Biosensors and PCR Insights
Biosensors and PCR Insights
Contents
Volume 92 [Link] ISSN 1726-5479
Issue 5
May 2008
Research Articles
Study of Room Temperature H2S Gas Sensing behavior of CuO-modified BSST Thick Film
Resistors
H. M. Baviskar, V. V. Deo, D. R. Patil, L. A. Patil ............................................................................... 24
Gas Sensing of Fluorine Doped Tin Oxide Thin Films Prepared by Spray Pyrolysis
A. A. Yadav, E. U. Masumdar, A. V. Moholkar, K. Y. Rajpure, C. H. Bhosale................................... 55
Design and Fabrication of Dual Mode Pyroelectric Sensor: High Sensitive Energymeter for
Nd: YAG Laser and Detector for Chopped He-Ne Laser
S. Satapathy, Puja Soni, P. K. Gupta, V. K. Dubey and K. B. R. Varma ........................................... 61
Vanadium Doped Tungsten Oxide Material - Electrical Physical and Sensing Properties
Shishkin N. Y., Cherkasov V. A., Komarov A. A., Bashkirov L. A., Bardi U., Gunko Y. K.,
Taratyn Y. A........................................................................................................................................ 69
Synthesis of Antimony Doped Tin Oxide and its Use as Electrical Humidity Sensor
B. C. Yadav, Preeti Sharma, Amit. K. Srivastava and A. K. Yadav.................................................... 99
Online Corrosion and Force Monitoring for Inner Containment Concrete Structures
K. Kumar, C. S. Unnikrishnan Nair, H. T. Jegadish, S. Muralidharan, A. K. Parande,
M. S. Karthikeyan and N. Palaniswamy ............................................................................................. 108
Active Bio-Sensor System, Compatible with Arm Muscle Movement or Blinking Signals in
BCI Application
Saeid Mehrkanoon, Mahmoud Moghavvemi...................................................................................... 144
Authors are encouraged to submit article in MS Word (doc) and Acrobat (pdf) formats by e-mail: editor@[Link]
Please visit journal’s webpage with preparation instructions: [Link]
Abstract: DNA based biosensors have recently gained much importance for detection of target genes
responsible for diseases, in food industry, environment and in agriculture. This article describes
different types of DNA based biosensors, their advantages and basic principle of operating system. The
DNA biosensors provide fast, simple, economical, sensitive and selective detection of target genes by
hybridization with specific probe. Various new strategies for DNA based biosensors have described
along with recent trends and challenges in development of technology. Electrochemical biosensor has
more advantages due to electrochemical behaviour of the labels towards the hybridization reaction on
electrode surface or in solution in the presence of redox indicators. PCR free DNA biochip is emerging
new tools in the field of diagnosis. Copyright © 2008 IFSA.
1. Introduction
Biosensors have become very popular from last 20 years. New research and developments in the field
of biosensor play important roles in daily life. In recent years, biosensors have been increasingly used
for continuous monitoring of biological and synthetic processes used in industrial and clinical
chemistry. Biosensor is becoming popular in the field of food analysis [1], bioterrorism [2-3],
environmental [2-4] and in the area of human health monitoring and diagnostics [5-7]. There is vast
exponential potential of biosensors. Presently, most fascinating and prospective sensors are
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immunosensors [8-9] based on affinity reactions between antibody and antigen and DNA sensors [10-
16] based hybridization of complementary ssDNA oligonucleotides.
In general, biosensor is small device employing biological recognition properties for a selective
bioanalysis. Such devices rely on the intimate coupling of a biological recognition element with a
physical transducer to convert the biological signals into an electrical signal or other signals,
proportional to the concentration of analytes [17]. Biosensors eliminate the need of the sample
preparation and hence offer great promise for several on-site analytical applications of rapid and low-
cost measurements.
A basic biosensor assembly 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 by adsorption, cross-linking or covalent binding. The transducers are
based upon the parameters of measurement. It may be amperometric (current measurement at constant
potential) [18], potentiometric (potential measurement at constant current) [19], piezoelectric
(measurement of changes in mass) [20], thermal (measurement of changes in temperature) [21] or
optical (detect changes in transmission of light) [22]. The usual analytical techniques require a number
of steps, much labor, time and expensive instruments whereas biosensors are quick, simple,
economical and may be used in small hospitals and laboratories of remote areas where sophisticated
instrument facilities are not available.
2. DNA Biosensor
The detection of specific DNA sequence is of significance in many areas including clinical,
environmental and food analysis [7, 23, 24]. The analysis of gene sequences and the study of gene
polymorphisms play a fundamental role in rapid detection of 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 genetically
modified organism (GMO) or pathogenic bacteria.
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 [25, 26].Recent advances in developing such devices opens a new opportunities for DNA
diagnostics. 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. Unlike
enzyme or antibodies, nucleic acid recognition layers can be readily synthesized and regenerated for
multiple use. 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 [27-29].
The current method for the identification of specific DNA sequence in biological samples are based on
isolation of double stranded (ds) genomic DNA and further polymerase chain reaction (PCR) to
amplify the target sequence of DNA. The PCR products can be subjected to electrophoresis or may be
adsorbed onto a suitable membrane and exposed to a solution containing DNA probe (Southern Blot).
The DNA probe is either chemically or enzymatically labeled with radioactive material,
chemilumnophore or ligands such as biotin etc as the nucleic acid itself has not able to provide any
signal. Recent advances in the field of biomolecular techniques can be used to fabricate new
generation miniaturized biosensor. The Table 1 summarized the advantage and disadvantages of
different types of DNA biosensors:
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DNA optical biosensors are based on a fiber optic to transducer the emission signal of a fluorescent
label. Fiber optics are devices that carry light from one place to another by a series of internal
inflections. The operation of fiber-optic DNA biosensors involves placement of an 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 [30-31]. The first DNA optical biosensor,
developed by Krull and coworkers using fluorescent indicator ethidium bromide [30, 32].Watts group
developed a fiber-optic DNA sensor array for the simultaneous detection of multiple DNA sequences
[33].The hybridization of fluorescent labeled complementary olgonucleotides was monitored by
observing the increase in fluorescence. A different optical transduction, based on evanescent wave
devices, can offer real-time label-free optical detection of DNA hybridization. The different types of
optical biosensors are as follows:
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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 strepatavidin,
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 [34, 35].
Surface plasmon resonance (SPR) is an quantum optical-electrical phenomenon arising from the
interaction of light with metal surface. Under specific conditions the energy carried by photons of light
is transferred to packets of electrons (photons) on a metal surface. Energy transfer occurs only at
specific resonance wavelength of light [33].
These biosensors are based on monitoring changes in surface optical properties (change in resonance
angle due to change in the interfacial refractive index) resulting from the surface binding reaction.
Such devices thus combine the simplicity of SPR with the sensitivity of wave guiding devices. The
resonance conditions are influenced by the material adsorbed onto the thin metal film. A linear
relationship is found between resonance energy and mass concentration of molecules such as proteins,
sugars and DNA. The SPR signal which is expressed in resonance units is therefore a measure of mass
concentration at the sensor chip surface [36-38].It has been reported that alkane thiol modified
oligonucleotide can be immobilized onto gold surface to detect DNA hybridization using SPR based
detection in agro food industry [39].
3.3. Quantum–Dot
An ultrasensitive nanosensor based on fluorescence resonance energy transfer (FREET) 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 FREET donor-acceptor assembly. Quantum dot also functions as to concentrate the signal by
confining several targets at nanoscale domain. Unbound DNA strand produce no fluorescence but on
biding of even small amount of target DNA (≤ 50 copies) may produce very strong FREET signal.
Several FREET based DNA probes (molecular beacons and TaqMan probes) whose fluorescence
signals change as a result of hybridization or enzymatic reactions have been developed for separation
free (unhybridized DNA strand) detection of target DNA [40-43].
DNA nanosensor consists of two target specific DNA probes i.e. reporter and capture probe. The
reporter probe is labeled with fluorophore whereas capture probe is labeled with biotin which binds
with streptavidin conjugated with QD. The QD functions as target concentrator as well as FREET
energy donor. When target DNA is present in solution, it becomes sandwiched by reporter and capture
probes. Several sandwiched hybrids are then captured by a single QD through biotin-streptavidin
binding and concentrate at nanoscale domain [44]. The fluorophore acceptor and QD donor close
proximity causing fluorescence from acceptor by means of FREET on illumination of the donor. The
detection of acceptor emission indicates the presence of target DNA. The unhybridized probe do not
participate in FREET and do not give fluorescence, therefore, it is not necessary to remove. The CdSe-
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ZnS core-shell nanocrystal can be used as donor and Cy5 (fluorophore) as acceptor for development of
QD based DNA nanosensor [44].
The fluorescent dyes used as the standard labels for this type of optical biosensors are very expensive
and it can rapidly photo-bleach (the dye is photochemically converted to a non-fluorescent compound).
An alternative to the fluorescence detection used in many systems is chemiluminescence format, which
overcomes the use of fluorescent dyes.
The immobilization step requires a well-defined probe orientation and accessible to the target for
hybridization. Depending upon the nature of the physical transducer, 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 transducers by covalent linkage to the gold surface via
functional alkanethiol-based monolayers. The other method of attachment of DNA probe is to
biotinylate DNA probe and attachment through biotin-avidin interaction on electrode surface [45-47,
61]. The avidin modified polyaniline electrochemically deposited onto a Pt disc electrode for direct
detection of E. Coli by immobilizing a 5’ biotin labeled probe using a differential pulse voltametric
technique in the presence of methylene blue as a DNA hybridization indicator [14, 46]. Similarly,
electrochemical DNA biosensor based on polypyrrole-polyvinyl sulfonate coated onto Pt disc
electrode was also fabricated using biotin-avidin binding [47].
The discovery of carbon nanotubes (CNTs) in DNA analysis plays an important role by development
of electrochemical DNA biosensor. CNT not only enables immobilization of DNA molecules but also
used as powerful amplifier to amplify signal transduction of hybridization. CNT also works as novel
indicator of hybridization. The application of arrayed CNT into DNA chip require small amount of
sample and development of CNT based biosensor play major role on DNA based diagnostics in
hospitals or at home [62].
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. The
unique structural, hybridization and recognition features of solution-phase PNA can be readily
extrapolated onto transducer surfaces in connection with the design of highly-selective DNA
biosensors. Such use of surface-confined PNA recognition layers imparts remarkable sequence
specificity onto DNA biosensors including detection of single-base mismatches [60].
The hybridization is commonly detected by the increase 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, offering greater discrimination
between single strand (ss) and dsDNA [27, 29, 48, 49, 51, 52, 63]. The use of an intercalator
ferrocenyl naphthalene diimide that binds to the DNA hybrid more tightly than usual intercalators and
displays small affinity to the single-stranded probe [64]. The electrochemical DNA biosensor may be
labeled based and lebeled free.
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In label based electrochemical biosensor specific organic dyes, metal complexes or enzymes are used
for hybridization detection. The use of enzyme labeled probe offers great promise for electrochemical
detection of DNA hybridization [28]. On addition of substrate to the enzyme modified electrode
surface, the electrochemical activity of the product simplifies the detection of hybridization [54].
Redox- active molecules such as daunomycin, methylene blue which is inserted between the dsDNA
and gives signal which can be used for hybridization detection [54, 65]. Redox-active molecules based
two commercialized DNA chips have been introduced in molecular diagnosis market in the trade name
of eSensorTM produced by Motorola Life sciences [25], Inc. and GenlyserTM by Toshiba [26].
Contrary to indirect detection techniques, where labeling is a requirement to translate the hybridization
event into a signal, in direct detection techniques, a target molecule or any other object from the
system does not need to be labeled [55, 54].Although label-dependent methods achieve the highest
sensitivities, eliminating the labeling steps simplifies the readout, the speed and ease of nucleic acid
assays.
In a label-free method the immobilized probe recognizes a complementary sequence if the target is
present in the sample. Next, the transducer converts the biological interaction into a measurable signal,
proportional to the degree of hybridization that is to the amount of target molecule in the sample.
Label-free strategies reduce analysis times and cost. They are also free from unfavorable effects from
the labels, such as its instability and steric hindrances.
Recently, a new label-free electrochemical detection technique has been developed which is faster and
simpler [14, 15, 45, 47, 53]. It is possible to exploit changes in the intrinsic electroactivity of DNA
(guanine oxidation peak of hybridization). To overcome the limitations of the probe sequences
(absence of G), guanines in the probe sequence were substituted by inosine residues (pairing with C)
and the hybridization was detected through the target DNA guanine signal [15, 53].Changes in the
guanine oxidation, and of other intrinsic DNA redox signals, have thus been used for detecting
chemical and physical damage. A greatly amplified guanine signal, and hence hybridization response,
was obtained by using the Ru(bpy)3 redox mediator. Direct, label-free, electrical detection of DNA
hybridization has also been accomplished by monitoring changes in the conductivity of conducting
polymer molecular interfaces (DNA-modified polypyrrole films). Eventually, it would be possible to
eliminate these polymeric interfaces and to exploit different rates of electron-transfer through ss DNA
and ds DNA for probing hybridization (including mutation detection via the perturbation in charge
transfer through DNA).
have been used for investigating other DNA interactions, including real-time detection of protein-
DNA binding or monitoring of enzymatic cleavage reactions.
Oligonucleotide QCM sensor for the microalgae Alexandrium minutum was developed by
immobilizing probe complementary strand of a partial sequence of the gene encoding microalgae that
produces neurotoxins responsible for paralytic shellfish poisoning on European and Asian coasts [68].
After hybridization in situ by using a 27 MHz quartz crystal microbalance the frequency changes
under controlled hydrodynamic conditions. The hybridization ratio between hybridized complementary
DNA and immobilized DNA probe was 47%. Piezoelectric biosensor was also developed for
Salmonella typhimurium [69].
Genomic DNA of [Link] hybridizes with the same rate constant on the QCM biosensor as in
homogeneous solution. A high hybridization rate was obtained when nucleic acids are hybridized in a
thin film, micro volume reaction on a non porous surface [70]. A DNA piezoelectric sensor was also
developed for detection of genetically modified organisms (GMO) by immobilizing DNA probe on the
sensor surface of a QCM device and hybridization of probe with target DNA was monitored in
solution. The above technique is sensitive and specific for detection of GMO and provides a useful
tool for screening and analysis of food [71].
A piezoelectric sensor for determination of genetically modified soybean Roundup Ready (RR
soybean) by immobilizing probes related to 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS)
gene onto gold piezoelectrodes. The hybridization reaction of probe and target DNA (non amplified by
PCR) was monitored in solution. The piezoelectric sensor can be used for genetically modified RR
soybean without amplification [72]. A coupling of DNA piezoelectric biosensor and PCR to detect a
point mutation in a human gene (apolipoprotein-E polymorphism) was established by immobilizing
biotinylated probe on the streptavidin coated gold surface of a quartz crystal. The hybridization of
probes with complementary, non complementary and mismatched DNA of synthetic as well as
amplified PCR samples from human blood DNA was carried out and the device was able to distinguish
polymorphism [65].
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7. DNA Biochip
Microarrays, DNA arrays, gene chips or biochips are same terminology often being intermixed to
describe analysis of complex DNA samples and expression of genes [59, 60, 77, 78, 79, 80]. The most
attractive features of these devices are the miniaturization, speed and accuracy. Accordingly, this DNA
microchip technology offers an enormous potential for rapid multiplex analysis of nucleic acid
samples, including the diagnosis of genetic diseases, detection of infectious agents, measurements of
differential gene expression, drug screening or forensic analysis. Such use of DNA microarrays is thus
revolutionizing many aspects of genetic analysis.
The biochips are fabricated from glass, silicon or plastic supports, and comprise thousands of
10-100 µm reaction zones onto which individual oligonucleotides have been deposited. This results in
high densities (up to 106 sites/cm2) in connection with typical 1-2 cm2-size chips. The exact number of
probes varies in accordance with the application. The actual construction of gene chips involves the
immobilization or synthesis of an array of DNA probes on a solid support. High-density DNA arrays
often require the use of physical delivery (e.g. microjet deposition technology), involving the
dispension of picoliter volumes onto discrete locations on the chip. It is essential to activate the surface
for a covalent attachment of the oligonucleotide probes.
Successful implementation of DNA chip technology requires development of methods for fabricating
the probe arrays, detecting the target hybridization, algorithms for analyzing the data, and
reconstructing the target sequence. Such array technology thus integrates molecular biology, advanced
microfabrication / micromachining technologies, surface chemistry, analytical chemistry, software,
robotics and automation. The automation of gene chip systems greatly facilitates their production and
accelerates their operation, while eliminating human errors. The detection of the DNA hybridization
(at the individual spots) relies on the signal generated by the binding event. The most common
application of DNA/oligonucleotide microarray is gene expression analysis. In this technique, RNA
isolated from two samples are labeled with two different fluorochromes (generally the green cyanine 3
and the red cyanine 5 (Cy3, Cy5)) before being hybridised to a microarray consisting of large numbers
of cDNAs / oligonucleotides orderly arranged onto a glass microscope slide. After hybridization under
stringent conditions, a scanner records, after excitation of the two fluorochromes at given wavelengths,
the intensity of the fluorescence emission signals that is proportional to transcript levels in the
biological samples. The microarray data are analyzed using specific software that enables clustering of
genes with similar expression patterns, assuming that they share common biological functions.
SPR, Quantum-Dot and piezoelectric biosensors are the emerging area of molecular diagnosis. The
Intelligent Opto sensors interfacing based on universal frequency-to-digital converter has opened new
opportunities for development of DNA biosensors [82]. Some success has been achieved in the
commercialization of optical fiber sensors. However, they still suffer from competition with other
mature sensor technologies and new ideas are being continuously developed and tested not only for the
traditional measurands but also for new applications [83-84].
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The use of DNA biostrip and biochip technologies eliminates the role of PCR. Future biosensors will
require the development of new reliable devices or the improvement of the existing ones in order to
allow superior transduction, amplification, processing, and conversion of the biological signals.
Efficient biosensors will not necessarily function as a stand-alone detector, but will form an integral
part of an analytical system. Compact and portable devices will constitute another future area of
intensive multidisciplinary sensor research. Further, increase of interest to 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.
Acknowledgment
The author is thankful to Department of Science and Technology, Ministry of Science and
Technology, Govt. of India, Delhi for funding project on DNA based biosensor to IGIB.
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Sensors & Transducers Journal, Vol. 92, Issue 5, May 2008, pp. 122-133
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Sensors & Transducers Journal, Vol. 92, Issue 5, May 2008, pp. 122-133
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