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Biosensor dr manju jha


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  • 1. Presented by : Dr. MANJU JHA II-year Resident, M.D. Biochemistry 09-10-2012 J.L.N.Medical College,Ajmer.
  • 2. HISTORY19621969197019751975197519761980198219831984198719901992199619982000- first description of a biosensor of : an amperometric enzyme electrode (Glucose sensor) by Clark. first potentiometric biosensor : urease immobilized on an ammonia electrode to detect urea by Guilbault & Montalva. ion-selective Field Effect Transistor (ISFET) by Bergveld. fibre-optic sensor with immobilized indicator to measure carbon-di-oxide or oxygen by Lubbers & Optiz. first commercial biosensor (Yellow Spring Instrumental Biosensor) . first microbe based biosensor (first Immunosensor).. first bedside artificial pancrease. first fibre-optic pH sensor for in-vivo blood gases by Peterson. first fibre-optic based biosensor for glucose. first surface plasmon resonance (SPR) immunosensor. first mediated Amperometric biosensor. Blood Glucose biosensor launched by Medi-sense Exac Tech. SPR based biosensor by Pharmacia BIA Core. hand-held blood biosensor by i-STAT.. launching of Gluco-card. blood glucose biosensor launched by Life-scan Fast Take. nanotechnology biosensor, chip,quantum dots etc..
  • 3. What is a BIOSENSOR ? A Biosensor may be defined as : a compact analytical device having a biological or biologically-derived sensing element either integrated within or intimately associated with a physicochemical transducer which is then detected by an electronic component and translated into a measurable electronic signal.
  • 4. IUPAC Definition - Biosensor is a self-contained integrated device which is capable of providing specific quantitative or semi-quantitative analytical information using a biological recognition element (biochemical receptor) which is in direct spatial contact with a electrochemical transducer element.
  • 5. WORKING PRINCIPLE OF A BIOSENSOR The interaction of the analyte with the bioreceptor is designed to produce an effect measured by the transducer, which converts the information into a measurable effect, such as an electrical / electronic signal.
  • 6. (e) (f) (d) (a) (b) (c) The interaction of the analyte (a) with the bioreceptor, which identifies the stimulus (b) is designed to produce an effect measured by the transducer (c), which converts it to an electrical signal. The output from the transducer is amplified (d), processed (e) and displayed (f).
  • 7. Main components- A. Biological recognition element- It is the sensitive biological element or biological material (tissue ,microorganisms , organelles , cell receptors , enzymes, antibodies, nucleic acids, etc.) or biomimetic component that interacts (binds or recognises) the analyte under study. The biologically sensitive elements can also be created by biological engineering.
  • 8. B. Transducer- The transducer or the detector element transforms the signal resulting from the interaction of the analyte with the biological recognition element into another signal that can be more easily measured and quantified. C. Amplifier, Microprocessor and Display- Biosensor reader device with the associated electronics or signal processors that are primarily responsible for the display of the results.
  • 9. Biological elements and Transducers commonly used in BIOSENSORSTransducer elements Biological elements 1. Enzymes 2. Antibodies 3. Hormone receptors 4. Cells 5. Cell organelle 6. Tissues 7. Membranes 8. Micro organisms 9. Nucleic acids 10. Biomimetic materials 1. Electrochemical Amperometric Potentiometric 2. Electrical Conductometric Ion-sensitive 3. Photochemical Calorimetric Fluorescence Reflectance 4. Optical Fiber optic SPR Luminometric Fluorometric 5. Piezoelectric Acoustic MCL Ultrasonic QCM
  • 10. Biological recognition element— (Classification acc to Biological Signalling method) A. Catalytic biosensor: kinetic devices that measure steady state concentration of a ‘tranducer - detectable species’ formed / lost due to a biocatalytic reaction. Monitored quantities arei. rate of product / acid ( ↑ or ↓pH) formation, ii disappearance / consumption of reactant, iii. inhibition of reaction ,etc.
  • 11. Biocatalysts used are---- 1.EnzymesEnzyme-based biosensors use their catalytic activity and binding capabilities for specific detection . The catalytic activity of the enzymes provides these types of biosensors with the ability to detect much lower limits than with normal binding techniques. This catalytic activity is related to the integrity of the native protein structure.
  • 12. Most common – Glucose oxidase, Urease, Alcohol oxidase etc. Commercial example is GLUCOSE SENSOR using oxidase (GOD) . Clarks (1962) Glucose + O2 -- glusoce oxidase  Gluconic acid + H2O2 (1) H2O2  O2 + 2H+ + 2e- O2 + 4H+ + 4 e-  2H2O -and current flows. (3) at anode (2) at cathode There are three measurement routes-pH change (acid production) - O2 consumption (fluorophore monitor) - H2O2 production (electrochemical)
  • 13. 2. Micro-organisms – Microorganisms such as bacteria and fungi can be used as biosensors to detect specific molecules or the overall state of the surrounding environment. Cell behaviour such as cell metabolism, cell viability, cell respiration, and bioluminescence can be used as indicators for the detection. Furthermore, proteins that are present in cells can also be used as bio-receptors for the detection of specific analytes. Rhodococcus erythropolis (in collagen) used to measure BOD.
  • 14. 3. Cell / Tissue samples. Use of cell as a biosensor occurred in 1977 by Rechnitz . Rechnitz coupled Streptococcus faecium on the surface of an ammonia gas sensing membrane electrode . This Rechnitz electrode was capable of detecting the amino acid ARGININE. 4. Organelles – Mitochondra , Cell walls etc. 5. Membranes .
  • 15. 6. Bio-mimetic materials A bio mimetic biosensor is an artificial or synthetic sensor that mimics the function of a natural biosensor . These can include aptasensors , where apta -sensors use aptamers as the biocomponent. Aptamers are synthetic single stranded nucleic acid that can be designed to identify or recognize amino-acids, oligosaccharides , peptides , and proteins . Aptamers have high affinity , high selectivity, cheaper & easy to synthesize .
  • 16. B. Affinity biosensors: device in which receptor molecules bind analyte molecules causing physicochemical change that is detected by a transducer. Receptors used are- 1. Antibody / Antigen (Immunosensor) The high specificity between an antibody and an antigen can be utilized in this type of sensor technology. Biosensors utilizing this specificity must ensure that binding occurs under conditions where nonspecific interactions are minimized . Binding can be detected either through fluorescent labelling or by observing a refractive index or reflectivity changes. 2. Hormone receptor/Antagonist-
  • 17. 3. Nucleic acid -The complementary relationships between nucleic acid ‘bases’ in the DNA form the basis of specificity in nucleic acid based biosensors. These sensors are capable of detecting trace amounts of microorganism DNA by comparing it to a complementary strand of known DNA. By unwinding the target DNA strand, adding the DNA probe, and annealing the two strands , the probe will hydrolyze to the complementary sequence on the adjacent strand . If the probe is tagged with a fluorescent compound , then this annealing can be visualized under a microscope. eg. DNA Chip.
  • 18. (Target probe ) (Capture probe) General DNA biosensor scheme--Target DNA is captured at the recognition layer (A), and the resulting hybridization is transduced into a measurable electronic signal (B). It is used for genome mutation detection & clinical diagnosis.
  • 19. Biosensor construction-I. Recognition element immobilization (Immobilization of biological receptor)- Biological receptors, i.e. enzymes, antibodies, cells or tissues with high biological activity, can be immobilized in a thin layer at the transducer surface by using different procedures. (a) Entrapment behind a membrane - viscous aqueous solution trapped by membrane permeable to analyte. (b) Entrapment of biological receptors within a polymeric matrixMembranes— Cellophane, Cellulose acetate, Polyurethane membranes . Gel entrapment– Agarose, Gelatin, Agar gel , Polyacrylamide gel. MicroencapsulationEncapsulation inside Liposomes, or absorbed in fine carbon . particles that are incorporated in a gel or membrane
  • 20. (c) Entrapment of biological receptors within self-assembled monolayers (SAMs) or bilayer lipid membranes (BLMs). (d) Adsorption: direct adsorption onto membrane or transducer; can also be adsorbed onto pre -adsorbed proteins . (e) Covalent binding (via –COOH, -NH2, -OH ) , or cross linking (via glutaraldehyde ) to transducer or membrane surface. Receptors are immobilized either alone or are mixed with other proteins, such as bovine serum albumin (BSA), either directly on the transducer surface, or on a polymer membrane covering it .
  • 21. II. Inner & Outer membranes-These serve three important functions - (a) Protective barrier -- Membrane prevents large molecules, such as proteins or cells of biological samples, from entering and interfering with the reaction layer. It also reduces leakage of the reacting layer components into the sample solution. This function of the outer membrane is important, for example, for implanted glucose sensors, since its glucose oxidase is of nonhuman origin and may cause immunological reactions.
  • 22. (b) Diffusional outer barrier for the substrate – The thinner the membrane, the shorter the biosensor response time so such a diffusional barrier also makes the sensor response independent of the amount of active enzyme present and improves the sensor response stability. (c) Biocompatible and biostable surfaces – Biosensors are subject to modifications when they are in direct contact with biological tissues or fluid , i.e. implanted in vivo or, more generally, in biologically active matrices, such as cell cultures.
  • 23. BIOCOMPATIBILITY- If the implantation of the biosensor does not materially affect the normal functioning of the host medium and if the medium does not materially affect the normal operation of the biosensor , then the biosensor is considered to be biocompatible.
  • 24. Detection or Measurement mode (Transducer)— 1. Electrochemical- translate a chemical event to an electrical event by measuring current passed. a. Amperometric- most common : movement of electron produced in a redox reaction. In 2000 a wearable non-invasive Glucose monitor has been introduced. SONY has developed a biofuel cell using sugar as the fuel and enzymes as catalysts to power a Walkman. b. Potentiometric - electrical potential producing phenomenon.
  • 25. 2. Electrical – a. Conductometric b. Ion-sensitive – 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.
  • 26. 3. Photochemical – translate chemical event to a photochemical event, measure light intensity & wave length. a. Calorimetric – measure heat changes of a reaction. Isothermal , Heat conduction , or Isoperibol . b. Fluorescence - property of fluorescence is used. eg. DNA Microarray c. Reflectance – use the property of reflection which is detected by photo detector.
  • 27. 4. Optical – There is light output during the reaction or a light absorbance difference between the reactants & products. It uses property of fluorescence , phosphorescence , refraction, and dispersion spectrometry etc. a. Fiber optic (optrode/optode) b. Surface plasmon resonance (SPR) c. Luminometric d. Fluorometric
  • 28. 5. Piezoelectric– These devices are used to detect the specific angle at which electron waves are emitted when the substance is exposed to laser light or crystals, such as quartz ,which vibrate under the influence of an electric field . It translates a mass change from a chemical adsorption event to electrical signal. The change in frequency is proportional to the mass of absorbed material. a. Acoustic (Surface acoustic wave) b. Microcantilever c. Ultrasonic d. Quartz crystal microbalance (QCM)
  • 29. Nanobiosensors Nanotechnology will enable us to design sensors that are much smaller, less power hungry, and more sensitive than current micro- or macro sensors. Examples— Quantum dots (QD's) QCM Microcantilever Nanoparticles Nanotubes(CNTs) Nanowires
  • 30. Ideal Biosensor Characteristics 1. Sensitivity: high ΔS/ Δcanalyte (S = signal) 2. Simple calibration (with standards) 3. Linear Response: ΔS/ Δcanalyte constant over large concentration range . 4. Accuracy must be there. 5. No hysteresis—signal independent of prior history of measurements . 6. Response time / Recovery time - less . 7. Selectivity—response only to changes in target analyte concentration
  • 31. Ideal Biosensor Characteristics – 8. Long-term Stability—not subject to fouling, poisoning, or oxide formation that interferes with signal; prolonged stability of biological molecule . 9. Dynamic Response-rapid response to variation in analyte concentration . 10. Biocompatibility—minimize clotting, platelet interactions, activation of complement when in direct contact with bloodstream. 11. Must be cost effective , smaller or portable in size .
  • 32. Applications 1. Medicala. Biosensors are used in both clinical and laboratory use in medical care. Glucose monitoring in diabetes patients . Medtronic glucose sensor - implants in major vein of heart. b. Tumor cells are used as biosensors to monitor the susceptibility of chemotherapeutic drugs. c. Routine analytical measurement of folic acid, biotin, vitamin B12 and pentothenic acid . d. Micro- and nanoscale biosensors— Genome mutation detection , cancer detection & clinical diagnosis. Bacterial-UTI , Human Immunodeficiency Virus (HIV) Detection, Hepatitis and Anthrax detection.
  • 33. 2. Bio / Pharmaceutical research- a. Quality assurance b. Study of biomolecules and their interaction c. Protein engineering . d. Drug discovery , evaluation monitor the manufacturing of biological activity of new compounds (research field). e. Biosensors are used for measuring concentration of various metal ions by specific protein concentration or by using genetically modified organisms. f. Aptamers are used for the detection of proteins –Thrombin , IgE , HIV – tat protein , Lysozyme , Abrin toxin.
  • 34. 2. Bio / Pharmaceutical research- g. Biosensors are used in monitoring of the glutamate and acetyl choline , which is the main cause in neurodegenerative diseases. h. Microbiology: bacterial and viral analysis . i. Biosensors are used in analysing micro dialysis samples. j. Biosensors are used in biotechnological process such as to determine proteins or peptides. k. Biosensors are also used in determining intracellular proteins and also plasmids. l. Detection of cancer biomarkers – CEA , PSA , CA-125 & Tumour necrosis factor .
  • 35. 3. Industrial / Agricultural – a. Biosensors used in process control will be able to measure materials present in the process. b. Use of biosensors in industry will improve manufacturing techniques, this will allow for usage of wider variety of sensing molecules. c. Biosensors are used in controlling the industrial processes. d. Microbial sensor measure Ammonia & Methane.
  • 36. 4. Environmental- a. Biosensors are used in detecting environmental pollutants and monitoring of Mines, Industries and toxic gases. b. Biosensors are used in the BOD measurement during waste water treatment. c. Biosensors are used in the detection of poly aromatic hydrocarbons present in water. d. Environmental applications e.g. the detection of pesticides and river water contaminants such as heavy metal ions. e. Detection and determination of organophosphates .
  • 37. 5. Food industry- a. Quality assurance in food industries , ex. E. Coli, Salmonella. b. Food & drink production analysis. c. Biosensors are used for detection of food freshness marker determining parameters in wine industry. d. Determination of drug residues in food, such as antibiotics and growth promoters, particularly meat and honey. e. Detection of toxic metabolites such as mycotoxins.
  • 38. 6. Biodefence- a. Detection of pathogens (SARS in 2003). b. Remote sensing of airborne bacteria / virus , e.g. In counter bio-terrorist activities. c. Detection system for biological welfare agent eg. Bacillus anthracis (anthrax) spores. d. Determining levels of toxic substances before and after bioremediation. e. Crime detection.