Dna Based Biosensors In Diseases Diagnosis Biology Essay

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The advent of fast and easy DNA testing has given the space for the Science to develop small and easy-to-handle equipments called Biosensors. DNA based biosensors have been proven very useful and are accorded with much importance in detecting the target genes responsible for diseases. This article enlists different types of biosensors, their basic principle of operating system, the preparation of DNA microarrays, lab-on-a-chip and their role in diseases diagnosis. DNA biosensors provide swift, sensitive, simple, economical and selective detection of DNA hybridization. New strategies for DNA biosensor are enumerated and are used meticulously in recent trends and for future directions. Carbon nanotubes (CNTs) amplify the electrochemical signal when used with DNA hybridization. Electrochemical, piezoelectric, SPR, optical DNA biosensors are used to detect various viruses like hepatitis virus, HCMV, HIV, orthopox virus etc. and also for the diagnosis of various diseases like cancer, tuberculosis, COPD, genetic diseases (sickle cell anemia i.e. due to single point gene mutation), cystic fibrosis, diabetes etc. The methodologies of detecting such diseases using different types of DNA based biosensors and gene chips are described in this article. PCR free DNA chips, cell- omic sensors and nanosensor are emerging tools in the field of diagnosis. Recent advances in developing such devices provide myriads of new opportunities for DNA diagnostics.

Introduction

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A rapidly developing area of biotechnology arousing intense scientist interest is that of biosensor. Biosensor has become popular in the field of food analysis [1], bioterrorism [3], environmental [2-3] and in the area of human health monitoring and diagnostics[4-6]. Recent advances are being mad in all areas of biosensors technology. Presently, most fascinating and prospective sensors are immunosensors based on affinity reactions between antibody and antigens and DNA biosensors based on the hybridization between DNA probes and their complementary DNA strands.

In general, biosensor is an analytical device which employs biological recognition properties for a selective analysis. Such sensors integrate a biological element with a physiochemical transducer to produce an electronic signal proportional to the concentration of analytes [7].

A basic biosensor assembly includes a biological element, transducer and detector. The sensing material may be antibodies, enzymes, whole cell or nucleic acids that form a recognition layer which is integrated with the transducer via immobilization by cross linking, adsorption or covalent binding. Transducers may be amperometric (measuring the current at constant potential) [8], potentiometric (measuring the potential at constant current) [9], piezoelectric (measuring the changes in mass), thermal (measuring the changes in temperature) [10] or optical (detects changes in transmission of light) [11]. The nature of interaction between the analyte and the biological material used in the biosensors may b of two types. Bioaffinity sensors rely on the selective binding of the target analyte to a surface-confined ligand partner (e.g. antibody, nucleic acids). In contrast, in biocatalytic sensors, an immobilized enzyme is used for recognizing the target substrate (sensor strips with immobilized glucose oxidase used for personal monitoring of diabetes. A number of steps, much labor, time and costly instruments are required in usual analytical technique whereas biosensors are economical, fast and simple and can be used in small laboratories and hospitals of remote areas which are devoid of sophisticated instruments facilities.

Figure 1. A biosensor showing four components: a biological sensing element, a transducer, a signal conditioner and a data processor

DNA Biosensors

Nucleic acid recognition process is the basis of DNA Biosensors. These are being developed with a rapid pace with an ambition for inexpensive testing for genetic and infectious disease and for detecting DNA damage and interactions. The study of gene polymorphisms and the analysis of gene sequences play a fundamental role in rapid detection of genetic mutations, opens up new opportunities for reliable diagnosis even before any symptoms of a disease appear. Thus recent advances in developing such devices offer the opportunities for DNA diagnostics.

DNA biosensors are made by immobilizing single stranded (ss) DNA probes on different transducers for measuring the hybridization between the DNA probes and their complementary DNA strands [12-13].

The current methods to identify specific DNA sequence in Biological samples depends on the isolation of double stranded (ds) DNA and further polymerase chain reaction (PCR) to amplify the target sequence of DNA. The PCR product is then subjected to electrophoresis or adsorbed onto a suitable membrane and exposed to a solution containing DNA probe.

Surface Chemistry and Biochemistry

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The immobilization of DNA probe onto the transducer plays an important role in the performance of the DNA Biosensor. The immobilization step must lead to a well-defined probe orientation, readily accessible to the target. The mode of immobilization is the determining factor for the environment of immobilized probes at the solid surface. On the basis of nature of physical transducer, various schemes may be used for attaching the DNA probe to the surface. These include the use of thiolated DNA for self assembly onto gold transducers (gold electrodes or gold coated piezoelectric crystals) covalent linkage to the gold surface via functional alkanethiol-based monolayer, the use of biotylated DNA for complex formation with a surface-confined strepavidin or avidin, covalent coupling (carbodiimide) to the functional groups on carbon electrodes or a simple adsorption onto carbon surfaces.

Introduction of peptide nucleic acid (PNA) has opened up many exciting opportunities for DNA biosensors. PNA is a DNA mimic in which the sugar-phosphate bone is replaced by a pseudo-peptide one. Such use of surface-confined PNA recognition layers imparts remarkable sequence specificity on DNA biosensors and offers other advantages.

DNA dendrimers may also be used for imparting high sensitivity onto DNA Biosensors. These are tree-like superstructures which possess numerous ss arms that can hybridize to their complementary DNA sequence. The immobilization of these dendritic nucleic acids onto physical transducer gives an amplified response [14].

Recent advances in the field of biomolecular techniques may be used to design new generation miniaturized biosensor.

Types of DNA based Biosensors

Type

Biological Element

Transducer

Advantages

Disadvantages

1.Optical

Fiber optics

Laser Interferometry

DNA

Optical fiber

Highly sensitive

Costly equipment and not portable

Susceptibility to turbidity interference

2.Electrochemical

Conductometric

Potentiometric

Amperometric

DNA

Carbon paste electrodes

Fast, low cost

Highly buffered solution may interfere

3. Piezoelectric

DNA

Quartz Crystals

Fast, highly sensitive

4. DNA chips

DNA

Quantitative

Optical DNA based Biosensor

Optical methods are the most commonly used for the detection of analytes. DNA optical biosensors are based on a fiber optic to transduce the emission signal of a fluorescent label. Fiber optics is devices that carry light from one place to another by a series of internal inflections. The methodology of fiber-optic DNA bio-sensors involves placement of a single stranded DNA 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 [15 - 16].

The first DNA optical bio-sensors were developed by Krull and Co workers using fluorescent indicator ethidium bromide. Watts group developed a fiber-optic DNA sensor array for the simultaneous detection of multiple DNA sequences [17]. The hybridization of fluorescent labeled complementary oligonucleotides was monitored by observing the increase in fluorescence. A real time label free optical detection of DNA hybridization can be offered by a different optical transduction based on evanescent wave devices. The different types of optical biosensors include:

1.1 Surface Plasmon Resonance (SPR)

It is a quantum optical electrical phenomenon based on the interaction of light with metal surface. Only at specific resonance wavelength of light, the energy carried by photons of light is transferred to packets of electrons (photons) on a metal surface [17].

These biosensors rely on monitoring change in surface optical properties (change in resonance) angle due to change in the interfacial refractive index) resulting from the surface binding reaction. Thus such devices combine the simplicity of SPR with the sensitivity of wave guiding devices. The SPR signal that is expressed in resonance units is therefore a measure of mass concentration at the senor chip surface [18-20].

1.2 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. MB probes offer high sensitivity and specificity and direct monitoring capability. A biotinylated molecular beacon probe was developed to prepare a DNA sensor using a bridge structure. MB was biotinylated at quencher site of the stem and linked on a glass through streptavidin that act as a bridge between MB and glass matrix. The fluorescence change was measured by confirmation change of MB in the presence of complementary target DNA [21-23].

Quantum - Dot

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It is an ultra sensitive nanosensor based on fluorescence resonance energy transfer (FREET) that can detect very low concentration of DNA. In these neon sensors, quantum dots (QDs) 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 target concentrator as well as FREET energy donor [24]. DNA nanosensor contains 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 that binds with streptavidin conjugated with QD [25]. The fluorophore acceptor and QD donor in close proximity produce fluorescence from acceptor by means of FREET on illumination of the donor. The presence of target DNA is indicated by the detection of acceptor emission. The un-hybridized probe does not give fluorescence. The CdSe - Zns core shell nanocrystal can be used as donor and Cy5 (fluorophore) as acceptor for developing QD based DNA nanosensors [25].

For this type of optical bio sensors fluorescent dyes used as standard labels are very expensive and can rapidly photo bleach. An alternate used is chemiluncinscence format, which overcomes the use of fluorescent dyes.

A Fiber-optic DNA biosensor array

A new method of preparing the fiber-optic DNA biosensor and its array for the simultaneous detection of multiple genes is described. The optical fibers were made into fiber-optic DNA biosensors by adsorbing and immobilizing the oligonucleotide probe on its end but were first treated with poly-l-lysine. The fiber-optic DNA biosensor array was well prepared by assembling the fiber-optic DNA biosensors in a bundle in which each fiber carried a different DNA probe. Hybridization of fluorescent- labeled cDNA of Rb1 gene, N-ras gene and Rb1 p53 gene to the DNA array was monitored CCD camera. A good result was achieved [61].

2. Electrochemical DNA Bio sensors

These are very useful devices for sequence specific biosensing of DNA. The inherent miniaturization of such devices and advance micro fabrication technology make them excellent tool for DNA diagnostics. Electrochemical detection of DNA hybridization usually involves monitoring a current response at fixed potential. Detection of hybridization even is commonly done via the increased current signal of a redox indicator (that recognizes the DNA duplex) or from other hybridization induced changes in electrochemical parameters (e.g. conductivity or capacitance) [26-28].

The discovery of carbon nano tubes (CNTs) plays an important role in development of electrochemical DNA sensors. Various CNT based electrochemical are developed because the combination of unique electrical, thermal, chemical, mechanical and 3-D spatial properties of CNTs with DNA hybridization offers the possibility of creating DNA bio sensors with specificity, simplicity, high sensitivity and multiplexing. Two major groups in which CNTs divided are - single walled CNTs (SWCNTs) that are comprised of a single graphite sheet rolled with a tube and multi walled CNTs (MWCNTs) that are concentric & closed graphite tubes [29].

CNT enables immobilization of DNA molecules and also used as powerful amplifier to amplify signal transduction of hybridization [30]. Two types are generally used to immobilize the CNT on electrodes - aligned and non-aligned.

Two approaches are generally used for the immobilization of bio molecules onto CNTs that are non covalent attachment (physical absorption) and covalent binding (some cross linker agents (1-ethyl - 3-3 dimethylaminopropyl) carbodilimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS)] or affinity binding (avidin - biotin interaction).

CNT also act as novel indicator of hybridization. The application of arrayed CNT into DNA chip requires small amount of sample and development of CNT base biosensor has an important role in DNA based diagnostics in hospitals or at home [30]. Various methods are used for immobilizations step i.e. for attaching the DNA probe onto the solid surface that are - (a) 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.(b) Attachment of biotinylated DNA probe through biotin avidin interaction on electrode surface for e.g. avidin modified polyaniline electro chemically deposited onto a Pt disc electrode for direct detection of E. Coli by immobility a 5' biotin labeled probe using a differential pulse Voltametric technique in the presence of methylene blue as an DNA hybridization indicator [31,32]. The electrochemical DNA biosensors may be labeled free and labeled based.

Label Free

In this direct detection technique the target molecule does not need to be labeled [27]. The elimination of labeling steps simplifies the readout the speed and ease of nucleic acid assays. Thus recently increased attention has been given to new label free electrochemical detection schemes. 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 were substituted by inosine residues (pairing with C) and the hybridization was detected through the target DNA guanine signal. Changes in the guanine oxidation and of other intrinsic DNA redox signals detect the chemical and physical damage [33].

Label Based

In label based electrochemical biosensor specific organic dyes, enzymes or metal complexes are used for hybridization detection. Redox active molecules such as methylene blue, dacinomycin that is inserted between the dsDNA and gives signal which is used for detecting hybridization [26] (e.g. of two commercialized DNA chips based on redox active molecules are eSensor TM produced by Motorola life sciences [34], Inc. and Genlyser TM by Toshiba)[35].

Piezoelectric DNA Biosensor

These are the mass sensitive devices rely on quartz crystal that oscillate at a defined frequency when oscillation voltage is applied. Increased attention has been given to piezoelectric method due to their simplicity, cost, sensitivity and real time label free detection. The quartz crystal microbalance is an extremely sensitive piezoelectric device that monitors the hybridization events. These biosensors DNA probe is immobilized on the surface of oscillation crystal. The increased mass due to hybridization reaction results in change in oscillating frequency [36-37].

A Piezoelectric sensor for determination of genetically modified soyabean roundup ready [RR soyabean] by immobilizing probe related to 5-enolpyrllvylshikimate 3-phosphate synthase (EPSPS) gene onto gold piezoelectrodes [38].

For detecting a point mutation in a human gene (apolipoprotein-E polymorphism) a combination of DNA piezoelectric biosensor and PCR was developed by immobilizing biotinylated probe on the streptavidin coated gold surface of quartz crystal. The hybridization probes with complementary, non-complementary and mismatched DNA of synthetic as well as amplified PCR samples from human blood DNA was taken out and the device was able to distinguish polymorphism [39].

Colorimetric or Strip type DNA sensor

Using these sensors the direct detection of DNA hybridization is possible [40-42]. The dry-reagent strip type biosensor has been developed for visual detection of double stranded DNA within a short time [43]. Oligonucleotides conjugated gold particle is used as probe. The main advantage of these biosensors is not requiring any instruments, multiple incubation and washing steps.

Integral part of strip consists of gold particles reporters with oligo (dT) attached to their surface. Biotinylated PCR products are hybridized with poly (dA) tailed oligo and applied on the strip and immersed in the appropriate buffer. As the buffer migrates upward, the nanoparticles that are linked through target DNA through poly (dA/dT) hybridization are rehydrated. Immobilized streptavidin then capture the hybrid in the control zone of the strip. The test is 8-10 times more sensitive than ethidium bromide in agarose gel electrophoresis. The detection limit is as low as 2 fmol of amplified DNA products.

DNA Biochips

Microarrays, DNA arrays, gene chips or biochips are same terminology often being intermixed. DNA microarrays are small, solid supports which themselves are usually microscopic slides, but can also be silicon chips or nylon membranes onto which the sequences from thousands of different genes are immobilized, or attached, at fixed locations. The DNA is spotted, or actually synthesized directly onto the support. DNA microarrays detect the change in gene expression levels, genomic gained and losses, mutations in DNA and infectious agents, diagnosis of genetic diseases, drug screening or forensic analysis.

Development of methods for fabricating the probe arrays, detecting the target hybridization, algorithms for analyzing the data and reconstructing the target sequence are required for successful implementation of DNA chip technology. Such array technology thus integrates molecular biology, advanced micro fabrication / micromachining technologies, surface chemistry, analytical chemistry, software, robotics and automation.

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 hybridized to a microarray consisting of large numbers of cDNAs/oligonucleotides orderly arranged onto a glass microscopic slide. After hybridization, a scanner records and 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 [33, 44].

Figure 2. For obtaining gene-expression profile data from a cDNA microarray, RNA is first extracted from an infected cell. Then the RNA is reverse transcribed and labeled. The prepared RNA is hybridized to the chip. The hybridized chip is scanned and image processed to provide corresponding gene-expression profiles.

A new ultrasensitive electronic sensor has been developed by Singapore scientists that would speed up DNA testing for disease diagnosis and biological research. The novel electronic sensor array would be rapid, accurate and cost-efficient. Excellent sensitivity has been shown by the Nanogap Sensor Array at detecting trace amounts of DNA. By saving time and lowering expenses, newly developed Nanogap Sensor Array offers a scalable and viable alternative for DNA testing. The presence of DNA is translated into an electrical signal by biosensor for computer analysis. The distinctively designed sensor chip has the ability to detect DNA more efficiently. The novel vertical nanostructure design and two different surfaces of the sensor allow ultrasensitive detection of DNA [45].

Lab-on-a-chip

Integration of the sample preparation and DNA array detection in the so-called 'Lab-on-a-Chip' configuration is the another active field. The objective of this technology is to fully integrate multiple processes, including sample collection and pretreatment with the DNA extraction, amplification, hybridization and detection, on a microfluidic platform. The ability to perform all the steps of the biological assay on a single self-contained microchip gives significant advantages in terms of speed, cost, sample/reagent consumption, contamination, efficiency and automation. Transportation of the laboratory to the sample source will be enabling by such miniaturization of analytical instrumentation (as desired for point-of-care testing). The preparation of these credit-card sized microlaboratories is commonly based on advanced microfabrication and micromachining technologies, using processes common in the manufacture of electronic circuitry [14].

Cell-omic sensors

Cell based detection systems can be combined with the microarray probes generating the hybrid arrays of cells within arrays of DNA/protein probs. This allows multiparameters analysis [46].

Applications of DNA Biosensors

Biosensors plays a distinguished role in the field of environmental quality, food analysis, study of biomolecules and their interactions, drug development, crime detection, medical diagnosis, quality control, industrial process control, detection system for biological warfare agents, manufacturing of pharmaceuticals and replacement organs. The applications of DNA biosensor can be classified into three broad categories: sequencing, mutation detection and matching detection [47]. Their main use is for diseases diagnosis. Numerous diseases can be diagnosed and variety of infectious agents can be detected using DNA biosensors.

1. Viral diseases

By DNA microarrays

Either viral detection were being carried by immunological techniques (i.e. use of enzyme-linked immunosorbent assays (ELISAs) for the detection of circulating virus-specific antibodies) or PCR - based techniques (i.e. reverse transcriptase (RT) - PCR is used to detect the presence of specific viral genes). Both these approaches possess some limitations. Immunological tests need specific antisera and the production of antisera is laborious and time-consuming task whereas PCR is prone to failure in its ability to identify multiple viruses simultaneously [48]. Therefore, recent advances in DNA and protein microarray methodology fulfill the need of a rapid and sensitive detection of viral infections (also identify multiple viruses in parallel).

DNA microarrays for viral analysis can be divided into - viral chips and host chips. Each not only detects and identifies but also monitor the viral populations.

In 1999, the first viral DNA microarray for the temporal profiling of viral (human cytomegalovirus, HCMV) gene expression was described. Viral replication or de novo protein synthesis was blocked by treatment of infected cells with cycloheximide or ganciclovir and then the expression profiles of viral genes was generated using microarray. Using this approach, the HCMV genes were classified to immediate-early, early or late expression classes, on the basis of their expression profile in response to the drug treatments. This can be used as an identifying hybridization signature for the molecular staging of an infection [49].

Orthopoxvirus causes smallpox and has two subtypes - variola major and variola minor, of differing pathogenicity. This problem of orthopoxvirus subtype discrimination was solved by producing an array capable of correctly identifying the four of the orthopoxvirus species by laassri etal. [50].

HIV genotyping was done using chip technology [51]. A unique signature that is derived from viral is provided by viral chips.

Host chip is used for examining the host response i.e. changes in host gene expression. This provides a molecular signature of infection. Cummings and Relman exposed an idea of host chips [52].

Van't wout etal. examined HIV - 1 infection in CD4+ T-cells to detect changes in host gene expression that were specific to HIV infection [53].

Proinflammatory genes and genes involved in endoplasmic reticulum stress pathways, cell cycle, and apoptosis were the host gene signatures identified.

Detection of hepatitis B virus

Hepatitis B virus (HBV) is one of the causative agents of viral hepatitis which is leading cause of liver cancer. Infection of HBV is a public health problem of worldwide significance with acute and chronic clinical consequences. Acute HBV infection may lead to liver failure or may progress to chronic liver disease. Some chronically infected individuals may subsequently suffer cirrhosis and liver failure or develop hepatocellular carcinoma. Effective antiviral therapy may inhibit or retard the progression to severe liver disease.

By DNA optical biosensor

Bacterial alkaline phosphatase (phoA) gene and hepatitis B virus (HBV) DNA were used as target DNA. For capturing the target gene onto streptavidin - coated magnetic beads, a biotinylated DNA probe was used. A calf intestine alkaline phosphatase - labeled DNA probe was used for subsequent enzymatic chemiluminescence's detection. The detection cycle was less than 30 min, excluding the DNA hybridization time that was about 100 min. at fematomole or picogramme levels both phoA gene and HBV DNA could be detected. No response signal was obtained when in sample target DNA did not exist [54].

By Piezoelectric DNA biosensor

HBV nucleic acid probe was immobilized onto the coated gold surface of quartz crystal using polyethyleneimine adhesion, glutaraldehyde cross-linking (PEI-Glu) method or the physical adsorption method. Better results were obtained with the coated crystal with the PEI - Glu method to immobilized HBV nucleic acid probe than physical adsorption method with respect to sensitivity, reproducibility and stability. The increased mass, associated with the hybridization reaction, results in change in oscillating frequency. The frequency shifts of hybridization have better linear relationship with the amount of HBV DNA, when the amount was in range of 0.02-0.14 microgram/ml [55].

By electrochemical DNA biosensor

An electrochemical DNA biosensor was developed rely on the recognition of target DNA by hybridization detection. Glassy carbon electrode (GCE) modified with label free21mer single-stranded oligonucleotides related to hepatitis B virus sequence via covalent immobilization was used and [Cu(dmp)(H2O)Cl2] (dmp = 2,9-dimethyl-1,10-phenanthroline) as an electrochemical indicator, whose sizes are comparable to those of the small groove of native double-duplex DNA. The method, that is simple and low cost, allows the accumulation of copper complex within the DNA layer. Electrochemical detection was performed by cyclic voltammetry and differential pulse voltammetry over the potential range where the [Cu(dmp)(H2O)Cl2] was active. The detection of hybridization is accomplished by using [Cu(dmp)(H2O)Cl2], where electroactivity and strong association with the immobilized dsDNA segment lead to significantly enhanced voltammetric signal.

The differential pulse voltammograms for the cathodic signals of [Cu(dmp)(H2O)Cl2] at a bare GCE, and at ss- and dsDNA-modified GCEs are also recorded. The peak currents of [Cu(dmp)(H2O)Cl2] increased in the order of bare GCE, ssDNA/GCE, and dsDNA/GCE. After hybridization process, a greater peak current was observed from dsDNA/GCE than at ssDNA/GCE, because that more [Cu(dmp)(H2O)Cl2] molecules are concentrated or bound to dsDNA helix than to ssDNA. Thus, [Cu(dmp)(H2O)Cl2] can be used as an electroactive indicator for recognition of the surface hybridization process.

The sensitivity of the electrochemical hybridization assay was investigated by varying the target oligonucleotides concentration. The different current value obtained in the DPV response of [Cu(dmp)(H2O)Cl2] after hybridization of probe with target is recorded with three repetitive measurements. The current response at about 0.485V increased in proportion to the amount of the target sequence used [56]

Detection of hepatitis C 3a virus

An electrochemical DNA biosensor, using a gold electrode modified with a self-assembled monolayer composed of a peptide nucleic acid (PNA) probe and 6-mercapto-1-hexanol was developed. The sensor relies on covalent attachment of the14-mer PNA probe that is related to the hepatitis C virus genotype 3a (pHCV3a) core/E1 region on the electrode. Covalently self-assembled PNA could selectively hybridize with a complementary sequence in solution to form dsPNA-DNA on the surface. Upon hybridization of the self-assembled probe with the target DNA in the solution, the increase of peak current of methylene blue (MB) was observed and used to detect the target DNA sequence. Some hybridization experiments with noncomplementary oligonucleotides were carried out to determine whether the suggested DNA sensor responds selectively to the target. Diagnostic performance of the biosensor is described and the detection limit was found to be 5.7 Ã- 10−11 M with a relative standard deviation of 1.4% in phosphate buffer solution, pH 7.0. This sensor exhibits high reproducibility and could be used to detect the target DNA for seven times after the regeneration process [57].

Cystic fibrosis

Mikkelsen's team, that pioneered the use of redox indicators, demonstrated utility of electrochemical DNA biosensor for detecting the cystic fibrosis F508 deletion sequence associated with 70% of cystic fibrosis patients. A detection limit of 1.8 fmol was demonstrated for the 4000-base DNA fragment in connection to a Co(bpy)33+ indicator. High selectivity towards the disease sequence (but not to the normal DNA) was achieved by performing the hybridization at an elevated (43°C) temperature [14].

3. Diabetes

Diabetes is a worldwide public health problem. The diagnosis and management of diabetes requires a tight monitoring of blood glucose levels. Thus millions of diabetics test their blood glucose levels daily by making glucose the most commonly tested analyte. The challenge is to provide such reliable and tight glycemic control. Electrochemical biosensors for glucose thus play a leading role. Amperometric enzyme electrodes, based on glucose oxidase (GOx) bound to electrode transducers, have thus been found the subject of substantial research [58].

Glucose sensors are generally used to measure the blood glucose level of diabetes patients. Using the newest DNA chip technology, scientists at Joslin Diabetes Center have discovered a new gene implicated in the cause of type 2 diabetes they created a defect in one of these genes called ARNT in mice, the mice developed alterations in insulin secretion that were like those in humans with type 2 diabetes.

The ARNT (aryl hydrocarbon receptor nuclear translocator) gene is a member of a family of transcription factors required for normal embryonic development and also is involved in response to conditions of hypoxia and certain environmental toxins, such as dioxin. Transcription factors like ARNT regulate the expression and activity of many other genes in the cell and thus serve as master regulators of cell function. ARNT is a component of the response to toxins and hypoxic stress and thus it is also at a potential site to integrate genetic and environmental insults.

The first use of DNA chips is represented by this study to study islet of diabetic patients and the first demonstration of an important role for ARNT and altered gene expression in impaired beta-cell function in the pathogenesis of human type 2 diabetes.

Type 2 diabetes is the most common human metabolic disease, affecting almost 200 million people worldwide, and is increasing at epidemic rates in the U.S. and worldwide. The pathogenesis of type 2 diabetes includes two defects: insulin resistance and impaired functioning of the insulin-producing beta cells in the pancreas. Both of these two defects have some element of genetic programming. In 2 to 5 percent of patients with a form of type 2 diabetes known as maturity onset diabetes of the young (MODY), defects involving genes necessary for beta-cell function have been found. Even so the genetic defects of this beta-cell defect remain unknown in the majority of patients with the common variety of type 2 diabetes.

4. Tuberculosis

An electrochemical biosensor for the determination of short sequences from the Mycobacterium tuberculosis (MTB) DNA was developed. The sensor based on the modification of the carbon-paste transducer with 27- or 36.mer oligonucleotide probes and their hybridization to complementary strands from the MTB DNA direct repeat region. Chronopotentiometry is used to transduce the hybridization event, in connection with a Co(phen):* indicator. Short (5-15 min) hybridization periods permit convenient quantization of ng ml-' levels of the MTB DNA target. Similar results are observed using microfabricated carbonstrip transducers [59].

Label-free sensing technology (SPR) can be used as a novel approach for diagnosis of chronic obstructive pulmonary disease.

5. Genetic diseases

Piezoelectric DNA biosensor is also used for detecting the TaySachs genetic disorder. For such detection, a highly sensitive microgravimetric device was developed.

An oligonucleotides sensor was designed for the detection of point mutation associated with sickle cell disease. Sickle cell disease is a medical condition where the red blood cells assume an abnormal, rigid sickle shape. These sickle-shaped blood cells are tending to form clumps, which block blood flow in the blood vessels leading to the limbs and organs. Blocked blood vessels may cause severe pain, infections, and organ damage. It is known that the gene defect associated with sickle cell disease is the mutation of a single nucleotide (from A to T) of the β-globin gene which results in a valine instead of a glutamate to be expressed. This disease occurs when a person inherits two sickle cell genes from the parents. Those people who inherit a single sickle cell gene from a parent will not develop the disease, but will have sickle cell trait, which means they can pass the sickle cell gene on to their own children.

The sensor was based upon luminescence resonance energy transfer between a donor and an acceptor. Photon upconverting nanoparticles (NaYF4 doped with Yb3+ and Er3+) were used as the donor and a conventional fluorophore, N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), as the acceptor. The sensor could find the perfectly matched target, in the background of the mismatched target or other oligonucleotides of random sequences. The detection limit of this sensor for perfectly matched target was calculated to be 120 femtomoles, with no photobleaching. Oligonucleotide sensors of such design show high sensitivity and specificity.

A sandwich-type hybridization format was followed by using two shorter oligonucleotides with designed sequence to capture the longer target oligonucleotide. While designing oligonucleotide strands of DNA, proper care should be taken to avoid any loops and secondary structure in both short oligonucleotides. One of the short oligonucleotides was covalently bound to the photon upconverting nanoparticles, while the other was labeled with TAMRA which was chosen as the fluorophore in this study, because its excitation spectrum overlaps with the emission spectrum of the upconverting nanoparticles. Upon excitation by an infrared laser, the visible light would be emitted by the photon upconverting nanoparticles. Without the target oligonucleotide, separation of short oligonucleotide took place. Negligible energy was transferred between the photon upconverting nanoparticles and TAMRA. The addition of the target oligonucleotide took the fluorophore close to the nanoparticle. So, energy transfer took place between the photon upconverting nanoparticles and TAMRA as shown in where the NaYF4:Yb3+,Er3+ nanoparticle emission (537 nm) decreased while TAMRA emission (575 nm) increased as the amount of the target oligonucleotide increased. The presence of the target oligonucleotide can be detected by monitoring the TAMRA emission upon an infrared excitation [60].

Conclusion and Future prospects

Huge progress is observed particularly in the development of electrochemical DNA biosensors and arrays. Different types of electrodes immobilized with specific probes can be used for the diagnosis of various diseases. Carbon nanotubes based electrochemical biosensors can be developed for high sensitivity. SPR, Quantum Dot and Piezoelectric biosensors are the emerging area of molecular diagnosis. The use of DNA biochip technologies eliminates the role of PCR. New method has been developed for preparing fiber-optic DNA biosensor and its array. Future biosensors will require the development of new reliable and more sensitive devices or the improvement of the existing ones to achieve the goal of superiority (in transduction, amplification, processing) so that more efficient diagnosis will be done. Also compact and portable devices will be required to develop. Some success has been achieved in the DNA biosensors and new ideas are being continuously developed and tested for new applications.

Acknowledgement

I take this opportunity with much pleasure to thank all the people who have helped me through the course of my journey towards producing this data. I sincerely thank my supervisor, Dr. Deepshikha Pande Katare (Assistant Director - Amity Institute of Pharmacy), for her guidance, help, motivation and support in writing this review. Apart from the subject of my review, I learnt a lot from her, which I am sure, will be useful in different stages of my life.

I would like to express my gratitude to the other members: Mr. Jayendra Kumar for much help and Dr. Kumud Bala for her review and many helpful comments.

­

My sincere gratitude also goes to all those who instructed and taught me through the years.

I whole-heartedly thank Amity Institute of Pharmacy for providing me this opportunity. I would like to thank all the Faulty members of AIP for their caring and supportive attitude.