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WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Neha et al. World Journal of Pharmacy and Pharmaceutical Sciences Volume 2, Issue 1, 407-428. Review Article ISSN 2278 4357

DNA BASED BIOSENSORS IN DISEASE DIAGNOSIS

*Neha Mathur, Anjali Mahendru, Navjayot Brar, Deepshikha Pande katare Amity Institute of Pharmacy, Amity University, Noida, Uttar Pradesh. India

ABSTRACT
Article Received on 01 January 2012, Revised on 18 January 2012, Accepted on 28 January 2012

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

*Correspondence for Author: * Neha Mathur Amity Institute of Pharmacy, Amity University, Noida, Uttar Pradesh. , India. neha07.mathur@gmail.com,

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, selective, simple and economical 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- genomic 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. KEYWORDS: DNA microarrays, DNA chips, fibre optics, optical DNA sensor, diagnostics.

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INTRODUCTION 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 made 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 combine a biological element with a physiochemical transducer for the electronic signal output which is 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 interaction between the analyte and the biological material, used in biosensors may be of two types: a) Bioaffinity sensors: depend on the selective and specific attachment of the target molecule to the surface-attached ligand partner (e.g. antibodies, nucleic acids). b) Biocatalytic sensors: an immobilized enzyme is used as a tool to recognize 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.

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Sample analyte
Immobilized enzyme, Nucleic acid, Immunoagent, Whole cell Electrochemical, Optical, Piezoelectric

BIORECEPTORS

TRANSDUCER

AMPLIFIER

Signal

PROCESSOR

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 The immobilization of DNA probe onto the transducer plays an important role in the performance of the DNA Biosensor. Probe orientation should be well-defined and so that www.wjpps.com 409

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readily accessible to the target. The mode of immobilization is the determining factor for the type of environment of probes that are immobilized at the solid surface. On the basis of nature of physical transducer, various schemes can be opted for the DNA probes attachment to the surface such as thiolated DNA utilization for self binding onto gold transducers, the formation of a complex by the use of biotylated DNA with a surface-confined strepavidin or avidin, covalent binding to the gold surface through functional alkanethiol-based monolayer and coupling covalently (carbodiimide) to the functional groups on carbon electrodes or adsorption onto carbon surfaces. Introduction of peptide nucleic acid (PNA) has paved way for many exciting and new opportunities to DNA biosensors. Peptide Nucleic Acid is a DNA mimic, the only difference is that the sugar-phosphate bone is replaced by a pseudo-peptide one. Like use of surfaceconfined PNA recognition layers provides remarkable sequence specificity on DNA biosensors and offers other advantages.

DNA dendrimers may also be utilized for imparting extreme sensitivity onto DNA Biosensors. By shape, these are tree-like superstructures which possess numerous single stranded arms that are able to 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 1.Optical Fiber optics Laser Interferometry 2.Electrochemical Potentiometric Conductometric Amperometric Biological Element DNA Transducer Advantages Limitations

Optical fiber Highly sensitive Expensive equipment and not portable turbidity interference Interference of buffered solution highly

DNA

Carbon paste Cheap, Fast electrodes

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3. Piezoelectric

DNA

Quartz Crystals

highly sensitive, Fast Quantitative

4. DNA chips

DNA

1. 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 which transduces the emission signal to a fluorescent label and that can carry light from one region to another through a series of internal inflections.

The methodology of fiber-optic DNA bio-sensors involves placing of a single stranded DNA probe at the ending-site of fiber and assessing the fluorescent changes resulting from the combination of a fluorescent indicator with the double stranded DNA hybrid [15 16]. The first DNA optical bio-sensors were developed by Krull and Co workers using fluorescent indicator ethidium bromide. A fiber-optic DNA sensor array was developed by Watts group for the detection of multiple DNA sequences at one time [17]. The hybridization of fluorescent labeled complementary oligonucleotides was assessed by observing the increase in fluorescence. A real label free optical detection of DNA hybridization can be offered by a different type of 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 depend on change in surface optical properties (change in resonance angle because of alteration in interfacial refractive index) which results from the surface binding reaction. Thus, these devices integrate the simplicity of SPR with the sensitivity and specificity 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].

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1.2 Molecular Beacons (MBs) MBs are oligonucleotides possessing a stem and loop structure that are labeled with a quencher at one end and a fluorophore on the other end of the stem that converts into fluorescent upon hybridization. MB probes possess high sensitivity and specificity and direct monitoring capability. A biotinylated molecular beacon probe was developed to prepare a DNA sensor using a bridge structure. It 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 alteration in confirmation of MB in the presence of complementary target DNA [21-23]. 1.3 Quantum Dot 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

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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 to diagnose DNA. DNA hybridization is detected electrochemically by monitoring the current response at fixed potential. Detection of hybridization is also commonly done through the increased current of a redox indicator or from other changes induced by hybridization in electrochemical parameters such as capacitance or conductivity [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)/Nhydroxysuccinimide (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 covalently bonding to the gold surface through functional alkanethiol based www.wjpps.com 413

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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. 2.1 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 accorded to new label free electrochemical detection schemes. There is a possibility in exploiting the changes in DNAs intrinsic electroactivity (Guanine oxidation peak of hybridization). To deal with the drawbacks of the probe sequences i.e. absence of G, guanines were substituted by inosine residues (pairing with C) and detection of hybridization was done through the target DNA guanine signal. The change in the guanine oxidation and intrinsic DNA redox signals detects the chemical and physical damage [33]. 2.2. 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 Toshiba)[35]. 3.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 [3637].
TM TM

produced by Motorola life sciences [34], Inc. and Genlyser

by

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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 (apolipoproteinE 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]. 4. Colorimetric or Strip type DNA sensor Using these sensors the direct detection of DNA hybridization is possible [40-42]. The dryreagent 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, with oligo (dT) attached to their surface. Biotinylated PCR products are hybridized with poly (dA) tailed oligo, switched to the top of strip and immersed in the appropriate buffer. With the migration of buffer in upward direction, the nanoparticles that are linked through target DNA through poly (dA/dT) hybridization are rehydrated. Immobilized streptavidin then capture the hybrid in the controlled zone of the strip. The test is 8-10 times more sensitive than ethidium bromide in agarose gel electrophoresis. The detection limit is abysmally low of 2 fmol of amplified DNA products. 5. 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 may be spotted, or 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. Developing the methods for detecting target hybridization, designing probe arrays, data analysis and reconstructing the target sequence are required for successful implementation of

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DNA chip technology. Such array technology thus forms the basis of integration of molecular biology, surface and analytical chemistry, advanced micro fabrication, robotics, software and automation. In this technique, RNA extracted from two samples are labeled with two different fluorochromes before being hybridized to a biochip containing large numbers of cDNAs arranged orderly onto a microscopic slide. Generally the green cyanine 3 and the red cyanine 5 (Cy3, Cy5) are used as fluorochrome. After hybridization, a scanner records excitation of these two fluorochromes at given wavelengths and the intensity of the fluorescence emission signals that is proportional to hybridization. The data is analyzed using specific software that enables gathering of genes with similar expression patterns i.e. those with common biological functions [33, 44].

Infected cell

mRNA

cDNA

Image analysis DNA microarray


Figure 2. To obtain gene-expression profile data from a microarray, RNA is first isolated from an infected cell and then reverse transcribed and labeled. This prepared RNA is hybridized to the chip which is scanned and image processed to obtain corresponding gene-expression profiles. A new ultrasensitive electronic sensor has been developed by Singapore scientists that would speed up effectively DNA testing for disease diagnosis and biological research. The novel

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electronic sensor array would be faster, accurate and cost-efficient. Excellent sensitivity has been shown by the Nanogap Sensor Array in detecting the trace amounts of DNA. By saving time and cutting 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 and meticulously designed sensor chip has the ability to detect DNA efficiently. The novel vertical nanostructure design and two different surfaces of the sensor allow ultrasensitive detection of DNA [45]. Labona-chip (LOC) Lab Chip is a device which involves preparation of sample and detection of DNA array. The objective of this technology is to integrate multiple processes, including collection of sample and pretreatment of it with the DNA extraction, hybridization and detection, on single selfcontained microchip i.e. on a microfluidic platform. The capability to do all the processes on a single chip merits excellent advantages in terms of cost, speed, efficacy, effectiveness, contamination, sample consumption and automation. Laboratory transportation to the source of sample will be enabled by such miniaturization of analytical instrumentation. The development of these credit-card sized microlaboratories is commonly based on latest micromachining and microfabrication technologies, utilizing processes well known in the manufacture of electronic circuitry [14]. Cell-omic sensors It is a combination of cell based detection systems 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.

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1. Viral diseases By DNA microarrays Either viral detection were being carried by immunological techniques (i.e. enzyme-linked immunosorbent assays (ELISAs) for detecting circulating virus-specific antibodies) or PCR based techniques (i.e. RT PCR for detecting 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 capability to detect multiple viruses simultaneously [48]. Therefore, latest advances in DNA and protein based microarray methodology fulfill the need of a rapid and sensitive detection of viral infections (also identify multiple viruses in parallel). For analyzing viruses, DNA microarrays may be divided into host chips and viral chips. Each not only detects and identifies but also monitor the viral populations. In 1999, the first viral DNA microarray for the detecting viral (human cytomegalovirus, HCMV) gene expression profile was described. Viral replication or de novo protein synthesis was blocked by treating the infected cells with ganciclovir or cycloheximide and then the expression profiles of viral genes was generated using microarray. Using this approach, the HCMV genes were classified to immediate, early or late expression classes, on the basis of their expression profile in response to the various drug treatments. This can be used as a hybridization signature for the molecular staging of a disease [49]. Orthopoxvirus that causes smallpox has two subtypes that are variola major and variola minor, differing in their pathogenicity. This problem of orthopoxvirus subtype discrimination was solved by laassri et al. He said that this problem could be solved by developing an array that has an ability of correctly detecting the four of the orthopoxvirus species [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]. Vant wout examined HIV infection in T-helper cells to detect changes in host gene expression profile which were specific to HIV infection [53]. Proinflammatory genes and genes involved in endoplasmic reticulum (ER) stress pathways, cell cycle, and apoptosis were the host gene signatures identified.

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a) 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 menace for worldwide resulting 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 chemiluminescences 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. With the hybridization reaction, the mass is increasing that resulted 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 that is a glassy carbon electrode (GCE) modified with label free21mer single-stranded (ss) oligonucleotides that is related to HBV sequence and covalently immobilized onto the electrode. [Cu(dmp)(H2O)Cl2] (dmp = 2,9-dimethyl-1,10phenanthroline) is used as an electrochemical indicator. The method is simple, economical and allows the accumulation of copper complex within the DNA layer. Cyclic voltammetry and differential pulse voltammetry were used for electrochemical detection. The detection of www.wjpps.com

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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]. b) Detection of hepatitis C 3a virus An electrochemical DNA biosensor that was a gold electrode modified with a monolayer of a peptide nucleic acid probe and 6-mercapto-1-hexanol was used. A 14-mer PNA probe (related to the HCV genotype 3a (pHCV3a) core/E1 region) was immobilized onto the electrode. This self-assembled PNA could selectively hybridize with a complementary sequence in solution to give dsPNA-DNA on the surface, and this increased the peak current of methylene blue (MB) which was used for detecting target DNA sequence. Diagnostic performance of the biosensor was described and the detection limit was found to be 5.7 1011 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]. 2. Cystic fibrosis Mikkelsens team pioneered the utilization of redox indicators, demonstrated utility of electrochemical DNA biosensor for detecting the cystic fibrosis F508 deletion sequence which is associated with 70% of cystic fibrosis patients. For the 4000-base DNA fragment, 1.8 fmol was the detection limit in relation to a Co(bpy)33+ indicator. High selectivity for the disease

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sequence (not for normal DNA) was accomplished by doing the hybridization at high (43C) 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 commonly used to measure the blood glucose level of diabetes patients. Using the latest DNA chip technology, many scientists at Diabetes Center have discovered the implication of new gene in the cause of type 2 diabetes. They created an abnormality in one of these genes known as ARNT (aryl hydrocarbon receptor nuclear translocator gene which is a member of a family of transcription factors) in mice and the mice developed changes in insulin secretion which were same as in patients with type 2 diabetes. The ARNT is required for the development of normal embryo. It is also related to responses to hypoxic stress condition and certain environmental toxins, such as dioxin and thus for integrating genetic and environmental insults it is present at specific potential sites. The expression of many other genes in the cell is regulated by transcription factors like ARNT and thus they are the master regulators of cellular functions. The first use of DNA chips has been represented by this study, for studying an important role for ARNT and altered expression and activity of gene in impaired beta-cell function in human type2 diabetes. Type 2 diabetes is the very common metabolic disorder in human beings, affecting approximately 200 million people worldwide. The pathogenesis of type 2 diabetes includes two defects: impaired beta cells function in the pancreas and insulin resistance. Both of these two defects must be related to genetic programming and thus some defects in genes required for insulin-producing beta-cell function have been found. Even the genetic defects are not reported in the majority of type 2 diabetic patients.

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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 detecting single gene mutation associated with sickle cell anemia. 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 -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 exchange between a donor and an acceptor. The structure was built up by using photon upconverting nanoparticles, NaYF4 doped with Yb3+ and Er3+ as the donor and a conventional fluorophore, N,N,N',N'tetramethyl-6-carboxyrhodamine (TAMRA), as the acceptor. The sensor was able to find the perfectly matched target, among the mismatched targets or other oligonucleotides having random sequences. Detection limit of this sensor for perfectly matched target found to be 120 femtomoles, with no evidence of photobleaching. Oligonucleotide sensors of designed in a way to show high sensitivity and specificity.

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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 fiberoptic DNA biosensor. 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. REFERENCES 1. Firstenberg R.E. and Schaertel B.J. (1988). Biosensors in the food industry: Present and Future. Journal of food protection, 51, 811-820. www.wjpps.com 423

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