BIOELECTRONICS ASSIGNMENT ON DNA BIOSENSORS

SUBMITTED BY:MUDIT MISRA B.TECH(B.T.) VII SEMESTER SECTION A ROLL No.-33

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INTRODUCTION
The development of biosensors is a major thrust of the rapidly growing biotechnology industry, which encompasses a very diverse range of research efforts including genomics, proteomics, computational biology, and pharmaceuticals, among other activities. Advances in these areas are giving scientists new methods for unraveling the complex biochemical processes occurring inside cells, with the larger goal 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 years has enabled biotechnologists to begin packing their traditionally bulky sensing tools into smaller and smaller spaces, onto so-called biochips or biosensors. These chips or sensors are essentially miniaturized laboratories that can perform hundreds or thousands of simultaneous biochemical reactions. Biosensors enable researchers to quickly screen large numbers of biological analytes for a variety of purposes, from disease diagnosis to detection of bioterrorism agents. These are actually small devices which utilize biological reactions for detecting target analytes.Such devices intimately couple a biological recognition element (interacting with the target analyte) with a physical transducer that translates the biorecognition event into a useful electrical signal.Common transducing elements, including optical, 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 analyte to a surface-confined ligand partner (e.g. antibody, oligonucleotide). In contrazst, in biocatalytic devices-, an immobilized enzyme is used for recognizing the target substrate. For example, sensor strips with immobilized glucose oxidase have been widely used for personal monitoring of diabetes.

THE INTIMATE COUPLING OF BIORECOGNITION AND SIGNAL TRANSDUCTION

HISTORY
In 1953, Watson and Crick announced their discovery of the now familiar double helix structure of DNA molecules and set the stage for genetics research that continues to the present day (Nelson, 2000). The development of sequencing techniques in 1977 by Gilbert (Maxam, 1977) and Sanger (Sanger, 1977) (working separately) enabled researchers to directly read the genetic codes that provide instructions for protein synthesis. This research showed how hybridization of complementary single oligonucleotide strands could be used as a basis for DNA sensing. Two additional developments enabled the technology used in modern DNA-based biosensors. First, in 1983 Kary Mullis invented the polymerase chain reaction (PCR) technique (Nelson, 2000), a method for amplifying DNA concentrations. This discovery made possible the detection of extremely small quantities of DNA in samples. Second, in 1986 Hood and coworkers devised a method to label DNA molecules with fluorescent tags instead of radiolabels (Smith, 1986), thus enabling hybridization experiments to be observed optically. The rapid technological advances of the biochemistry and semiconductor fields in the 1980s led to the large scale development of biochips in the 1990s. At this time, it became clear that biochips were largely a "platform" technology which consisted of several separate, yet integrated components. Figure 1 shows the makeup of a typical biochip platform. The actual sensing component (or "chip") is just one piece of a complete analysis system. Transduction must be done to translate the actual sensing event (DNA binding, oxidation/reduction, etc.) into a format understandable by a computer (voltage, light intensity, mass, etc.), which then enables additional analysis and processing to produce a final, human-readable output. The multiple technologies needed to make a successful biochip -- from sensing chemistry, to microarraying, to signal processing -- require a true multidisciplinary approach, making the barrier to entry steep. One of the first commercial biochips was introduced by Affymetrix. Their "GeneChip" products contain thousands of individual DNA sensors for use in sensing defects, or single nucleotide polymorphisms (SNPs), in genes such as p53 (a tumor suppressor) and BRCA1 and BRCA2 (related to breast cancer) (Cheng, 2001). The chips are produced using microlithography techniques traditionally used to fabricate integrated circuits.

Today, a large variety of biochip technologies are either in development or being commercialized. Numerous advancements continue to be made in sensing research that enable new platforms to be developed for new applications. Cancer diagnosis through DNA typing is just one market opportunity. A variety of industries currently desire the ability to simultaneously screen for a wide range of chemical and biological agents, with purposes ranging from testing public water systems for disease agents to screening airline cargo for explosives. Pharmaceutical companies wish to combinatorially screen drug candidates against target enzymes. To achieve these ends, DNA, RNA, proteins, and even living 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. The particular sensing and transduction techniques chosen depend on factors such as price, sensitivity, and reusability.

DNA BIOSENSORS
These are based on nucleic acid recognition processes, are rapidly being developed towards the goal of rapid, simple and inexpensive testing of genetic and infectious diseases and for the detection of DNA damage and interactions. Unlike enzyme or antibodies, nucleic acid recognition layers can be readily synthesized and regenerated for multiple use.

SEQUENCE-SPECIFIC HYBRIDIZATION BIOSENSORS

Hybridization biosensors rely on the immobilization of a singlestranded(ss) DNA probe onto the transducer surface. The duplex formation can be detected following the association of an appropriate hybridization indicator or through other changes accrued from the binding event.

SURFACE CHEMISTRY AND BIOCHEMISTRY

The immobilization of the nucleic acid probe onto the transducer surface plays an important role in the overall performance of DNA biosensors and gene chips. The immobilization step should lead to a well-defined probe orientation, readily accessible to the target. The environment of the immobilized probes at the solid surface depends upon the mode of immobilization and can differ from that experienced in the bulk solution. Depending upon the nature of the physical transducer, various schemes can be used fir 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 monolayers, the use of biotylated DNA for complex formation with a surface-confined avidin or strepavidin, covalent (carbodamide) sequence associated with 70% of cystic fibrosis 5

patients . A detection limit of 1.8 3+ fmol was demonstrated for the 4000-base DNA fragment in connection to a Co(bpy)33 High 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 the indicator and its detection scheme. New redox indicators, offering greater discrimination between ss and dsDNA are being developed for attaining higher sensitivity. The use of a threading intercalator ferrocenyl naphthalene diimide (20) that binds to the DNA hybrid more tightly than usual intercalators and displays small affinity to the singlestranded probe has been very successful.

The use of enzyme labels also offers great promise for electrochemical detection of DNA hybridization. Heller's group demonstrated that a direct amperometrie monitoring of the hybridization event can be achieved in connection to the use of horseradishperoxidase labeled target. In this system, the hybridization event resulted in the `wiring' of the enzyme to the transducer (via an electronconducting redox hydrogel), hence leading in a continuous hydrogenperoxide electroreduction current. Willner's group illustrated that multiple amplifications 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-tree electrochemical detection schemes which offer faster and simpierassays. 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 sequence were substituted by inosine residues (pairing with Cs) and the hybridization 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 accomplished by monitoring changes in the conductivity of conducting polymer molecular 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 dsDNA for probing hybridization (including mutation detection via the perturbation in charge transfer through 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 DNA damage or interactions. Changes in the guanine oxidation, and of other intrinsic DNA redox signals, have thus been used for detecting chemical and physical damage . Coupling to functional groups on carbon electrodes, or a simple adsorption onto carbon surfaces. As in solution-based hybridization assays, conditions for interfacial hybridization events (e.g. ionic strength, temperature, presence of accelerators) have to be optimized. Chemical and thermally-induced dehybridisation of the resulting duplex 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 is a DNA mimic in which the sugarphosphate backbone is replaced with a pseudopeptide one. The unique structural, hybridization and recognition features of solution-phase PNA can be readily extrapolated onto transducer surfaces in connection with the design of highlyselective DNA biosensors. Such use of surface-confined PNA recognition layers imparts remarkable sequence specificity onto DNA biosensors (including detection of single-base mismatches) and offers other attractive advantages (including greater latitude in the selection of experimental conditions). DNA dendrimers can be used for imparting higher sensitivity onto DNA biosensors. These tree-like superstructures possess numerous singlestranded arms that can hybridize to their complementary DNA 7

sequence. A greatly increased hybridization capacity and hence a substantially amplified response is achieved by immobilizing these dendritic nucleic acids onto the physical transducer .

OPTICAL BIOSENSORS
Optical detection of DNA hybridization has attracted considerable attention. DNA. optical hiosensors commonly rely on a fiber optic to transduce the emission signal of a fluorescent label. Fiber optics are devices that carry light from one place to another by a series of internal inflections. The operation of fiber-optic DNA biosensors typically involves placement of a ssDNA probe at the end of the fiber and monitoring the fluorescent changes resulting from the association of :I fluorescent compound (indicator) with the double-stranded (ds) DNA hybrid. The first DNA optical hiosensor, developed by Kroll and coworkers, relied on the use of an ethidium bromide indicator. Extremely low (femtomolar) detection limits have been achieved in connection with other fluorescent indicators (e.g. PicoGreen). Walt's group developed a fiber-optic DNA sensor array for the simultaneous detection of multiple DNA sequences. Hybridization of fluorescentlylabeled complementary olgonucleotides was monitored by observing the increase in fluorescence that accompanied binding. A different optical transduction, based on evanescent wave devices, can offer real-time Libel-free optical detection of DNA hybridization. These biosensors rely on monitoring changes in surface optical properties (shift in resonance angle due to change in the interfacial refractive index) resulting from the surface binding reaction. Such devices thus combine the simplicity of surface plasmon resonance with the sensitivity of wave guiding devices. The coupling of chemiluminescence with sandwich hybridization, magnetic bead capture and flow injection operation has been used for the rapid detection of hepatitis B virus DNA. In situ, label-free, optical detection can be achieved throughchanges in other optical properties. For example, a novel nanoparticle-based colorimetric detection offers great promise for direct detection of DNA hybridization . In this case, a distance change, accrued from the hybridization event, results in changes of the optical properties of the aggregated functional gold nanoparticles. Another new innovative approach for the direct fluorescent detection of DNA hybridization relies on the use of molecular beacons (MBs) . MBs are oligonucleotides with a stem-and-loop structure. labeled with a fluorophore and a quencher on the two ends of the stem, that become fluorescent upon hybridization. In addition to their direct monitoring capability, MB probes offer high sensitivity and specificity.

Target

THE OPERATION OF MB BASED OPTICAL DNA BIOSENSORS. THE HYBRIDIZATION EVENT INDUCES CONFORMATIONAL REORGANIZATION THAT SEPARATES THE QUENCHER FROM THE FLUOROPHORE. [REPRINTED WITH PERMISSION FROM (16). COPYRIGHT 1999 AMERICAN CHEMICAL SOCIETY.]

ELECTROCHEMICAL BIOSENSORS
Electrochemical devices have also proven very useful for sequencespecific biosensing of DNA. The inherent miniaturization of such devices and their compatibility with advanced microfabrication technology make them excellent candidates for DNA diagnostics. Electrochemical detection of DNA hybridization usually involves monitoring a current response under controlled potential conditions (in a manner analogous to most hand-held meters used by sufferers of diabetes for measuring their blood glucose level). The hybridization event is commonly detected via the increased current signal of a redox indicator (that recognizes the DNA duplex) or from other hybridizationinduced changes in electrochemical parameters (e.g. conductivity or capacitance). Mikkelsen's team, that pioneered the use of redox indicators, demonstrated its utility for detecting the cystic fibrosis 2~1508 deletion sequence associated with 70% of cystic fibrosis patients. A detection limit of 1.8 3+ fmol was demonstrated for the 4000-base DNA fragment in connection to a Co(bPY)3 indicator. High 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 the indicator and its detection scheme. New redox indicators, offering greater discrimination between ss and dsDNA are being developed for attaining higher sensitivity. The use of a threading intercalator ferrocenyl naphthalene diimide that binds to the DNA hybrid more tightly than usual intercalators and displays small affinity to the singlestranded probe has been very successful. The use of enzyme labels also offers great promise for electrochemical detection of DNA hybridization. Heller's group demonstrated that a direct amperometric monitoring of the hybridization event can be achieved in connection to the use of horseradishperoxidase labeled 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 that multiple amplifications 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 labelfreeelectrochernical detection schemes which offer faster and simpler assays. 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 sequence were substituted by inosine residues (pairing with Cs) and the hybridization was detected through the target DNA guanine signal Cr 1). A greatly amplified guanine signal, and hence hybridization response, was obtained by using the Ru(bPY)3+2 redox mediator C24. Direct, label-free, electrical detection of DNA hybridization has also been accomplished 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. DNAmodified polypyrrole films (UL6). Eventually, it would be possible to 10

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 transfer through 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 DNA damage or interactions. Changes in the guanine oxidation, and of other intrinsic DNA redox signals, have thus been used for detecting chemical 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 medical technologies lead to the discovery of more genetic diseases, DNA sensing has become increasingly important for rapid genetic screening and detection. Coupled with wide-scale genetic and environmental testing, the threat of biological warfare and forensic applications, the development of a DNA hybridization biosensor is of intense interest to many researchers in past decade. The DNA biosensor involves the integration of DNA molecules (the probe DNA) with the electrical elements to create electronic readouts that transduce the Watson-Crick base pairing (hybridization) event between the probe DNA and th e interested g enomic s equence. T wo c rucial s teps a re i nvolved in t h e development of DNA biosensor: i) the controlled immobilization of probe DNA onto the transducer and ii) the DNA transduction which includes optical, microgravimetric and electrochemical methods. Immobilization of probe DNA through self-assembly of thiolated DNA onto the gold surface is ideal for the controlled immobilization of probe DNA due to the simplicity of this approach. Electrochemical method is chosen for the DNA transduction as it offers a simple, rapid, low cost point-of-care detection for selected genomic sequence and is suitable for fabrication of miniaturized devices. AC-Impedance spectroscopy and electrochemical methodologies are used employed to characterize the resultant DNA surfaces prior to and after hybridization on gold surfaces. Subsequently, electrochemical detection of DNA hybridization is achieved through a novel in situ electrochemical approach which utilizes the remarkable ability of DNA duplex to transport electron of a DNA intercalated redox-active molecule over

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long distance to and from the gold surfaces. This electrochemical detection scheme has proven to be successful as it is i) highly sensitive with good detection limits, ii) highly selective with the ability to distinguish single-base mismatches (including the most thermodynamic stable G-A mismatch) and iii) has a commercial viable assay time. Further to that, we will report the use of the same in situ electrochemical approach to assay the interaction of the anticancer drug with the immobilized DNA molecules.

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