1.M.Prasad Naidu MSc Medical Biochemistry, Ph.D.Research Scholar

2. Biosensor is an analytical device for the detection of an analyte that combines a biological component with a physico chemical detector 3. " A biosensor is a device that detects, records, and transmits information regarding a physiological change or the presence of various chemical or biological materials in the environment More technically, a biosensor is a probe that integrates a biological component, such as a whole bacterium or a biological product (e.g., an enzyme or antibody) with an electronic component to yield a measurable signal. Biosensors, which come in a large variety of sizes and shapes, are used to monitor changes in environmental conditions. They can detect and measure concentrations of specific bacteria or hazardous chemicals; they can measure acidity levels (pH). In short, biosensors can use bacteria and detect them, too. 4. the sensitive biological element (biological material (e.g. tissue, microorganisms, organelles, cell receptors, enzymes, antibodies, nucleic acids, etc.), a biologically derived material that interacts (binds or recognises) the analyte under study. The biologically sensitive elements can also be created by biological engineering. 5. the transducer or the detector element (works in a physicochemical way; optical, piezoelectric, electrochemical, etc.) that transforms the signal resulting from the interaction of the analyte with the biological element into another signal (i.e., transducers) that can be more easily measured and quantified; 6. Biosensor reader device with the associated electronics or signal processors is primarily responsible for the display of results This sometimes accounts for the most expensive part of the sensor device. The readers are usually custom designed and manufactured to suit the different working principles of biosensors. 7. First generation: the two components (biocatalyst & tranducer) may be easily separated & both may remain functional in the absence of the other Second generation :the two components interact in a more intimate fashion & removal of one of the two components affects the usual functioning of the other Third generation : the biochemistry & where the electrochemistry occurs at a semiconductor ,the term biochip may be applied to describe such instruments. 8. First generation instruments Glucose+o2-----------gluconic acid+H2o2 The rate of consumption of the substrate o2 can be measured by its reduction at a platinum cathode The rate of production of the product H2O2 can be measured by its oxidation at a platinum anode The rate of production of the product gluconic acid can be measured using a pH electrode 9. YSI MODEL 23 10. Second generation instruments SECOND GENERATION INSTRUMENTS can be constructed by designing an electrode surface that is capable of capturing electrons which are usually transferred in the oxidation reduction reactions. 11. Glucose + GO/FAD Gluconic acid+ GO/FADH2 GO FADH2+ 2M+ GO/FAD + 2M + 2H+ 2M 2M+ + 2e- 12. Exac tech glucometer 13. Third generation instruments These instruments involve the most intimate interactions of the biocatalyst and transducer A glucose biosensor operating on the principle of Exac Tech meter but in which the enzyme was directly reduced at the electrode surface (obviating the need for a mediator) is an example of such an instrument 14. Cell based biosensors Immobilised whole cells or tissues are used to produce biosensors. More recent immobilisation techniques have intended to use gentler physical methods such that cell viability is retained The advantage of this is that such cells may be involved in converting substrate into product via a complex multi enzyme pathway Without having to immobilise each of the enzymes & then provide them with expensive coenzymes 15. Eg.,Nocardia erythropolis immobilised in poly acrylamide on an oxygen electrode Cholesterol+ O2 cholest-4-en-3-one+ H2O2 The oxygen electrode measures the rate of oxygen uptake & this can be related to the cholesterol content of the biological sample Chol.oxidase N.erythropolis 16. Advantages Cheaper No requirement for a complex biocatalyst They have longer response times than do enzyme based sensors Disadvantages Cells contain many enzymes.Hence, care has to be taken to ensure selectivity of response Time taken for cell based biosensor to return to base line potential after use is more. 17. Enzyme immunosensors Several kinds of enzyme immunosensors have been developed They combine the molecular recognition properties of antibodies with the high sensitivity of enzyme based analytical methods The enzyme is used as a marker as it reacts with its substrate,giving changes that can be detected by a transducer. There is similarity between such methods & ELISA techniques. 18. A similar assay can be carried out for hCG . Catalase is used to label hCG & oxygen evolution noted by oxygen electrode In attempts to construct enzyme immunosensors, bioluminiscence,chemiluminiscence & fluorescence principles are exploited because of their great sensitivity A luminiscent immunoassay with catalase has been used to detect human serumalbumin at only 1 ng/cm3 19. Enzyme based biosensors are used in different analysers for quantification of glucose (PO2 electrode) ,urea, creatinine etc where the enzyme is immobilized on the sensor. Affinity sensors have immobilized molecules with specific high affinity binding properties like binding proteins, antibodies, aptamers (DNA SENSORS) 20. Oxygen electrode 21. O2+2e+2H2O--H2O2+2OH H2O2+2e------2OH ----------------------------------------- Total O2+4e+2H2O--4OH The reaction occuring at the anode is 4Ag+4Cl--- 4Agcl+4e The Overall electrochemical process is 4Ag+O2+4Cl+2H2O-4Agcl+4OH 22. Schematic diagram showing the main components of biosensor 23. The biocatalyst (a) converts the substrate to product. This reaction is determined by the transducer (b) which converts it to an electrical signal. The output from the transducer is amplified (c), processed (d) and displayed (e). 24. Principles of detection Photometric Electrochemical Ion channel switch Others,like piezoelectric thermometric etc 25. photometric Many optical biosensors based on the principle of surface plasma resonance(SPR) are evanescent wave techniques This utilises a property of gold & other materials specifically that a thin layer of gold on a high refractive index glass surface can absorb light producing electron waves (surface plasmons) on the glass surface. This occurs only at a specific angle & wave length of incident light and is highly dependent on the surface of gold , such that binding of a target analyte to a receptor on the gold surface produces a measurable signal 26. Interferometric reflectance imaging sensor(IRIS) The Interferometric Reflectance Imaging Sensor (IRIS) was developed by the Unlu research group at Boston University for the purpose of label-free biosensing. Using simple lenses and low-powered, coherent LEDs, the device offers exquisite sensitivity and reproducibility and is able to image with remarkable resolution beyond the classical diffraction limit. This relatively cheap solution also presents minimal hazards when compared to a laser illumination source. 27. Practical uses of this device include the detection of bacterial and viral infections in underdeveloped countries. When pathogen specific growth factors are introduced into a microarray, only spots with the targeted pathogens will grow and increase in concentration. In turn, this dictates a change in the reflected intensity compared to pre-growth. Thus, by measuring how reflectance changes over time, unknown pathogens and their growth rates can be easily characterized and identified. 28. Electro chemical biosensors Electrochemical biosensors are normally based on catalysis of reaction that produces or con sumes electrons (such enzymes are rightly called redox enzymes). The sensor substrate usually contains three electrodes; a reference electrode, a working electrode and a counter electrode. The target analyte is involved in the reaction that takes place on the active electrode surface, and the reaction may cause either electron transfer across the double layer (producing a current) or can contribute to the double layer potential (producing a voltage). 29. We can either measure the current (rate of flow of electrons is now proportional to the analyte concentration) at a fixed potential or the potential can be measured at zero current (this gives a logarithmic response). Further, the label-free and direct electrical detection of small peptides and proteins is possible by their intrinsic charges using biofunctionalized ion-sensitive field-effect transistors. 30. All biosensors usually involve minimal sample preparation as the biological sensing component is highly selective for the analyte concerned They enable the detection of analyte at levels previously only achieved by HPLC & MS & with out rigorous sample preparation 31. Ion channel switch The use of ion channels has been shown to offer highly sensitive detection of target biological molecules. By imbedding the ion channels in supported or tethered bilayer membranes (t-BLM) attached to a gold electrode, an electrical circuit is created . Capture molecules such as antibodies can be bound to the ion channel so that the binding of the target molecule controls the ion flow through the channel. This results in a measurable change in the electrical conduction which is proportional to the concentration of the target. 32. An Ion Channel Switch (ICS) biosensor can be created using gramicidin, a dimeric peptide channel, in a tethered bilayer membrane. One peptide of gramicidin, with attached antibody, is mobile and one is fixed. The magnitude of the change in electrical signal is greatly increased by separating the membrane from the metal surface using a hydrophilic spacer. 33. Ion channel switch Ion channels open Ion channels close 34. others Piezoelectric sensors utilise crystals which undergo an elastic deformation when an electrical potential is applied to them. An alternating potential (A.C.) produces a standing wave in the crystal at a characteristic frequency. This frequency is highly dependent on the elastic properties of the crystal, such that if a crystal is coated with a biological recognition element the binding of a (large) target analyte to a receptor will produce a change in the resonance frequency, which gives a binding signal. In a mode that uses surface acoustic waves (SAW), the sensitivity is greatly increased. This is a specialised application of the Quartz crystal microbalance as a biosensor. 35. Surface attachment of biolocal elements An important part in a biosensor is to attach the biological elements (small molecules/protein/cells) to the surface of the sensor (be it metal, polymer or glass). The simplest way is to functionalize the surface in order to coat it with the biological elements. This can be done by polylysine, aminosilane, epoxysilane or nitrocellulose in the case of silicon chips Another group of hydrogels, which set under conditions suitable for cells or protein, are acrylate hydrogel, which polymerize upon radical initiation. One type of radical initiator is aperoxide radical, typically generated by combining a persulfate with TEMED (Polyacrylamide gel are also commonly commonly used for protein electrophoresis) 36. Applications of biosensors Glucose monitoring in diabetes patients historical market driver related targets Remote sensing of airborne bacteria e.g. in counter- bioterrorist activities 37. Invivo biosensors Invivo miniaturized sensors are being developed for measurement of saO2,pH etc. Implantable subcutaneous glucose sensors are also being used to adjust the dose of insulin Intravascular sensors that release nitric oxide have been developed to decrease the possibility of thrombosis 38. Detection of pathogens Determining levels of toxic substances before and after bioremediation Detection and determining of organophosphate Routine analytical measurement of folic acid, biotin, vitamin B12 and pantothenic acid as an alternative to microbiological assay Determination of drug residues in food, such as antibiotics and growth promoters, particularly meat and honey. Drug discovery and evaluation of biological activity of new compounds. Protein engineering in biosensors Detection of toxic metabolites such as mycotoxins 39. Biosensors in food analysis There are several applications of biosensors in food analysis. In food industry optic coated with antibodies are commonly used to detect pathogens and food toxins. The light signal system in these biosensors has been fluorescence, since this type of optical measurement can greatly amplify the pathogens. A range of immuno- and ligand-binding assays for the detection and measurement of small molecules such as water-soluble vitamins and chemical contaminants (drug residues) such as sulfonamides and Beta-agonists have been developed for use on SPR based sensor systems, often adapted from existing ELISA or other immunological assay. These are in widespread use across the food industry. 40. Detecting Cancer and Health Abnormalities Tuan Vo-Dinh of Oak RidgeNationalLaboatory(OR NL) (left) and Bergein Overholt and Masoud Panjehpour, both of Thompson Cancer Survival Center of Knoxville, have developed a new laser technique for nonsurgically determining whether tumors in the esophagus are cancerous or benign. 41. Of these biosensors, the most publicized is the optical biopsy sensor developed by Tuan Vo-Dinh in collaboration with medical researchers at Thompson Cancer Survival Center in Knoxville. This sensor can tell whether a tumor in the esophagus is cancerou s or benign. In the past, determining accurately whether a patient has cancer of the esophagus has required surgical biopsy. However, laser-based fluorescence method has eliminated the need for biopsy, reducing pain and recovery time for patients. 42. Laser light of the appropriate wavelength is directed to the inner surface of the esophagus by means of a fiber-optic device that is swallowed by the patient. The epithelial cells and tissue inside the esophagus fluoresce when exc ited by the laser light. When the esophagus interior is illuminated with blue light [410 nanometers (nm)], the normal tissue emits light at wavelengths different from those emitted by the cancer cells. 43. the spectral properties of the light at wavelengths ranging from 400 to 700 nm can be analyzed at various positions in the esophagus by the soft ware developed. Emissions from normal cells and cancer cells can be distinguished quite accurately; the difference is expressed as the differential normalized fluorescence index. Tests on more than 200 patients show that, compared with the results of surgical biopsies, laser fluorescence diagnosis is accurate in over 98% of the cases. 44. Medical telosensors This "medical telesensor" chip on a fingertip can measure and transmit body temperature. 45. Medical telesensors A chip on our fingertip may someday measure and transmit data on body temperature. An array of chips attached to our body may provide additional information on blood pressure, oxygen level, and pulse rate. This type of medical telesensor, which is being developed at ORNL for military troops in combat zones, will report measurements of vital functions to remote recorders. 46. The goal is to develop an array of chips to collectively monitor bodily functions. These chips may be attached at various points on a soldier using a nonirritating adhesive like that used in waterproof band-aids These medical telesensors would send physiological data by wireless transmission to an intelligent monitor on another soldier's helmet. The monitor could alert medics if the data showed that the soldier's condition fit one of five levels of trauma. 47. The infra red microspectrometer The infrared microspectrometer developed at ORNL can be used for blood chemistry analysis, gasoline octane analysis, environmental monitoring, industrial process control, aircraft corrosion monitoring, and detection of chemical warfare agents. 48. ORNL has developed a sensitive detector for monitoring changes in the body's concentrations of calcium ions. It may be useful in diagnosing disease or exposure to chemical warfare agents. This biosensor consists of an optical fiber to which is attached a synthesized hybrid molecule. One half of the hybrid molecule binds calcium ions and the other half fluoresces when calcium ions are bound to the molecule. 49. Blood pressure and pulse rate may be measured by chips designed to detect pressure changes. Unlike a glass fiber, a silicone fiber is flexibleit can be squeezed or stretched, and the amount of compression or expansion can be measured by changes in light transm ission through the fiber. Thus, silicone fibers embedded in roads can be used to weigh trucks. If a silicone fiber on a chip can sense pressure at various positions in the body, it may be used for monitoring blood pressure, pulse rate, breathing (chest expansion), knee bending during physical rehabilitation, and foot pressure distribution. 50. Aging, diseases such as diabetes and Alzheimer's, and chemical warfare agents cause changes in metal ion concentrations in the body. If these changes could be detected and measured, the information could provide clues about changes in disease states a nd exposure to toxins. Tuan Vo-Dinh and his coworkers have developed a biosensor using a glass optical fiber and a hybrid molecule . One half of the hybrid molecule binds calcium ions and the other half fluoresces when calcium ions are bo und to the molecule. By attaching this molecule to the end of a very small diameter optical fiber, the conc of calcium ions in a solution can be measured. 51. ROLE OF BIOSENSORS IN POINTOFCAR E TESTING 52. Point of care testing is a laboratory testing conducted close to the site of patient care. Point of care testing is also commonly described as ancillary ,bed-side ,near-patient, satellite,remote & decentralised testing. 53. Role of biosensors in the management of diabetes mellitus Self monitoring of blood glucose(SMBG) SMBG is the most important day to day metabolic parameter that must be assessed by the person affected with diabetes All insulin treated patients regardless of type 1 or type 2 DM should optimally perform SMBG three or more times daily The most modern SMBG strips can be used with any whole blood specimen, whether arterial, venous or capillary & are suitablefor use in neonates 54. A common example of a commercial biosensor is the blood glucose biosensor, which uses the enzyme glucose oxidase to break blood glucose down. In doing so it first oxidizes glucose and uses two electrons to reduce the FAD (a component of the enzyme) to FADH2. This in turn is oxidized by the electrode (accepting two electrons from the electrode) in a number of steps. The resulting current is a measure of the concentration of glucose. In this case, the electrode is the transducer and the enzyme is the biologically active component. 55. The newest blood glucose detection systems use an electrochemical biosensor based on a glucose dehydrogenase reaction that is not affected by oxygen availability. Glucose dehydrogenase catalyses the formation of glucono-d-lactone from glucose while NAD+ is reduced to NADH The glucose dehydrogenase assays are highly specific for glucose & show good agreement with hexokinase assay. Glucose +NAD+ D-glucono-d-lactone+ NADH+H 56. Continuous glucose monitoring system(CGMS) CGMS involves placing under the skin a small needle that is attached by a wire to a control module Typically the needle is inserted under the skin overlying the abdomen The needle contains the electrochemical micro electrode sensor that is thinly coated with glucose oxidase beneath a biocompatable membrane The measurement of the interstitial fluid glucose is changed into an electronic signal & stored in a pager sized control module,which can be worn on the patients belt The patient does not have access to the CGMS glucose results while they are wearing the device 57. A glucose reading is taken every 10 sec & the measurements are averaged and recored (288 measurements per day) In studies of anesthetized dogs ,there was a 5-12 min delay between changes in blood glucose& interstitial fluid glucose levels. The researchers concluded that differences between plasma & interstitial fluid glucose will not be a significant obstacle in advancing the use of interstitial fluid as an alternative to blood glucose measurements 58. Biochips: a new generation of biosensors using DNA probes (DNA Biochip) have been developed Probe recognition is based on the molecular hybridization process, which involves the joining of a strand of nucleic acid with a complementary sequence. Biologically active DNA probes are directly immobilized on optical transducers which allow detection of Raman, SERS, or fluorescent probe labels. DNA biosensors could have useful applications in areas where nucleic acid identification is involved. The DNA probes could be used to diagnose genetic susceptibility and diseases. The Biochip using antibody probes has recently been developed to detect the p53 protein system. 59. Using methods to form short sequences from the DNA of interest, DNA fragments are produced that have one of the letters at their end and that differ acco rding to their sizes. As a result, DNAs containing 400 to 600 letters can be sequenced accurately. However, many hours are required to prepare the fragments and separate them by size (using gel electrophoresis). Using this method, the sequences of millions of DNA letters have been determined, enabling the identification of the site of a genetic mutation that causes such diseases as sickle cell anemia, Huntington's disease, fragile X syndrome (a serious type of mental retardation), and several hundr ed other inherited diseases or traits. 60. Serg probes ORNL's surface-enhanced Raman gene (SERG) probes can locate free DNA molecules that have hybridized to other DNAs fixed on a surface. The technique has use in medi cine, forensics, agriculture, and environmental bioremediation. 61. Serg probes 62. Another hybridization method being developed at Oak Ridge uses a process called surface-enhanced Raman spectroscopy (SERS). Hybridized DNA is transferred from the nylon membrane to a glass strip coated with tiny silver spheres. The dye labels attached to the DNA bases have unique Raman infrared spectra, but the normally weak Raman lines are greatly enhanced by the presence of the silver spheres. This enhancement allows DNA bases to be detected at sufficient sensitivity to be useful for sequenci ng studies. 63. DNA processing & analysis chip Conceptional design of DNA processing and analysis chip that Mike Ramsey and Bob Foote are developing. 64. To determine which DNA fragments make up a fingerprint, they must be separated and identified. The "lab on a chip" developed by Mike Ramsey and colleagues has been adapted to perform such separations within a few minutesmuch faster than standard gel procedures. Like microcircuits and computers operating in parallel, these chemical separations chips can be used in parallel for DNA analysis. 65. In one application, liquids containing DNA and a restriction enzyme are injected into different chambers etched into the chip. Electric fields pump the liquids through a microscopic channel into a reaction chamber, where the enzyme cuts the DNA into pieces of different lengths. The DNA snippets are then electrically pumped to the separation channel, wher e they are tagged with fluorescent dyes for detection. 66. The DNA fragments of various sizes are sorted in a liquid containing fibrous strands of a polymer. The DNA through fragments get tangled with the polymer strands, which slow them down as they pass. Small chunks of DNA find their way through the tangled web faster than the larger ones, so separation results. As the fragments ar e separated, they are illuminated with a laser light, causing them to fluoresce. The detected light intensities are fed to a computer, which sorts through signals from separated fragments to provide a sample analysis. 67. nanosensors NANOSENSORS: the development of nano-biosensors and in situ intracellular measurements of single cells using antibody-based nanoprobes has recently been reported. The nano-scale size of this new class of sensors also allows for measurements in the smallest of environments. One such environment that has evoked a great deal of interest is that of individual cells. Using these nanosensors, it is possible to probe individual chemical species and molecular signalling processes in specific locations within a cell. it has been shown that insertion of a nano-biosensor into a mammalian somatic cell not only appears to have no effect on the cell membrane, but also does not effect the cell's normal function. 68. Bruce Jacobson (left) and Carl Gehrs, manager of ORNL's Center for Biotechnology, examine a newly fabricated temperature control block for a genosensor chip designed by Mitch Doktycz. The genosensor chip can detect specific DNA sequ ences. 69. Atomic force microscopy This atomic force microscopy image shows two knots, or "protein bumps," formed on the DNA thread. Each knot results when a mutant enzyme binds to, rather than cleaves, the DNA strand. 70. The magic scissors enzyme, or restriction endonuclease, cuts a DNA sequence each time it occurs in the strand. But the mutant form of this enzyme simply binds to, rather than cleaves, the DNA strand. The AFM can image the resulting knot, or protein bum p, formed on the DNA thread. ORNL researchers can identify where the enzyme binds to the DNA within 100 base pairs, which is a very high resolution. They've shown that the distance from one bump to the next, as imaged by the AFM, was exactly as predicted from enzyme cutting experiments analyzed by gel electropho resis. 71. beams and mirrors can determine the shapes of human body parts. Its accuracy could facilitate the creation of clothes that fit. 72. Perhaps the most unusual of ORNL's biosensors is a new technique to measure human body surfaces. Such measurements, called anthropometry, are used by tailors, artists, and scientists. One of the finest minds in science to take a strong interest in anth ropometry was Leonardo da Vinci, who drew the famous Proportions of the Human Figure some 500 years ago. An ORNL scientist who has moved the field forward is Judson Jones of the Computer Science and Mathematic s Division. He has developed a technique using laser beams and mirrors to determine the shape of human body parts. He measures the topology of a solid surface, using amplitude-modulated laser radar, which measures arc lengths along complex and oft en inaccessible body contours. Because laser radar measures the phase and amplitude of a reflected, modulated laser beam, only one optical path is required between the sensor and the subject. Multiple images are combined by integrating information from different virtual viewpoints, any number of which can be created with strategically positioned mirrors. Arc lengths along arbitrary contours, surface areas, and volume estimates all become possiusing ble. The accuracy of the measurements is within 1 mm. Be cause creating "clothes that fit" may be accomplished a single camera with several mirrors, a blue jeans manufacturer has shown interest in this methodology. 73. Light emissions from microspheres and bacteria are seen through a fluorescence microscope. Shown are red- fluorescing S. aureus bacteria bound to 6.5-m spheres and one yellow orange- fluorescing E. coli bound to the larger 10-m sphere. 74. 100 different types of bacteria can be identified simultaneous ly because the stained bacteria all would fluoresce at one wavelength and of light the diameters of the spheres could be illuminated at another wavelength. Although all the spheres fluoresce when excited by one wavelength, the morphological resonances, which look like saw teeth superimposed on a fluorescence emission spectrum, can distinguish among diameters of many different-sized spheres. This approach satisfies one of today's challenges in biotechnology: multiplex biosensors to obtain more infor mation from one sample analysis. 75. In another approach to the use of immunosensors, microspheres of different sizes are labeled with antibodies that bind to different bacteria; thus, microspheres of one size have one particular antibody and microspheres of another s ize have a different antibody. The sizes of the microspheres are identified by their "morphological resonances" (shape-based light emissions when excited by a laser), and the bacteria that become bound are detected by the color of fluorescent dye with which they are stained. 76. bioreporters Genetically engineered bacteria can also be useful because of their ability to "tattle" on the environment. Such commonly used bacteria have been designed to give off a detectable signal, such as light, in the presence of a specific pollutant they like to eat. They may glow in the presence of toluene, a hazardous compound found in gasoline and other petroleum products. They can indicate whether an underground fuel tank is leaking or whether the site of an oil spill has b een cleaned up effectively. These informer bacteria are called bioreporters. 77. The key to protecting a military unit or community from dangerous bacteria is to detect them before they reach their intended victims. People can then be warned to leave an area or at least wear protective gear. Bacteria can be detected using "biosensors. 78. Critters on a chip Mike Simpson shows the newly developed "critters on a chip" in which bioluminescent bacteria signal the presence of pollutants. 79. "critters on a chip" technology in which light sensor s pick up and transmit information from chip bacteria that glow in the presence of trace levels of poisons, explosives, or pollutants. These types of biosensors are useful for monitoring efforts to clean up industrial spills because these light-emitt ing bacteria can "report" continuously the progress of biodegradation. Such bioreporters have proven successful using trichloroethylene, toluene, and various petroleum products in laboratory tests. They are now being tested on a much larger scale usi ng a lysimeter. 80. Thank You