Impedimetric graphene-based biosensor for the
detection of Escherichia coli DNA†
Nurulasma Zainudin,
a
Ab. Rahim Mohd Hairul,
a
Mashitah Mohd Yusoff,
a
Ling Ling Tan
b
and Kwok Feng Chong
*
a
A label-free impedimetric DNA biosensor based on graphene nanosheets has been developed for the
detection of Escherichia coli O157:H7 strain GZ-021210. Probe DNA (pDNA) of E. coli was immobilized
onto graphene nanosheets by the surface functionalization of graphene with 1-pyrenebutyric acid (PyBA)
followed by carbodiimide linkage. The hybridization of complementary DNA (cDNA) of E. coli with the
immobilized pDNA increased the electron transfer resistance of the graphene nanosheets, as observed
by electrochemical impedance spectroscopy (EIS). The E. coli DNA biosensor displayed a wide range of
linear response (1.0 10
10
M to 1.0 10
14
M), low detection limit (0.7 10
15
M), single-base
mismatch selectivity, high robustness and good reproducibility. The current work demonstrates an
important advancement in the development of a sensitive biosensor for E. coli detection.
Introduction
Escherichia coli, better known as E. coli, is a dangerous pathogen
that can cause stomach cramps, anaemia, haemorrhagic diar-
rhoea and kidney failure.
1–3
The contamination of food products
with E. coli is a chronic worldwide problem that causes million
dollar losses and presents a deadly threat to healthy living. The
largest E. coli outbreak in 2011 infected more than 3950 people
and caused 53 fatalities in Europe.
4
Therefore, it is essential to
develop a specic monitoring system for E. coli in food
products.
Conventional methods for E. coli detection include colony
counting and immunoassay. These methods, however, have
certain drawbacks such as being time consuming and requiring
skilled laboratory personnel. Various methods have been
proposed to improve the detection of E. coli, including optical,
5,6
calorimetric
7,8
and electrochemical methods.
9,10
Among these,
electrochemical methods are proven to be an effective approach
for rapid E. coli detection. These methods generally involve
probe immobilization onto a transducer; the bio-recognition
event is converted into electrical signal by the transducer. The
immobilized probe can be an antigen,
11
DNA
12
or enzyme.
13
Label-free electrochemical detection of E. coli was reported on
various transducers such as a self-assembled monolayer,
14
pol-
yaniline nanotubes,
15
indium tin oxide,
16
and an interdigitated
array microelectrode.
17
In this work, graphene nanosheets were
employed as transducers for E. coli detection due to their
remarkable electron mobility.
The Nobel Prize-winning isolation and characterization of
graphene in 2004 added a new dimension to many applications
such as electronics,
18
photonics,
19
energy
20
and sensors.
21
Gra-
phene is a two-dimensional array of sp
2
-hybridized carbon
atoms in a honeycomb lattice with exceptional mechanical,
thermal and electronic properties.
22,23
Though it is equipped
with fascinating electronic properties, graphene nanosheets are
hydrophobic in nature, hindering their application in aqueous
solution. Thus, surface functionalization is needed to tailor
graphene for various applications. In order to preserve high
electron mobility in graphene networks, surface functionaliza-
tion is performed by non-covalent p–p stacking of pyrene
derivatives. Xu et al. rst reported the functionalization of gra-
phene by 1-pyrenebutyrate in order to manipulate graphene
solubility in aqueous solution.
24
Since then, similar function-
alization approaches have been frequently employed to bind
various molecules onto graphene surfaces.
25–27
The functional-
ization of graphene by 1-pyrenebutanoic acid succinimidyl ester
for glucose oxidase immobilization was reported by Huang and
co-workers.
28
Other types of pyrene derivatives, such as perylene
tetracarboxylic acid, tetrakis(1-methyl-4-pyridinio)porphyrin,
etc., have also been employed.
29–31
Herein, we report the surface
functionalization of graphene by 1-pyrenebutyric acid (PyBA)
followed by E. coli DNA immobilization through carbodiimide
linkage. The hybridization between probe DNA and comple-
mentary DNA imparted charge transfer resistance to the gra-
phene surface, which was monitored by electrochemical
impedance spectroscopy (scheme 1).
a
Faculty of Industrial Sciences & Technology, Universiti Malaysia Pahang, 26300
Gambang, Kuantan, Pahang, Malaysia. E-mail: ckfeng@ump.edu.my; Fax: +60
95492766; Tel: +60 95492403
b
Southeast Asia Disaster Prevention Research Initiative (SEADPRI-UKM), LESTARI,
Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ay01836b
Cite this: DOI: 10.1039/c4ay01836b
Received 4th August 2014
Accepted 7th August 2014
DOI: 10.1039/c4ay01836b
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