b.tech. Biotechnology Notes (5)

8/6/2019 b.tech. Biotechnology Notes (5)

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BIOELECTRONICSASSIGNMENT

ONDNA

BIOSENSORS

SUBMITTED BY:-MUDIT MISRAB.TECH(B.T.)

VII SEMESTERSECTION A

ROLL No.-33


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LK23041035

INTRODUCTION

The development of biosensors is a major thrust of the rapidly growingbiotechnology industry, which encompasses a very diverse range of research efforts including genomics, proteomics, computationalbiology, and pharmaceuticals, among other activities. Advances inthese areas are giving scientists new methods for unraveling thecomplex biochemical processes occurring inside cells, with the largergoal of understanding and treating human diseases. At the same time,the semiconductor industry has been steadily perfecting the science of microminiaturization. The merging of these two fields in recent yearshas enabled biotechnologists to begin packing their traditionally bulkysensing tools into smaller and smaller spaces, onto so-called biochips

or biosensors. These chips or sensors are essentially miniaturizedlaboratories that can perform hundreds or thousands of simultaneousbiochemical reactions. Biosensors enable researchers to quickly screenlarge numbers of biological analytes for a variety of purposes, fromdisease diagnosis to detection of bioterrorism agents.

These are actually small devices which utilize biological reactions fordetecting target analytes.Such devices intimately couple a biologicalrecognition element (interacting with the target analyte) with aphysical transducer that translates the biorecognition event into auseful electrical signal.Common transducing elements, includingoptical, electrochemical or mass-sensitive devices, generate light,

current or frequency signals, respectively. There are two types of biosensors, depending on the nature of the recognition event.Bioaffinity devices rely on the selective binding of the target analyteto a surface-confined ligand partner (e.g. antibody, oligonucleotide). Incontrazst, in biocatalytic devices-, an immobilized enzyme is used forrecognizing the target substrate. For example, sensor strips withimmobilized glucose oxidase have been widely used for personalmonitoring of diabetes.

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THE INTIMATE COUPLING OF BIORECOGNITION AND SIGNAL TRANSDUCTION

HISTORY

In 1953, Watson and Crick announced their discovery of the nowfamiliar double helix structure of DNA molecules and set the stage forgenetics research that continues to the present day (Nelson, 2000). Thedevelopment of sequencing techniques in 1977 by Gilbert (Maxam,1977) and Sanger (Sanger, 1977) (working separately) enabledresearchers to directly read the genetic codes that provide instructionsfor protein synthesis. This research showed how hybridization of complementary single oligonucleotide strands could be used as a basisfor DNA sensing. Two additional developments enabled the technologyused in modern DNA-based biosensors. First, in 1983 Kary Mullisinvented the polymerase chain reaction (PCR) technique (Nelson,2000), a method for amplifying DNA concentrations. This discoverymade possible the detection of extremely small quantities of DNA insamples. Second, in 1986 Hood and coworkers devised a method tolabel DNA molecules with fluorescent tags instead of radiolabels (Smith,1986), thus enabling hybridization experiments to be observedoptically.

The rapid technological advances of the biochemistry andsemiconductor fields in the 1980s led to the large scale development of biochips in the 1990s. At this time, it became clear that biochips werelargely a "platform" technology which consisted of several separate, yetintegrated components. Figure 1 shows the makeup of a typical biochipplatform. The actual sensing component (or "chip") is just one piece of a complete analysis system. Transduction must be done to translatethe actual sensing event (DNA binding, oxidation/reduction, etc. ) into aformat understandable by a computer (voltage, light intensity, mass,etc. ), which then enables additional analysis and processing to producea final, human-readable output. The multiple technologies needed tomake a successful biochip -- from sensing chemistry, to microarraying,to signal processing -- require a true multidisciplinary approach, makingthe barrier to entry steep. One of the first commercial biochips wasintroduced by Affymetrix. Their "GeneChip" products contain thousandsof individual DNA sensors for use in sensing defects, or singlenucleotide polymorphisms (SNPs), in genes such as p53 (a tumorsuppressor) and BRCA1 and BRCA2 (related to breast cancer) (Cheng,2001). The chips are produced using microlithography techniquestraditionally used to fabricate integrated circuits.

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Today, a large variety of biochip technologies are either in developmentor being commercialized. Numerous advancements continue to bemade in sensing research that enable new platforms to be developedfor new applications. Cancer diagnosis through DNA typing is just onemarket opportunity. A variety of industries currently desire the ability to

simultaneously screen for a wide range of chemical and biologicalagents, with purposes ranging from testing public water systems fordisease agents to screening airline cargo for explosives. Pharmaceuticalcompanies wish to combinatorially screen drug candidates againsttarget enzymes. To achieve these ends, DNA, RNA, proteins, and evenliving cells are being employed as sensing mediators on biochips.Numerous transduction methods can be employed including

BIOCHIPS ARE A PLATFORM THAT REQUIRE, IN ADDITION TO MICROARRAY TECHNOLOGY,TRANSDUCTION AND SIGNAL PROCESSING TECHNOLOGIES TO OUTPUT THE RESULTS OF

SENSING EXPERIMENTS.

surface plasmon resonance, fluorescence, and chemiluminescence. Theparticular sensing and transduction techniques chosen depend onfactors such as price, sensitivity, and reusability.

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http://en.wikipedia.org/wiki/Image:Biochip_platform.jpg


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DNA BIOSENSORS

These are based on nucleic acid recognition processes, are rapidly

being developed towards the goal of rapid, simple and inexpensivetesting of genetic and infectious diseases and for the detection of DNAdamage and interactions. Unlike enzyme or antibodies, nucleic acidrecognition layers can be readily synthesized and regenerated formultiple use.

SEQUENCE-SPECIFIC HYBRIDIZATION BIOSENSORS

Hybridization biosensors rely on the immobilization of a single-stranded(ss) DNA probe onto the transducer surface. The duplexformation can be detected following the association of an appropriatehybridization indicator or through other changes accrued from thebinding event.

SURFACE CHEMISTRY AND BIOCHEMISTRY

The immobilization of the nucleic acid probe onto the transducersurface plays an important role in the overall performance of DNAbiosensors and gene chips. The immobilization step should lead to awell-defined probe orientation, readily accessible to the target. Theenvironment of the immobilized probes at the solid surface dependsupon the mode of immobilization and can differ from that experiencedin the bulk solution. Depending upon the nature of the physicaltransducer, various schemes can be used fir attaching the DNA probeto the surface. These include the use of thiolated DNA for self assembly onto gold transducers (gold electrodes or gold-coatedpiezoelectric crystals), covalent linkage to the gold surface viafunctional alkanethiol-based monolayers, the use of biotylated DNA forcomplex formation with a surface-confined avidin or strepavidin,covalent (carbodamide) sequence associated with 70% of cystic fibrosis

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patients . A detection limit of 1.8 3+ fmol was demonstrated for the4000-base DNA fragment in connection to a Co(bpy) 3 3High selectivity towards the disease sequence (but not to the normal

DNA) was achieved by performing the hybridization at an elevated(43C) temperature. Such use of the electrochemical transduction

mode requires that proper attention be given to the choice of theindicator and its detection scheme. New redox indicators, offeringgreater discrimination between ss and dsDNA are being developed forattaining higher sensitivity. The use of a threading intercalatorferrocenyl naphthalene diimide (20) that binds to the DNA hybrid moretightly than usual intercalators and displays small affinity to the single-stranded probe has been very successful.

The use of enzyme labels also offers great promise for electrochemicaldetection of DNA hybridization. Heller's group demonstrated that adirect amperometrie monitoring of the hybridization event can beachieved in connection to the use of horseradishperoxidaselabeled target. In this system, the hybridization event resulted in the`wiring' of the enzyme to the transducer (via an electron-conducting redox hydrogel), hence leading in a continuous hydrogen-peroxide electroreduction current. Willner's group illustrated thatmultiple amplifications can be achieved by coupling of a peroxidase

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enzyme label with the surface accumulation of the phenol reactionproduct.Increased attention has been given recently to new label-treeelectrochemical detection schemes which offer faster andsimpierassays. For example. it is possible to exploit changes in the

intrinsic electroactivity of DNA (e.g. the guanine oxidation peak)accrued from the hybridization event. To overcome the limitations of the probe sequences (absence of G), guanines in the probe sequencewere substituted by inosine residues (pairing with Cs) and thehybridization was detected through the target DNA guanine signal 23).A greatly amplified 2 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 accomplishedby monitoring changes in the conductivity of conducting polymermolecular interfaces, e.g. DNA-modified polypyrrole films C25?6).Eventually, it would be possible to eliminate these polymeric interfaces

and to exploit different rates of electron-transfer through ss and dsDNAfor probing hybridization (including mutation detection via theperturbation in charge transfer through DNA). Recent activity in thisdirection is encouraging .

The electrochemical response of the G nucleobase is also verysensitive to the DNA structure and can thus be used for probing DNAdamage or interactions. Changes in the guanine oxidation, and of otherintrinsic DNA redox signals, have thus been used for detectingchemical and physical damage .Coupling to functional groups on carbon electrodes, or a simpleadsorption onto carbon surfaces. As in solution-based hybridization

assays, conditions for interfacial hybridization events (e.g. ionicstrength, temperature, presence of accelerators) have to be optimized.Chemical and thermally-induced dehybridisation of the resultingduplex is often used for regenerating the interface.Recent advances in nucleic acid recognition can enhance the power of DNA biosensors. For example, the introduction of peptide nucleic acid(PNA) has opened up exciting opportunities for DNA biosensors. PNA isa DNA mimic in which the sugarphosphate backbone is replaced witha pseudopeptide one. The unique structural, hybridization andrecognition features of solution-phase PNA can be readily extrapolatedonto transducer surfaces in connection with the design of highly-

selective DNA biosensors. Such use of surface-confined PNArecognition layers imparts remarkable sequence specificity onto DNAbiosensors (including detection of single-base mismatches) and offersother attractive advantages (including greater latitude in the selectionof experimental conditions).DNA dendrimers can be used for imparting higher sensitivity onto DNAbiosensors. These tree-like superstructures possess numerous single-stranded arms that can hybridize to their complementary DNA

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sequence. A greatly increased hybridization capacity and hence asubstantially amplified response is achieved by immobilizing thesedendritic nucleic acids onto the physical transducer .

OPTICAL BIOSENSORS

Optical detection of DNA hybridization has attracted considerableattention. DNA. optical hiosensors commonly rely on a fiber optic totransduce the emission signal of a fluorescent label. Fiber optics aredevices that carry light from one place to another by a series of internal inflections. The operation of fiber-optic DNA biosensorstypically involves placement of a ssDNA probe at the end of the fiberand monitoring the fluorescent changes resulting from the associationof :I fluorescent compound (indicator) with the double-stranded (ds) DNAhybrid. The first DNA optical hiosensor, developed by Kroll andcoworkers, relied on the use of an ethidium bromide indicator.

Extremely low (femtomolar) detection limits have been achieved inconnection with other fluorescent indicators (e.g. PicoGreen). Walt'sgroup developed a fiber-optic DNA sensor array for the simultaneousdetection of multiple DNA sequences. Hybridization of fluorescently-labeled complementary olgonucleotides was monitored by observingthe increase in fluorescence that accompanied binding. A differentoptical transduction, based on evanescent wave devices, can offerreal-time Libel-free optical detection of DNA hybridization. Thesebiosensors rely on monitoring changes in surface optical properties(shift in resonance angle due to change in the interfacial refractiveindex) resulting from the surface binding reaction. Such devices thuscombine the simplicity of surface plasmon resonance with thesensitivity of wave guiding devices. The coupling of chemiluminescence with sandwich hybridization, magnetic beadcapture and flow injection operation has been used for the rapiddetection of hepatitis B virus DNA.In situ, label-free, optical detection can be achieved throughchanges inother optical properties. For example, a novel nanoparticle-basedcolorimetric detection offers great promise for direct detection of DNAhybridization . In this case, a distance change, accrued from thehybridization event, results in changes of the optical properties of theaggregated functional gold nanoparticles. Another new innovativeapproach for the direct fluorescent detection of DNA hybridizationrelies on the use of molecular beacons (MBs) . MBs areoligonucleotides with a stem-and-loop structure. labeled with afluorophore and a quencher on the two ends of the stem, that becomefluorescent upon hybridization. In addition to their direct monitoringcapability, MB probes offer high sensitivity and specificity.

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Target

THE OPERATION OF MB BASED OPTICAL DNA BIOSENSORS. THE HYBRIDIZATION EVENTINDUCES CONFORMATIONAL REORGANIZATION THAT SEPARATES THE QUENCHER FROM

THE FLUOROPHORE. [REPRINTED WITH PERMISSION FROM (16). COPYRIGHT 1999AMERICAN CHEMICAL SOCIETY.]

ELECTROCHEMICAL BIOSENSORS

Electrochemical devices have also proven very useful for sequence-specific biosensing of DNA. The inherent miniaturization of suchdevices and their compatibility with advanced microfabricationtechnology make them excellent candidates for DNA diagnostics.Electrochemical detection of DNA hybridization usually involvesmonitoring a current response under controlled potential conditions (ina manner analogous to most hand-held meters used by sufferers of diabetes for measuring their blood glucose level). The hybridizationevent is commonly detected via the increased current signal of a redoxindicator (that recognizes the DNA duplex) or from other hybridization-induced changes in electrochemical parameters (e.g. conductivity orcapacitance). Mikkelsen's team, that pioneered the use of redoxindicators, demonstrated its utility for detecting the cystic fibrosis2~1508 deletion sequence associated with 70% of cystic fibrosispatients. A detection limit of 1.8 3+ fmol was demonstrated for the4000-base DNA fragment in connection to a Co(bPY)3 indicator. Highselectivity towards the disease sequence (but not to the normal DNA)was achieved by performing the hybridization at an elevated (43C)temperature. Such use of the electrochemical transduction mode

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requires that proper attention be given to the choice of the indicatorand its detection scheme. New redox indicators, offering greaterdiscrimination between ss and dsDNA are being developed forattaining higher sensitivity. The use of a threading intercalatorferrocenyl naphthalene diimide that binds to the DNA hybrid more

tightly than usual intercalators and displays small affinity to the single-stranded probe has been very successful. The use of enzyme labels also offers great promise for electrochemicaldetection of DNA hybridization. Heller's group demonstrated that adirect amperometric monitoring of the hybridization event can beachieved in connection to the use of horseradishperoxidase labeledtarget. In this system, the hybridization event resulted in the 'wiring of the enzyme to the transducer(via an electron-conducting redoxhydrogel), hence leading in a continuous hydrogen-peroxideelectroreduction current. Willner's group illustrated that multipleamplifications can be achieved by coupling of a peroxidase enzyme

label with the surface accumulation of the phenol reaction product.Increased attention has been given recently to new label-freeelectrochernical detection schemes which offer faster and simplerassays. For example. it is possible to exploit changes in the intrinsicelectroactivity of DNA (e.g. the guanine oxidation peak) accrued fromthe hybridization event. To overcome the limitations of the probesequences (absence of G), guanines in the probe sequence weresubstituted by inosine residues (pairing with Cs) and the hybridizationwas detected through the target DNA guanine signal Cr1). A greatly amplified guanine signal, and hence hybridizationresponse, was obtained by using the Ru(bPY)3+2 redox mediator C24.

Direct, label-free, electrical detection of DNA hybridizationhas

also beenaccomplished by monitoring changes in

LIGATE AND LIGHT. SCHEMATICS DIAGRAM OF REAL-TIME MONITORING OF THE NUCLEIC ACID LIGATION PROCESS BY A MOLECULAR BEACON.

the conductivity of conducting polymer molecular interfaces. e.g. DNA-modified polypyrrole films (UL6). Eventually, it would be possible to

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eliminate these polymeric interfaces and to exploit different rates of electron-transferthrough ss and dsDNA for probing hybridization(including mutation detection via the perturbation in charge transferthrough DNA). Recent activity in this direction is encouraging.

The electrochemical response of the G nucleobase is also very

sensitive to the DNA structure and can thus be used for probing DNAdamage or interactions. Changes in the guanine oxidation, and of otherintrinsic DNA redox signals, have thus been used for detectingchemical and physical damage.

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TYPES OF DNA BIOSENSORS

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APPLICATIONS OF DNA BIOSENSORS

As we enter into the 21st century where advances in medicaltechnologies lead to thediscovery of more genetic diseases, DNA sensing has becomeincreasingly importantfor rapid genetic screening and detection. Coupled with wide-scalegenetic andenvironmental testing, the threat of biological warfare and forensicapplications, thedevelopment of a DNA hybridization biosensor is of intense interest tomanyresearchers in past decade. The DNA biosensor involves the integrationof DNAmolecules (the probe DNA) with the electrical elements to createelectronic readoutsthat transduce the Watson-Crick base pairing (hybridization) eventbetween the probeDNA and th e interested g enomic s equence. T wo c rucial s teps a re involved in t h edevelopment of DNA biosensor: i) the controlled immobilization of probe DNA ontothe transducer and ii) the DNA transduction which includes optical,microgravimetricand electrochemical methods. Immobilization of probe DNA throughself-assembly of thiolated DNA onto the gold surface is ideal for the controlledimmobilization of probeDNA due to the simplicity of this approach. Electrochemical method ischosen for theDNA transduction as it offers a simple, rapid, low cost point-of-caredetection forselected genomic sequence and is suitable for fabrication of miniaturized devices.AC-Impedance spectroscopy and electrochemical methodologies areused employed tocharacterize the resultant DNA surfaces prior to and after hybridizationon goldsurfaces. Subsequently, electrochemical detection of DNA hybridizationis achievedthrough a novel in situ electrochemical approach which utilizes theremarkable abilityof DNA duplex to transport electron of a DNA intercalated redox-activemolecule over

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long distance to and from the gold surfaces. This electrochemicaldetection scheme hasproven to be successful as it is i) highly sensitive with good detectionlimits, ii) highlyselective with the ability to distinguish single-base mismatches

(including the mostthermodynamic stable G-A mismatch) and iii) has a commercial viableassay time.Further to that, we will report the use of the same in situelectrochemical approach toassay the interaction of the anticancer drug with the immobilized DNAmolecules.

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b.tech. Biotechnology Notes (5)