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DNA Based Biosensors for Detection of Pathogens

Ashok Kumar, Sandip K. Dash, Darshan P. Sharma and Suman

Biosensor is a quick analytical device and plays an important role in daily life. In last few
decades, biosensors have been increasingly used for monitoring of biological and synthetic
processes used in industrial and clinical chemistry. Biosensor is becoming popular in the field
of food analysis (Eden-Firstenberg and Schaertel, 1988), bio-terrorism (Lindner, 2001 and
Burkle, 2003), environmental (Mascini, 2001) and in the area of human health monitoring and
diagnostics (Anjum and Pundir, 2007).There is vast exponential potential of biosensors in the
field of horticulture. Presently, no biosensor is available for detection of fungal disease causing
harm in fruits and vegetables. Most fascinating and prospective sensors are immunosensors
and DNA sensors (Prummond, 2003; Umek, 2001; Junhui, 1997 and Arora et al., 2006) based
on hybridization of complementary ssDNA oligonucleotides.
In general, biosensor is small device rely on the intimate coupling of a biological recognition
element with a physical transducer to convert the biological signals into a electrical signal or
other signals, proportional to the concentration of analytes (Sharma et al., 2003). Biosensors
eliminate the need of the sample preparation and can be used on-site for analytical applications.
A basic unit of biosensor includes a receptor, transducer and processor. The sensing elements
may be whole cells, antibodies, enzymes or nucleic acids forming a recognition layer that is
integrated with transducer via immobilization. The transducers are based upon the parameters
of measurement which may be amperometric (current measurement at constant potential) (Ho
et al., 2004), potentiometric (potential measurement at constant current) (Wang et al., 2001),
piezoelectric (measurement of changes in mass) (Bunde et al.,1998), thermal (measurement
of changes in temperature) (Xie, 2000) or optical (detect changes in transmission of light)
(Mebravar, 2000). The usual analytical techniques require a number of steps, time and expensive
instruments whereas biosensors are quick, simple, economical and may be used in small
places of remote areas where expensive instrumental facilities are not existing.
32 Molecular Approaches for Plant Fungal Disease Management

1. DNA BIOSENSOR
The detection of specific DNA sequence is of significance in many areas including clinical,
environmental, horticulture and food analysis. The analysis of gene sequences and genetic
mutations, offering the possibility of performing reliable diagnosis even before any symptoms
of a disease appear. In environmental and food areas, the detection of specific DNA sequences
can be used for the detection of pathogenic bacteria, fungus or genetically modified organism
(GMO).
DNA biosensors and gene chips are of major interest due to their tremendous promise for
obtaining sequence-specific information in a faster, simpler and cheaper manner compared to
the traditional hybridization. DNA biosensors, based on nucleic acid recognition processes, are
rapidly being developed towards the assay of rapid, simple and economical testing of genetic
and infectious diseases. DNA sensors can be made by immobilizing single stranded (ss) DNA
probes on different electrodes using electroactive indicators to measure the hybridization between
DNA probes and their complementary DNA strands. The different types of DNA biosensors
are as follows:

1.1. Optical DNA Biosensors

Optical DNA biosensors are based on a fiber optic to transduce the emission signal of a
fluorescent label. Fiber optics are devices that carry light from one place to another by a series
of internal reflections (Byoungho, 2003). The operation of fiber-optic DNA biosensors involves
placement of a ssDNA probe at the end of the fiber and monitoring the fluorescent changes
resulting from the association of a fluorescent indicator with the double-stranded (ds) DNA
hybrid. The hybridization of fluorescent labeled complementary oligonucleotides was monitored
by observing the increase in fluorescence. The different types of optical biosensors are as
follows:

1.1.1. Molecular Beacons (MBs)

MBs are oligonucleotides with a stem-and-loop structure, labeled with a fluorophore at
one end and a quencher on the other end of the stem that become fluorescent upon hybridization.
In addition to their direct monitoring capability, MB probes offer high sensitivity and specificity.
A biotinylated molecular beacon probe was developed to prepare a DNA biosensor using a
bridge structure. MB was biotinylated at quencher site of the stem and linked on a biotinylated
glass through avidin or streptavidin, which acted as bridge between MB and glass matrix. The
fluorescence change was measured by confirmation change of MB in the presence of
complementary target DNA (Weihong et al., 2001).

1.1.2. Surface Plasmon Resonance (SPR)

These biosensors are based on monitoring the changes in the surface optical properties
(change in resonance angle due to change in the interfacial refractive index) resulting from the
surface binding reaction. Thiol modified oligonucleotide can be immobilized onto gold surface
to detect DNA hybridization using SPR (Wang et al., 2004; Yu et al., 2004 and Rella et al., 2004).
 DNA Based Biosensors for Detection of Pathogens 33

1.1.3. Quantum–Dot
An ultrasensitive nanosensor based on fluorescence resonance energy transfer (FRET)
can detect very low concentration of DNA and do not require separation of unhybridized
DNA. Such type of technique is based on quantum-dots (QDs) which are linked to specific
DNA probes to capture target DNA. The target DNA strand binds to a fluorescent-dye
(fluorophore) labeled reporter strand and thus forming FRET donor-acceptor assembly. Unbound
DNA strand produce no fluorescence but on binding of even small amount of target DNA (50
copies) may produce very strong FRET signal (Chun-Yang Zhang et al., 2005).

1.2. Piezoelectric DNA Biosensor

Piezoelectric DNA biosensor is based on quartz crystal that oscillate at a defined frequency
at applied oscillating voltage. Piezoelectric method have recently emerged as most attractive
due to their simplicity, cost, sensitivity and real time label-free detection (Bunde et al., 1998).
The quartz crystal microbalance (QCM) is an extremely sensitive mass-measuring device, that
allows dynamic monitoring of hybridization events. QCM hybridization biosensors consist of
an oscillating crystal with the DNA probe immobilized on its surface. The increased mass,
associated with the hybridization reaction, results in a decrease of the oscillating frequency

1.3. Strip Type DNA Sensor

A novel nanoparticle based colorimetric detection offers great promise for direct detection
of DNA hybridization. In this case, changes of the optical properties of the aggregated functional
gold nanoparticles occur on hybridization. The dry-reagent strip type biosensor has been
developed for visual detection of DNA within a short time (Sato et al., 2007 and Glynou et al.,
2003). Oligo nucleotide conjugated gold nanoparticle is used as probe for detection of target
DNA through hybridization. The advantage of this type of biosensors is that it does not require
any instrument, multiple incubation and washing steps as performed in most assays.

1.4. Electrochemical DNA Biosensors

Electrochemical devices are very useful for sequence-specific biosensing of DNA. The
miniaturization of devices and advanced technology make them excellent tool for DNA
diagnostics. Electrochemical detection of DNA hybridization usually involves monitoring a
current at fixed potential. Electrical modes were developed for detection of both label-free and
labeled objects. The immobilization of the nucleic acid probe onto the transducer surface plays
an important role in the overall performance of DNA biosensors and gene chips.
Various methods can be used for attaching the DNA probe to the solid surface such as the
use of thiolated DNA probe for self assembled monolayers (SEM) onto gold surface by covalent
linkage (Suman Kumar, 2008; Malhotra et al., 2005; Patel et al., 2009; Kumar, 2009; Patel
et al., 2010 a and Patel et al., 2010b). In our laboratory, efforts are being made to develop
DNA based biosensors for infectious diseases such as bacterial meningitis (damage of brain
covering membrane meninges) due to infection of Neisseria meningitidis, typhoid fever caused
by Salmonella typhi and Rheumatic Heart Disease (damage of heart valves) caused by
Streptococcus pyogenes infections.
34 Molecular Approaches for Plant Fungal Disease Management

The other method of attachment of DNA probe is to biotinylate DNA probe and attachment
through biotin-streptavidin interaction on electrode surface. Similarly, electrochemical DNA
biosensor based on polypyrrole-polyvinyl sulfonate coated onto Pt disc electrode was also
fabricated using biotin-avidin binding. The CNT based biosensor play major role on DNA
based diagnostics in hospitals or at home (Pingang et al.,2006). The knowledge of peptide
nucleic acid (PNA) has opened a new research area of DNA biosensors. PNA is a DNA mimic
in which the sugar phosphate backbone is replaced with a pseudopeptide. PNA has remarkable
sequence specificity onto DNA biosensors including detection of single-base mismatches.
The hybridization is commonly detected by the change in current signal due to redox
indicator (that recognizes the DNA duplex) or from other hybridization-induced changes in
electrochemical parameters (e.g. conductivity or capacitance). New redox indicators, are
offering greater discrimination between single strand (ss) and dsDNA. The electrochemical
DNA biosensor may be either labeled based or labeled free.Further, increase of interest on
DNA based sensors can be expected in near future together with a commercial production of
these devices and their wide use. However, basic research is still necessary to improve the
sensor technologies, sensing strategies as well as analytical instrumentations and procedures.

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