equilibrium binding amounts of euro-
pium metal ion or adsorption capacity (Q) of proposed polymer
2.6.1. Vinyl group modication of r-GO dendrite nanostructure were calculated according to the equation (Lo et al., 2012):
The above-synthesized product (1.0 g) was well dispersed in
10 mL of water by sonication for 1 h and reuxed with SOCl2 (
Q = (C o C e ) V / W g g 1) (1)
(60 mL, 0.82 mol) at 80 C for 72 h. The nal product (vinyl group
modied r-GO dendrite) was separated by centrifugation, washed where Co and Ce (g L ) are the initial and equilibrium con-
1

with anhydrous THF and dried in vacuum oven. centrations of the template, respectively, V (mL) is the volume of
the initial solution, and W (g) is the weight of the MIP/NIP@r-GO
2.6.2. Polymerization onto the surface of vinyl group modied r-GO dendrite. In the cross-reactivity experiment, metal ions (Cd 2,
dendrite Cu 2, Pb 2, Gd 3, Tm 3, Hg 2, As 3, Ca 2) were chosen as
The vinyl group modied r-GO dendrite was dispersed in DMSO structural analogs. The MIP/NIP@r-GO dendrite modied PGE was
(0.010 g, 1.0 mL DMSO) and drop-coated onto the surface of PGE. incubated in the solutions of these structural analogs and SWSV
Separately, CuCl2 (0.017 g, 0.1 mmol) and bipyridyl (0.031 g, current was measured. Similarly, for the competitive binding as-
0.2 mmol) were dissolved in 0.5 mL DMSO and mixed in a glass say, some biologically important molecules viz., glucose (glu), uric
vial followed by addition of ascorbic acid (0.0176 g, 0.1 mmol) as acid (UA), urea, ascorbic acid (AA), dopamine (DA) as well as metal
reducing agent. There is instant color change from blue to green ions were mixed with xed concentration of Eu 3 (9.12 mg L  1)
takes place. Then, the pre-polymer mixture [template (Eu 3, and SWSV study was performed. The concentration of each in-
0.045 g, 1.0 mL DMSO) and cross-linker (N, N- bisacryl amide, terfering compound was taken 100fold higher than template
0.077 g, 0.5 mmol)] was added to the same glass vial. Finally, in- concentration.
itiator (2.0 mmol, 0.029 mL) was added to this mixture followed
by N2 purging for 10 min. Then, 2.0 L of the pre-polymer mixture
was added onto the r-GO dendrite modied PGE and kept in an 3. Result and discussions
electric oven for 30 min at 50 C. After polymerization, an adduct
polymer layer was formed on the surface of r-GO dendrite mod- 3.1. Characterization of r-GO dendrite nanostructure
ied PGE (Scheme 1 and Fig. 1A). Similarly, non-imprinted poly-
mer (NIP) modied PGE, used for control a purpose was also made 3.1.1. Powder XRD and FT-IR study
in an identical manner, but in the absence of the template mole- Fig. 1B shows the XRD patterns of the r-GO (curve 1), silane
cule. The morphological study of polymer modied PGEs surface group functionalized Fe3O4 nanoparticle (silane@Fe3O4) (curve 2),
were done by FE-SEM analysis and discussed in Supporting in- r-GO conjugated with silane modied Fe3O4 (r-GO/silane@Fe3O4)
formation (Section S1 and Fig. S1). (curve 3), and r-GO dendrite monomer (r-GO/silane@Fe3O4/r-GO)
(curve 4), respectively. According to the literature, two strong
2.6.3. Analysis of europium metal ion using MIP@r-GO dendrite diffraction peaks at 25.93 and 42.5 in curve 1 were due to dif-
modied PGE fraction from (002) and (100) plane of r-GO (JCPDS File no. 41-
The electrochemical behavior and the determination of Eu were 1487) synthesized by reduction method (Teymourian et al., 2013).
performed by CSV, DPSV, SWSV and EIS methods. Three main It is already reported that the Fe3O4 (curve 2) have six character-
steps were involved in the electrochemical measurement of Eu 3 istic peaks at 30.15, 35.52, 43.17, 53.7, 57.1, and 62.7 corre-
i.e. extraction, washing and analysis step. At rst the adduct sponding to diffraction from planes (220), (311), (400), (422), (511)
modied PGE was placed in 0.1 M NaOH solution under dynamic and (440), respectively. These diffraction peaks belong to the
condition for 30 min in order to extract the template, resulting in characteristic crystalline spinel structure (JCPDS 88-0315) of Fe3O4
Eu-imprinted r-GO dendrite modied-PGE (MIP@r-GO dendrite- (Peng et al., 2010). When the Fe3O4 is modied with APTES, the
PGE). Then the electrode was removed from the extraction solvent intensity of the peaks changes but crystal phase is not altered
and repeatedly washed with distilled water (n 3, 0.5 mL) to avoid (shown in curve 2 and 3), which indicates the successful mod-
any analyte carry over or non-specic adsorption. For qualitative ication of Fe3O4 nanoparticles with silane and r-GO. XRD pattern
analysis, CSV was recorded from 1.5 to  0.3 V at various scan of r-GO/silane@Fe3O4 (curve 3) and r-GO dendrite (curve 4) shows
rates (0.050.5 V s  1) for xed concentration of analyte (9.12 the combination of peaks present in curve 1 and 2. This suggests
mg L  1). However, for quantitative analysis DPSV and SWSV were the formation of r-GO/silane@Fe3O4 nanocomposites. The mod-
recorded for different concentration of analyte in the potential ication of the amino group of silane@Fe3O4 with -COOH group of
window of 1.1 to 0.4 V. All the experiments were carried out in r-GO results in the formation of r-GO/silane@Fe3O4 composite
an electrochemical cell containing 10.0 mL KCl solution (pH 7.0). material. As compared to curve 3, curve 4 showed little en-
The limit of detection (LOD) was calculated as three times the hancement in the peak intensity at 25.93 and 42.5 which belongs
standard deviation of the blank measurement (in the absence of to the (002) and (100) plane of r-GO, which is due to the attach-
template) divided by the slope of the calibration plot between Eu ment of another layer of r-GO to the r-GO/silane@Fe3O4 composite.
concentration and DPSV and SWSV current (Skoog et al., 1998). So, the XRD results conrm the successful formation of
Sample preparation of soil and food are described in Supporting r-GO/silane@Fe3O4/r-GO dendrite monomer.
information (Section S2). To further conrm the interaction between Fe3O4 and r-GO, the
synthesized composites were further investigated by FT-IR spec-
2.6.4. Batch binding and cross-reactivity study troscopy. Fig. 1C shows the FT-IR spectrum of silane@Fe3O4 (curve
The adsorption experiment was performed to evaluate the 1), r-GO (curve 2), r-GO/silane@Fe3O4 (curve 3), and r-GO dendrite
binding capacity of the MIP@r-GO dendrite towards europium (curve 4). In curve 1, two bands appear at 3400 cm  1 (NH
metal ion. For this, ve milligrams of MIP@r-GO and NIP@r-GO stretching) and 1690 cm  1 (NH bending) corresponds to the
dendrites were added to the europium metal ion solutions (10 mL) presence of NH2 group in silane@Fe3O4 nanoparticle. In curve 2,
 E. Roy et al. / Biosensors and Bioelectronics 68 (2015) 726735 731

Fig. 2. FE-SEM images of (A) r-GO, (B) silane@Fe3O4, (C) r-GO/silane@Fe3O4, (D) r-GO dendrite and TEM images of (E) r-GO/silane@Fe3O4 and (F) r-GO dendrite.

presence of CO (1735), CH2 (2800 cm  1), OH stretching 3.1.2. FE-SEM, TEM and EDAX analysis
(3420 cm  1) and bending (1399 cm  1) conrms the functional The morphology and structure of r-GO (A), silane@Fe3O4 (B),
groups present in the r-GO sheet. When the r-GO sheet get con- r-GO/silane@Fe3O4 (C), and r-GO/silane@Fe3O4/r-GO
jugated with silane@Fe3O4, amide I (1644 cm  1) and amide II (D) composites were investigated by FE-SEM analysis and por-
(1456 cm  1) bands were appears in the spectra (curve 3), suggests trayed in Fig. 2. The r-GO has sheet-like morphology with char-
the amide linkage of silane@ Fe3O4 and r-GO. However, the ex- acteristically crumpled, rough and porous architecture and the
istence of NH2 stretching band in the curve 3, attributed to the layers of r-GO are interacted with each other to form 3D network
presence of free NH2 groups in the r-GO/silane@Fe3O4, which gets (Fig. 2A). However, the spherical microstructures of magnetic na-
disappeared in the r-GO dendrite spectra (curve 4). The FT-IR study noparticles were clearly observed in Fig. 2B. The closure observa-
conrms the successful formation of each product and conjugation tion reveals that these microstructures are eventually composed of
of various nanomaterials to each other. very tiny spherical nanoparticles of size approximately 48 nm.
732 E. Roy et al. / Biosensors and Bioelectronics 68 (2015) 726735

Fig. 2C shows a typical FE-SEM image of r-GO/silane@Fe3O4, where silane@Fe3O4 nanoparticles are sandwiched between layers of
the nanoparticles are homogeneously and tightly contacted on the graphene sheets.
surface of r-GO, which may cause an increase in the surface area of To further evident about the morphology of synthesized na-
r-GO. Fig. 2D shows an FE-SEM image of r-GO dendrite, where the nocomposite TEM analysis was also performed (Fig. 2E and F). As

Fig. 3. (A) Cyclic voltammetric and (B) square wave volatmmetric response for ferrocynide on (1) bare, (2) silane@Fe3O4, (3) r-GO, (4) r-GO/silane@Fe3O4, (5) r-GO dendrite
modied PGEs. (C) Magnetic hysteresis loops of (1) r-GO, (2) silane@Fe3O4, (3) r-GO/silane@Fe3O4, (4) r-GO dendrite. (D) Binding capacity of MIP and NIP@r-GO dendrite.
(E) Control plate with lter paper disks without any nanoparticles, (F) Disk diffusion tests with silane@Fe3O4, (G) r-GO, (H) r-GO/silane@Fe3O4 and (I) r-GO dendrite.
 E. Roy et al. / Biosensors and Bioelectronics 68 (2015) 726735 733

shown in Fig. 2E, the nanoparticle is clearly visible onto the surface 45 mm) treated lter paper (Fig. 3I) and minimum around
of r-GO but after the additional layer of r-GO, they appear less silane@Fe3O4 sample solution treated lter paper (Fig. 3F). Liu
prominent (Fig. 2F). This suggests the sandwich type structure of et al. (2011) have reported a three-step possible interaction me-
r-GO dendrite, as drawn in Scheme 1 and Fig. 2A. chanism of graphene-based nanomaterials with E. coli. Accord-
The similar results were also obtained by EDAX analysis of ingly, E. coli cells may rst anchor on the surfaces of graphene-
these materials. The EDAX spectrum of r-GO shows only the peak based nanomaterials during incubation in the aqueous system.
of carbon and oxygen (Fig. S2A), whereas, the amino silane func- After cells adhering to graphene surfaces, the sharp edge of gra-
tionalized Fe3O4 shows peaks for Fe, Si, N, and C elements. In the phene nanosheets may destroy the integrity of the cell membrane,
spectra of r-GO/silane@Fe3O4, all the peaks due to Fe, Si, and N resulting in the leakage of intracellular materials and nally cell
are present (Fig. S2B). But the intensity of C peak is little bit death. Liu et al. also illustrated that graphene-based nanomaterials
increased. This suggests the binding between r-GO and were capable of inducing reactive oxygen species (ROS) in-
silane@Fe3O4 (Fig. S2C). Similar behavior of C peak was also dependent oxidative stress toward E. coli cells. The results found in
observed in the spectra of r-GO/silane@Fe3O4/r-GO, where peak this work, match well with the explanation given by Liu et al.
intensity is higher than previous spectra which have only one Herein, the maximum inhibition was found with r-GO dendrite
sheet of r-GO (Fig. S2D). nanomaterials. This may be due to availability of more surface area
of r-GO dendrite to interact with E. coli for cell deposition.
3.2. Multifunctional behavior of r-GO dendrite
3.2.4. Adsorption capacity of MIP@r-GO dendrite for europium
3.2.1. Electrocatalytic activity To investigate the binding afnity of MIP@r-GO and NIP@r-GO
Graphene and their derivatives are very popular for electro- dendrite for the template Eu 3, adsorption test was performed.
catalytic activity due to the presence of enormous -electrons in The adsorption capacity of MIP@r-GO and NIP@r-GO dendrite are
the structure. Herein, we have also performed a comparative study shown in Fig. 3D. In this case, the experimental adsorption capa-
of the electrochemical behavior of all the prepared materials. city (Q) of the MIP@r-GO dendrite was found 90.0 g/g for Eu 3.
Fig. 3A and B shows, the CV and SWSV runs of bare pencil graphite Different from MIP@r-GO dendrite; the NIP@r-GO dendrite
electrode (PGE) (curve 1), silane@Fe3O4 (curve 2), r-GO (curve 3), showed a low binding afnity toward Eu 3 and the adsorption
r-GO/silane@Fe3O4 (curve 4) and r-GO dendrite (curve 5) within capacity was 5.9 g/g. The result could be explained that special
the potential window of  0.8 to 1.2 V (for CV),  0.10 to cavities of Eu 3 were formed during the imprinting process and
0.80 V (for DPSV and SWSV) for 1.0 mM ferrocyanide in 1 M KCl the MIP@r-GO dendrite showed higher binding afnity for Eu 3.
solution. It can be clearly observed that, in each technique, the On the contrary, as for NIP@r-GO dendrite, there was a lack of
lowest current was observed for bare PGE, which enhanced after recognition sites and the nonspecic adsorption had a dominant
modication (curve 1). After modication with silane@Fe3O4 and effect. Therefore, it had low adsorption capacity. To further conrm
r-GO the peak current increases, but the current enhancement is the binding interaction between MIP and template, UVvisible
more in r-GO modied PGE compared to the silane@Fe3O4 mod- study was also performed and discussed in Supporting informa-
ied PGE. This may be due to poor conductivity of silane com- tion (Section S3 and Fig. S3).
pounds coated on the Fe3O4 nanoparticle. The current further
enhanced with attachment of r-GO to the nanoparticle (curve 4), 3.3. Role of MIP@r-GO dendrite for electrochemical determination of
owing to the conjugation of silane@Fe3O4 nanoparticle to the r-GO EU 3
sheets. The highest current was observed for r-GO dendrite, which
is almost double to the r-GO or r-GO/silane@Fe3O4 nanoparticle Under optimized analytical parameters (like accumulation po-
(curve 5). From the study it can be concluded that the tential, accumulation time, scan rate and pH of the supporting
silane@Fe3O4 nanoparticle acts as a facilitator material for con- electrolyte, SI Section S4 and Fig. S4), electrochemical behavior of
jugation of two r-GO sheets, which prevents the stacking of two Eu 3 was studied on MIP@r-GO dendrite modied PGE. CSV of
r-GO sheets resulting in enhanced available surface area which in Eu 3 (9.12 g L  1) was done using the MIP-modied PGE at dif-
turn results in enhancement of electrocatalytic activity. ferent scan rate (0.050.5 V s  1) in the potential window of 1.5
to  0.3 V. As shown in Fig. 4A, a single, irreversible cathodic peak
3.2.2. Magnetic properties at 0.75 V peak was observed, which is shifted towards more
The magnetization curves of r-GO, silane@Fe3O4, negative potential with increase in scan rate. The reduction reac-
r-GO/silane@Fe3O4, and r-GO dendrite were measured at room tion process was attributed to the one-electron transfer reaction
temperature (Fig. 3C). The S-shaped hysteresis-loops without re- attributed to Eu 3/Eu 2 pair (Alizadeh and Amjadi, 2013).
manence are observed for all samples, indicating the super- Electrochemical impedance spectroscopy (EIS) was also used to
paramagnetic behaviors of nanomaterials (Zhou et al., 2013). The study the characteristics behavior of different modied and un-
saturation magnetization of r-GO, silane@Fe3O4, modied-PGEs. Fig. 4B shows the EIS spectra of bare (a), r-GO
r-GO/silane@Fe3O4, and r-GO dendrite were 25.6, 210.2, 151.8, and dendrite (b), adduct@r-GO dendrite (c), and MIP@r-GO dendrite
180.2 emu/g, respectively. For r-GO dendrite, the reduction in the modied-PGEs (d) for 9.12 g L  1 Eu 3. Along with this, the re-
saturation magnetization might be due to the low amount of binding behavior of MIP@r-GO dendrite was also studied for
magnetic nanoparticle loading on r-GO. However, the material still 19.12 g L  1 Eu 3 (curve e). In the EIS spectra, the semicircle
possess high magnetization and can be rapidly and completely diameter of Nyquist plot corresponds to the electron transfer
separated from the mixture by an external magnetic eld within limited process and the diameter is equivalent to the electron
15 s. transfer resistance (Rct). The EIS spectra of bare and r-GO dendrite
coated PGE exhibited an almost straight line, which represents the
3.2.3. Antibacterial properties low solution resistance and high conductivity of the electrodes
In this work, the antibacterial effect of the silane@Fe3O4, r-GO, (curve a and b). But, after polymerization on the surface of PGE, an
r-GO/silane@Fe3O4 and r-GO dendrite samples were studied on E. obvious interfacial Rct was observed (curve c). The resistance gets
coli. Fig. 3EI exhibit the growth of E. coli and inhibition zone of decreased after extraction of template from adduct polymer,
different sample. Herein, it clearly observed that the zone of in- which makes the electron-transfer easier. As a result, the Rct of
hibition was at a maximum around the r-GO dendrite (around MIP@r-GO dendrite coated PGE was decreased obviously due to
734 E. Roy et al. / Biosensors and Bioelectronics 68 (2015) 726735

Fig. 4. (A) CSV runs of Eu 3 (9.12 g L  1) at different scan rates (0.050.5 V s  1), (B) EIS spectra of bare (a), r-GO dendrite (b), Adduct@r-GO dendrite (c), MIP@r-GO dendrite (d) and
Rebinding of Eu 3 on r-GO dendrite (e), (C) SWSV response on MIP@r-GO dendrite modied PGE with different concentration of Eu 3 (run 1, 0.059 to run 8, 47.6 mg L  1) and
(D) Selectivity study of MIP@r-GO dendrite modied PGEs with imprinting factor in bracket.

extraction of Eu 3 from the adduct layer (curve d). Furthermore, deviation (RSD %) of nine replicates of current measurements for xed
when the Eu 3 gets rebind to the MIP-cavities, Rct value again concentration of Eu 3 was found 2.4%, supports the repeatability of
increases (curve e). It demonstrated that when Eu 3 rebind to the the proposed sensor. Along with this, the stability of the sensor was
MIP-cavities, a compact lm generated onto the surface of PGE, also explored and found that a single electrode can be used for 90 days
hindering the electron transfer and increase in resistance. without any alteration in current if stored at the room temperature
(Fig. S5B).
3.4. Estimation of europium in aqueous and real samples To illustrate the feasibility of the developed sensor for routine
analysis, the electrode was applied to determine Eu 3 con-
Under optimized experimental conditions, the relation be- centration in different type of samples like water, food, urine and
tween the response of the electrode (Ip) and concentrations of Eu human blood serum by standard addition method. The obtained
was studied. The corresponding SWSV and DPSV run on MIP@r-GO results were summarized in Table S1, where, the recoveries are
dendrite modied PGE in KCl solution (1 M, pH 7.0) were illu- ranged from 95.0% to 103.5% (RSD from 0.3% to 2.1%), which re-
strated in Fig. 4C and S5A (runs 18). The calibration equation veals that the developed electrode can be used for rapid and
obtained with DPSV and SWSV techniques with their limit of de- highly sensitive determination of Eu 3 in real samples. Stability
tection (LOD) were given below: and reproducibility of the proposed sensor were also discussed in
Supporting information (Section S5).
1. DPSV (concentration range: 0.9929.12 mg L  1): Ip (mA)
(8.910 70.011) C (mg L  1) (26.29 71.98); n 8; R2 0.99, LOD 3.5. Cross-reactivity study
0.30 mg L  1.
2. SWSV (concentration range: 0.05947.6 mg L  1): Ip (mA) Selectivity of the proposed sensor was investigated by cross-
(18.032 71.450) C (mg L  1) (102.810 733.480); n 8; reactivity and competitive binding study. As displayed in Fig. 4D,
R2 0.99, LOD 0.019 mg L  1. both MIP and NIP@r-GO dendrite modied PGE gives almost
negligible current response for all of the metal ions. Imprinting
It was also observed that the concentration more than 29.12 mg L  1 factor (IF Current on MIP/current on NIP) values were also cal-
in DPSV and 47.6 mg L  1 in SWSV does not cause further increase in culated based on these data. It can be seen that much higher IF for
current. The behavior is attributed to the saturation of binding Eu 3 were exhibited than that for the other interfering com-
sites present in the MIP-modied PGE surface. The precision of the pounds on MIP@r-GO dendrite. To further illustrate the molecular
method was estimated in terms of repeatability. The relative standard selectivity of the fabricated MIP@r-GO dendrite, binary
 E. Roy et al. / Biosensors and Bioelectronics 68 (2015) 726735 735

competitive selectivity tests were also carried out. Keeping the Appendix A. Supplementary material
Eu 3 constant concentration and adding 100 fold high con-
centration of interfering compounds in that solution did the test. Supplementary data associated with this article can be found in
We noticed that 100% response was observed for Eu 3, even in the online version at [Link]
presence of very high concentration of interfering compounds (Fig.
S6). Therefore, we can conclude that MIP had an appreciable se-
lectivity toward the imprinted template Eu 3. References

Alizadeh, T., Amjadi, S., 2013. Talanta 106, 431439.

4. Conclusion Cao, H., He, J., Deng, L., Gao, X., 2009. Appl. Surf. Sci. 255, 79747980.
Chang, L., Wu, S., Chen, S., Li., X., 2011. J. Mater. Sci. 46, 20242029.
Chen, N., Chen, L., Cheng, Y., Zhao, K., Wu, X., Xian, Y., 2015. Talanta 132, 155161.
Herein, a multifunctional nanocomposite of magnetic nano- Ge, S., Sun, M., Liu, W., Li, S., Wang, X., Chu, C., Yan, M., Yu, J., 2014. Sens. Actuators B
particle and r-GO has been successfully synthesized. The 192, 317326.
Hummers, W.S., Offeman, R.E., 1958. J. Am. Chem. Soc. 80, 1339.
silane@Fe3O4 gets sandwiched between layers of graphene and
Jiang, B., Yang, K., Zhang, L., Liang, Z., Peng, X., Zhang, Y., 2014. Talanta 122, 278284.
made the structure a dendrite i.e. multi-branched. The application Kong, H.X., 2013. Curr. Opin. Solid State Mater. Sci. 17, 3137.
of r-GO dendrite is explored in the eld of inhibition of bacterial Li, L., Luo, C., Li, X., Duan, H., Wang, X., 2014. Int. J. Biol. Macromol. 66, 172178.
Li, Y., Li, X., Dong, C.K., Qi, J.Y., Han, X.J., 2010. Carbon 48, 34273433.
growth as well as fast and selective magnetic separation of euro-
Liang, P., Hu, B., Jiang, Z.C., Qin, Y.C., Peng, T.Y., 2001. J. Anal. At. Spectrom. 16,
pium ion. The r-GO dendrites were superparamagnetic and the 864866.
magnetization was 180.2 emu/g. Along with these, the r-GO den- Liu, S., Zeng, T.H., Hofmann, M., Burcombe, E., Wei, J., Jiang, R., Kong, J., Chen, Y.,
drite shows a high electrocatalytic behavior towards ferrocyanide 2011. ACS Nano 5, 69716980.
Lo, S.-F., Wang, S.-Y., Tsai, M.-J., Lin, L.-D., 2012. Chem. Eng. Res. Des. 90, 13971406.
molecule. Finally, the dendrite was used as a platform for the Mao, Y., Bao, Y., Gan, S.Y., Li, F.H., Niu, L., 2011. Biosens. Bioelectron. 28, 291297.
synthesis of europium imprinted polymer. The detailed estimation Martn, A., Escarpa, A., 2014. Trends Anal. Chem. 56, 1326.
of lanthanide in the presence of other metal ions and co-existing Metin, O., Aydogan, S., Meral, K., 2014. J. Alloy Compd. 585, 681688.
Peng, Q., Liu, Y., Zeng, G., Xu, W., Yang, C., Zhang, J., 2010. J. Hazard. Mater. 177,
compounds was contemplated. And multifunctional behavior of 676682.
r-GO dendrite was very well explored. Qiu, H.M., Luo, C.N., Sun, M., Lu, F., Fan, L., Li, X., 2012. Carbon 50, 40524060.
Qu, J.-C., Ren, C.-L., Dong, Y.-L., Chang, Y.-P., Zhou, M., Chen, X.-G., 2012. Chem. Eng.
J. 211212, 412420.
Richter, M.M., Bard, A.J., 1996. Anal. Chem. 68, 26412650.
Acknowledgment Skoog, D.A., Holler, F.T., Neiman, T.A., 1998. Principal of Instrumental Analysis, Fifth
ed. Harcourt Brace College Publisher, Orlando.
Teymourian, H., Salimi, A., Firoozi, S., Korani, A., Soltanian, S., 2014. Electrochem.
Authors are thankful to Department of Science and Technology, Acta 143, 196206.
Government of India for sanction of Fast Track Research Project for Teymourian, H., Salimi, A., Khezrian, Somayeh, 2013. Biosens. Bioelectron. 49, 18.
Young Scientists to Dr. Rashmi Madhuri (Ref. no.: SB/FT/CS-155/ Wang, D., Gan, N., Zhang, H., Li, T., Qiao, L., Cao, Y., Su, X., Jiang, S., 2015. Biosens.
Bioelectron. 65, 7882.
2012) and Dr. Prashant K. Sharma (Ref. no.: SR/FTP/PS-157/2011). Xu, W.P., Zhang, L.C., Li, J.P., Lu, Y., Li, H.H., Ma, Y.N., Wang, W.D., Yu, S.H., 2011. J.
Dr. Sharma (FRS/34/2012-2013/APH) and Dr. Madhuri (FRS/43/ Mater. Chem. 21, 45934597.
2013-2014/AC) are also thankful to Indian School of Mines, Zeng, Y., Zhou, Y., Zhou, T., Shi, G., 2014. Electrochim. Acta 130, 504511.
Zeng, Y., Zhou, Y., Kong, L., Zhou, T., Shi, G., 2013. Biosens. Bioelectron. 45, 2533.
Dhanbad for grant of Major Research Project under Faculty Re- Zhou, D., Zhang, T.L., Han, B.H., 2013. Microporous Mesoporous Mater. 165,
search Scheme. We are also thankful to Board of Research in Nu- 234239.
clear Sciences (BRNS), Department of Atomic Energy, Government Zhou, Y., Qu, Z.-b, Zeng, Y., Zhou, T., Shi., G., 2014. Biosens. Bioelectron. 52, 317323.
Zubir, N.A., Yacou, C., Motuzas, J., Zhang, X., da Costa, J.C.D., 2014. Sci. Rep. 4, 4594.
of India for major research project. S.P. & E.R. are also thankful to
[Link]
Indian School of Mines, Dhanbad for Junior Research Fellowship.
D.K. thankful to University Grant Commission for providing post-
doctoral fellowship (Dr. DS Kothari) [F.4-2/2006 (BSR)/13-899/
2013].