SAS 2009 – IEEE Sensors Applications Symposium
New Orleans, LA, USA - February 17-19, 2009
Design of a real time biorecognition system to
detect foodborne pathogens-
DNA Biosensor
Vijayalakshmi Velusamya, Khalil Arshaka1, Olga Korostynskaa, Kamila Oliwab, Catherine Adleyb2
a
Electronic and Computer Engineering Department, University of Limerick, Limerick, Ireland
b
Microbiology Laboratory, Department of Chemical and Environmental Sciences, University of Limerick, Limerick, Ireland
1
khalil.arshak@ul.ie, 2catherine.adley@ul.ie
Abstract—In recent years, there has been numerous research
papers reported on the use of DNA biosensors for the detection of
foodborne pathogens. However, none of the papers to date reflect
the detection of foodborne pathogens directly in food using a
handheld DNA biosensor.
It has been shown in our recent work that DNA sequences
named BCFomp1/BCRomp1 can be used for the specific
detection of the Bacillus cereus (B. cereus) group species (spp).
Analysis of these DNA probes using standard PCR analysis
showed that the minimum level of detection was 103 CFU/ml. The
lowest number of bacterial cell per reaction tube that can be
amplified was 5 CFU and the minimum quantity of DNA that can
be amplified was found to be 1pg.
The prime intention of this paper was to pioneer the design
and fabrication of a single-strand (ss) DNA biosensor for the
detection of the B. cereus group spp. Cyclic voltammetry (CV)
was used to develop and test a model DNA-based biosensor. The
electrically conducting polymer, polypyrrole was used as a
platform for immobilizing DNA on the gold electrode surface.
The model DNA biosensor generated unique CV signals between
complementary and noncomplementary oligonucleotides and it
proved to be effective.
Keywords: Bacillus cereus spp; foodborne pathogen detection;
DNA; cyclic voltammetry; biosensor; conducting polymers.
I. INTRODUCTION
Foodborne diseases are a worldwide growing health
problem involving a wide spectrum of illnesses caused by
microbial, viral, parasitic or chemical contamination of food.
Food diarrhoeal diseases can lead to serious illnesses and in
some cases to death. Some diseases are caused by toxins from
the “disease-causing” microbe, others by the human body’s
reactions to the microbe itself. Apart from Clostridium
botulinum, Campylobacter jejuni, Escherichia coli O157:H7,
Listeria monocytogenes, Salmonella spps, Shigella spps, the
most frequently isolated bacterial foodborne pathogens are the
B. cereus group spp [1], which include: Bacillus cereus,
Bacillus mycoides, Bacillus pseudomycoides, Bacillus
weihenstephanensis, Bacillus thuringiensis, Bacillus
anthracis [2, 3].
9781-4244-2787-1/09/$25.00 ©2009 IEEE
Growth of B. cereus results in production of several highly
active toxins. Therefore, consumption of food containing >106
bacteria/gm may result in emetic and diarrhoeal syndromes.
The most common source of this bacterium is found in liquid
food products, milk powder, mixed food products and is of
particular concern in the baby formula industry [4]. However,
they can also be found in other foods such as turkey, beef, rice
and noodles. The emetic toxin type is also known to grow well
in mashed potatoes, rice dishes and vegetable sprouts [5].
Although the safety of food has dramatically improved
overall, the progress is uneven and foodborne outbreaks from
microbial contamination, chemicals and toxins are common in
many countries [6]. International trade statistics (2007) by
World Trade Organization (WTO) reported that Europe has
accounted for 46% of world exports of agricultural products,
where food represents 80% of agricultural exports [7]. Trading
of contaminated food between countries increases the potential
for outbreaks and consequently, health risks posed by
microbial pathogens in food are of major concern to all
governments. In November 2007, the U.S. Food and Drug
Administration (FDA) developed a comprehensive ‘Food
Protection Plan’, in which it was outlined that food must be
considered as a potential vehicle for intentional contamination
[8]. Such intentional contamination of food could result in
human or animal illnesses and deaths, as well as economic
losses. It has been reported in EU legislation on
microbiological criteria for foodstuffs, that “foodstuffs should
not contain micro-organisms or their toxins or metabolites in
quantities that present an unacceptable risk for human health”,
as laid down in Regulation (EC) No 2073/2005 [9]. Recently,
the World Health Assembly (WHA) established a global
surveillance system for public health emergencies of
international concern by adopting the International Health
Regulations (IHR) which came into force in June 2007 [10].
These current legislations on food and health provide an
enhanced scientific activity into the area of food pathogen
detection. The role of pathogen detection technology is vital
and is the key to the prevention and identification of problems
related to health and safety. Therefore, giving importance to
develop a DNA biosensor for the rapid detection of bacterial
pathogens in food, we first designed DNA probes, which are
specific to the B. cereus group spp. A probe is a strand of
nucleic acid that serves as a starting point for DNA
replication. The best immobilization platform, the suitable
immobilization technique and the other important factors to
design a DNA biosensor also have been discussed.
II. DNA BIOSENSOR
Biosensors create new opportunities in the food industry
sector by forming new methodologies for microbial
monitoring. Fig.2 shows schematic diagram of a biosensor.
Biosensors provide the opportunity for rapid, in situ tests that
can replace lengthy laboratory assays. They can be designed to
monitor and quantitate a number of biological and biochemical
effects as a real-time detection system [11]. .
Fig 1: Schematic diagram of a biosensor.
Generally, DNA biosensors are constructed by the
immobilization of the oligonucleotide sequence (probe) onto a
transducer that is able to convert the biological event into
measurable signal [12]. Sometimes the probe can be free in a
solution but in all cases the principle of specific hybridization,
between single stranded DNA (ssDNA) known as the probe
and “target” sequence which must be detected, plays a key
role in DNA-based biosensors [13]. The converted signal is a
response to hybridization of the probe and target sequence can
be optical, electrochemical or pizoelectrical [14, 15].
Innovative methodologies for bacterial detection are being
developed to improve sensitivity and speed in detection. In
particular, focus was aimed at the polymerase chain reaction
(PCR) methodologies, including multiplex and real-time PCR
analysis as an answer to the traditional culture based methods,
which required 5-7 days for microbial detection. Similarly, the
solid phase microarray format emerged as the preferred
method for high throughput, high parallel hybridization testing
for DNA and RNA samples. Currently available microarray
technologies suffer from certain limitations that prohibit the
exploitation of the full range of life science applications. The
outcomes are not promising; the majority of the methods
developed require enrichments in culture media before the
analysis can begin. Problems associated with the established
fluorescence-based optical detection technique include the
high equipment costs and the need to use sophisticated
numerical algorithms to interpret the data [16]. DNA
biosensor is one of the proposed solutions for the detection of
foodborne pathogens. Over the past decade research efforts
have advanced the development of DNA biosensors but
improvements are still needed before DNA biosensors become
a real and reliable choice.
III. DESIGNING THE BIORECOGNITION
INTERFACE
For a biosensor to detect DNA, it is necessary to have a
biorecognition element which is selective for the sequence of
DNA to be detected. It is also important that this
biorecognition element is integrated with the signal transducer.
Integration is most commonly achieved by immobilising ss-
DNA on the electrode surface. Conducting polymers are more
suitable materials to immobilize the DNA. Conducting
polymers have unique properties which make them appealing
alternatives for specific materials currently employed for the
fabrication of biosensors. Common conducting polymers are
poly(acetylene)s, poly(pyrrole)s, poly(thiophene)s,
poly(terthiophene)s, poly(aniline)s, poly(fluorine)s, poly(3-
alkylthiophene)s, polytetrathiafulvalenes, polynapthalenes,
poly(p-phenylene sulfide), poly(p-phenylenevinylene)s,
poly(3,4-ethylenedioxythiophene), polyparaphenylene,
polyazulene, polyparaphenylene sulfide, polycarbazole and
polydiaminonaphthalene [17]. Among the various conducting
polymers polyaniline, polythiophene, and polypyrrole, are
biocompatible[18]. However, polyaniline and polypyrrole are
the most extensive used in biosensors for foodborne pathogen
detection. Polypyrrole is used mostly in biosensors and
immunosensors because of its best biocompatibility and the
ease of immobilization of various biologically active
compounds [18, 19]. To detect bio-analytes at a physiological
pH, biosensing materials must be electroactive in neutral
environments, unlike polyaniline and polythiophene. To
overcome this problem, polypyrrole is attractive because it can
be more easily deposited from neutral pH aqueous solutions of
pyrrolemonomers [20]. DNA can form a strong bond with
polypyrrole based on the interchanging of dopant DNA
molecules [21] and hence it has attracted attention for
application of DNA biosensors [22]. Recently, conducting
polymer nanocomposites with the enhanced properties has
been developed to overcome the inherent limitations of pure
conducting polymers [23].
The method of immobilization also plays a vital role in the
fabrication of a DNA biosensor. The followings are the
important factors associated with immobilising the DNA
probe onto the electrode:
(1) Selection of a suitable electrode material for
immobilizing the DNA;
(2) Choosing a suitable immobilization method;
(3) Designing linkers of suitable length for coupling
DNA to the surface.
Conducting polymers are excellent platforms for the
immobilisation of biomolecules at electrodes [24] since; they
are known to provide better signal transduction, enhanced
sensitivity, selectivity, durability, biocompatibility, direct
electrochemical synthesis, and flexibility for the
immobilization of biomolecules, including DNA [25]. The
commonly used immobilization methods are physical
adsorption, electrochemical adsorption, covalent attachment
and avidin-biotin.
IV. MATERIALS AND METHODS
A three-electrode cell comprising a gold working electrode
(2-mm diameter), a platinum wire counter electrode and a
Ag/AgCl reference electrode were connected to a CHI620
Electrochemical Analyser. The electrochemical deposition
technique, cyclic voltammetry (CV), was used to develop and
test a model DNA-based biosensor. Pyrrole (Sigma-Aldrich)
was electropolymerised using CV onto gold electrodes. The
electrically conducting polymer, polypyrrole (PPY) was used
as a platform for immobilizing DNA on the gold electrode
PPY with target complementary and non complementary
oligonucleotides were also carried out. Changes in the current
using the CV for 10 cycles were determined.
The electropolymerization of PPY onto gold are shown in
Fig.2. It is noticeable that the current for cycle 10 (443µA)
was higher than that for cycle 5 (407µA) and cycle 1 (380µA),
which indicates a successful deposition PPY on the gold. The
CVs for electrodeposition process and hybridization with
complementary and non complementary oligonucleotides are
demonstrated in Fig.3.
500
400
300

Current (µA)
surface. This is due to the fact that PPY can be more easily
200
deposited from neutral pH aqueous solutions of
pyrrolemonomers. All CVs were recorded using a potential
between 0.0-0.7 V at a scanning rate of 50 mV/s. 100

Specific probes for the B. cereus group spp. 0

(BCFomp1/BCRomp1) were designed. The BCFomp1 with 0 100 200 300 400 500 600 700 800
20-mer oligonucleotides and BCRomp1with 21-mer -100
oligonucleotides were used as probes in the hybridization Potential (mV)
experiment. The complementary ssDNA targets for BCFomp1
and BCRomp1 (20 and 21 bases, respectively) were tested and Fig.2. Electrochemical Polymerization of 0.05-M Pyrrole/0.5-M KCl onto
the non complementary (21 oligonucleotide) sample was also Gold electrode. CVs for 1st, 5th, and 10th cycle between 0.0 - 0.7V at a
tested. The recognition elements specific for the B. cereus scanning rate of 50 mV/s.
group spp. were derived from the motB gene that encodes an
outer membrane protein.
500
V. RESULTS AND DISCUSSION
One of the most prevalent pathogens that cause foodborne
400
outbreaks is the B .cereus group spp. which is generally found
in different types of foods and more commonly found in milk
products. It has been found that DNA sequences designed in
300
our laboratory named BCFomp1/BCRomp1 can be used for
the specific detection of the B. cereus group spp.[26]. Analysis
Current (µA)

of these primers using standard PCR analysis showed that the

200
minimum level of detection was 103 colony forming units per
ml (CFU/ml). The lowest number of bacterial cell per reaction
tube that can be amplified was 5 CFU and the minimum
100
quantity of DNA that can be amplified was found to be 1pg.
Low detection limit, high sensitivity and rapid speed of
detection still remains challenging. 0
0 100 200 300 400 500 600 700 800
To develop a DNA biosensor, conducting polymer
(polypyrrole) was used as a platform for the immobilization of -100
DNA on the gold electrodes. Successful electropolymerization Pote ntial (mV)
of the conductive polymer was achieved using a 0.05-M
Pyrrole/0.5-M KCl solution. The preparation of the modified Fig.3. Comparative CV’s of electrodeposition of polypyrrole and
Gold-DNA-PPY biosensor was achieved by electrodeposition complimentary and noncomplimentary oligonucleotides targeting the B.
cereus motB gene. (a) CVs after 10 cycles between 0.0 - 0.7 V at a scanning
of 0.05-M PPY in 1μg of each probe specific for the B. cereus rate of 50 mV/s (b) Complimentary oligonucleotide target and (c) Non
group spp. The hybridization on embedded DNA probes into complimentary oligonucleotide target.
 The model DNA biosensor generated unique CV signals
between complementary and noncomplementary
oligonucleotides. The current peak for 10th cycle during the
electrodeposition event is 443µA. There is a clear change in
the current after hybridization of the complementary
oligonucleotide (326.4µA) and for the noncomplementary
oligonucleotide (265.9µA). The drop in current after each
event is clearly noticeable.
Specificity was demonstrated after distinctive signals of
complementary sequence that yielded a higher current signal
than that obtained for a noncomplementary sequence.
Hybridization can be performed in less than 5 minutes. The
results have shown that the DNA biosensor was successful in
the rapid and specific detection of the B. cereus group spp.
The work presented here forms the model for the design and
fabrication of a handheld label free DNA biosensor for the
real-time detection of B. cereus spp, found in variety of food
products.
In our current work, attention is focused to develop a
biological sensor to detect and monitor the B.cereus group
spp. which is commonly found in milk products. A biosensor
based on the primers BCFomp1/BCRomp1 is used to identify
a unique signature (characteristics specific for B. cereus group
spp.) with more accuracy. The concept of the proposed
nanotechnology-based sensors system is depicted in Fig.4.
Individual sensors are based on the unique DNA signature of
each member of the group. Among all other different species
which may be present in milk products, it will allow for the
identification of the B. cereus group spp only. Based on sensor
identification of these pathogens, it will be possible to detect
them directly from the food source.
Fig. 4. Concept of the proposed nanotechnology-based sensors system
Improving the sensitivity, selectivity, response time, simplicity
and reducing the cost of food monitoring assays are important
goals. Other objectives include designing specific primers
which will be used to distinguish between the different species
within the B.cereus group spp.
VI. CONCLUSION
This paper reports on the development of a handheld
biosensor to detect and monitor B. cereus group spp. which is
commonly found in milk products.
Foodborne pathogen detection is an emerging area which
has led to the rapid development of DNA based biosensors. A
ssDNA biosensor was successfully demonstrated and it proved
to be effective. The novel use of conducting polymers for
sensor fabrication has allowed to develop DNA biosensors
which have characteristically low detection limits, allowing
rapid detection of pathogens. However, it is important to
consider the characteristics of the sensor. Also the method of
DNA immobilization plays a vital role in sensor design. The
hybridization event was clearly distinguishable from
complementary and noncomplementary sequences using CV
technique within 5 minutes. The use of a handheld DNA
biosensor platform is essential for the real-time detection of
specific pathogens from mixed food matrices.

ACKNOWLEDGEMENTS
This research work is funded by Science Foundation
Ireland (SFI) Research Frontiers Programme, ID no:
07RPF-ENEF500. Dr. O. Korostynska would like to
acknowledge the support received from the Irish Research
Council for Science, Engineering and Technology
(IRCSET): funded by the National Development Plan.
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