BIOSENSORS

1. Biosensors 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 biologicalelement (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. Biosensorreaderdevice 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. Firstgeneration: the two components (biocatalyst & tranducer) may be easily separated & both may remain functional in the absence of the other • Secondgeneration :the two components interact in a more intimate fashion & removal of one of the two components affects the usual functioning of the other • Thirdgeneration : 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

14. 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

15. 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

16. Eg.,Nocardiaerythropolisimmobilised 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

17. 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.

18. 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.

20. 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

21. Enzymebasedbiosensors are used in different analysers for quantification of glucose (PO2 electrode) ,urea, creatinine etc where the enzyme is immobilized on the sensor. • Affinitysensors have immobilized molecules with specific high affinity binding properties like bindingproteins, antibodies, aptamers (DNA SENSORS)

22. Oxygen electrode

23. 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

24. Schematic diagram showing the main components of biosensor

25. 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).

27. Principles of detection • Photometric • Electrochemical • Ion channel switch • Others…,like piezoelectric thermometric etc

28. 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

29. 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 LED’s, 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.

30. 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.

31. Electro chemical biosensors • Electrochemical biosensors are normally based on catalysis of reaction that produces or con sumeselectrons (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).

32. 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.

33. All biosensors usually involve minimalsample 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

34. 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.

35. 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.

36. Ion channel switch Ion channels open Ion channels close

37. 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.

38. 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 acrylatehydrogel, 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)

40. Applications of biosensors • Glucose monitoring in diabetes patients ←historical market driver related targets • Remote sensing of airborne bacteria e.g. in counter-bioterrorist activities

41. Invivo biosensors • Invivo miniaturized sensors are being developed for measurement of saO2,pH etc. • Implantablesubcutaneous glucose sensors are also being used to adjust the dose of insulin • Intravascularsensors that release nitric oxide have been developed to decrease the possibility of thrombosis

42. 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

43. 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.

44. Detecting Cancer and Health Abnormalities • Tuan Vo-Dinh of Oak RidgeNationalLaboatory(ORNL) (left) and BergeinOverholt and MasoudPanjehpour, 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.

45. 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-basedfluorescence method has eliminated the need for biopsy, reducing pain and recovery time for patients.

46. 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 excited 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.

47. 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, laserfluorescencediagnosisisaccurateinover98%ofthecases.

48. Medical telosensors • This "medical telesensor" chip on a fingertip can measure and transmit body temperature.

49. 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 bloodpressure, oxygenlevel, and pulserate. • This type of medical telesensor, which is being developed at ORNL for militarytroops in combat zones, will report measurements of vital functions to remote recorders.

50. 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.