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Biosensors ביו-חיישנים – עקרונות ויישומים מרפואה ביתית ועד לניטור סביבתי Dr. Ronen Almog מכון טכנולוגי חולון R. Almog Outline •Biosensors •Detection principles: •Electrochemical •Optical •Mechanical •Lab on a chip/BioMEMS •Examples: •Diabetes - glucose monitoring •Water toxicity detection R. Almog Elements of a biosensor R. Almog Biosensor definition An integrated device consisting of •a biological recognition element and •a transducer capable of detecting specific biological/chemical compound and converting it into an electronic signal. R. Almog Biosensors applications and importance •Medical/health monitors •Homeland security •Pharmaceutical industry •Food industry Biosensors Features •Simplicity •Miniaturization •Selectivity •Rapid •Sensitivity R. Almog Biological recognition elements •Enzymes and their substrates •Antibodies and their antigens •Nucleic acids and their complementary sequences •Whole cell A-T G-C R. Almog On Size and Scale R. Almog Enzymes • Large protein molecules. • Catalyze chemical reactions. • Used as tools to perform various biochemical reactions in the cell. Participates actively in the transformation of chemical A (the substrate) to chemical B (the product) but remains unchanged at the end of the reaction. R. Almog Immunosensors: Antibody – Antigen recognition Antibody - proteins, produced by the immune system of higher animals in response to the entry of “foreign” materials into the body, eg. viruses, bacteria Bind tightly to the foreign material (the antigen) that provoked the response and mark it for attack by other elements of the immune system. Antibodies are also very specific- they need to be, in recognizing and binding to the foreign substance only and not to materials native to the organism. If an antigen is present in that medium, it will be bound by the antibody to form a larger, antigen-antibody complex. This will change some physicochemical parameter (usually mass or an optical parameter) of the environment at the transducer surface of the sensor and that change is subsequently detected. R. Almog Antibody structure R. Almog The transducer Chemical change Physical change The key part of a biosensor is the transducer which makes use of a physical change accompanying the reaction. The most common are: 1. Electrochemical-Amperometric : Measures currents generated when electrons are exchanged between a biological system (in solution) and an electrode in a constant potential. 2. Optical detection - refractive index change or fluorescence 3. Mechanical detection 4. Conductometric/Impedimetric : detect changes in conductivity/impedance between two electrodes. R. Almog Transducers - sensing methods in biosensors R. Almog MEMS - MicroElectroMechanichal sensors Use: Bioassay of prostate-specific antigen (PSA) R. Almog Optical sensors - Biacore example R. Almog Reasons for Miniaturization R. Almog Biochips Microelectronic-inspired devices that are used for delivery, processing, analysis, or detection of biological molecules and species. These devices are used to detect cells, microorganisms, viruses, proteins, DNA and related nucleic acids, and small molecules of biochemical importance and interest. BioMEMS Biomedical or biological applications of MEMS (micro electro mechanical systems) R. Almog R. Almog Lab-on-a-chip R. Almog Burns et al. Science 282, 484, 1998 BioChip/BioMEMS Materials •Silicon • Glass, Quartz • Polymers – Poly (dimethylsiloxane) (PDMS) – Poly (methyl methacrylate) (PMMA) – Teflon, etc. Considerations • Biocompatibility, ideal for biomedical devices • Transparent within the visible spectrum • Rapid fabrication • Photo-definable • Chemically modifiable R. Almog Biochip - microfluidics Key Attributes of Biochips 1. Small length scale 2. Small thermal mass 3. Laminar flow 4. High surface-to-volume ratio R. Almog The Oxygen electrode Clark’s Glucose electrode R. Almog Biosensors History R. Almog Technology evolution 6 analyses from a drop of blood in about one minute. R. Almog Diabetes - Glucose Biosensor • The user carries a wallet sized case that contains the testing equipment • A lancet pierces the skin on the finger • The user places this blood sample on a test strip and inserts it into the reader • Electrochemical detection R. Almog http://www.diabetesuffolk.com/Managing%20Diabetes/Meters/LifeScan%20Ultrasmart.asp Alcohol Test – Drager Alcotest 7110 הינשוף The Alcotest 7110 Standard is a highly developed measuring instrument for precise determination of breath alcohol concentration . Two different and independent measuring systems: • Infrared spectroscopy – λ=9.5µm • Electrochemical measurement www.draeger-safety.com/ R. Almog Example - insect MEMS hybrid A radio-controlled beetle 6 electrodes affixed to the brain and muscles 1.3g electronic module Max weight: 3g The University of California, Berkeley IEEE MEMS, January 2009 R. Almog Example: Toxichip R. Almog Water toxicity detection motivation 1. Homeland security THE THREAT: Intentional poisoning of a drinking water source 2. Enviromental pollution THE NEED: A rapid early toxicity warning device 3. Pharmaceutical screening applications R. Almog Toxicity detection systems Toxicity bioassays The only question need to be asked is “Is the sample toxic?” Standard toxicity bioassays, mostly designed for environmental purposes, are unsuitable for our needs: size, response time. R. Almog The goal To develop a portable system that can detect the presence of unknown acute toxicity chemicals in drinking water within 20 minutes. R. Almog Whole-cell biosensor: The biological material is an intact, living, functioning cell. E. Coli bacteria Toxichip whole cell biosensors: •Bacterial cells •Genetically modified •Bioluminescent •Tailored to respond to different cell stress factors Three elements in the solution 1. Reporter cells: live cells “tailored” to detect toxicity. We use Escherichia coli (E. coli) bacteria as a whole cell sensor. 2. Biochips: disposable, credit card size, containing the cells. 3. Analyzer: a small mobile instrument into which the chip will be inserted, and which will provide the reading. R. Almog Biochip array E. Coli bacteria R. Almog Bioluminescence The emission of light by a living organism as the result of a chemical reaction. Chemical energy is converted to light energy. Marine organisms Fungee Insects Emission spectrum: Visible - blue-green (490-500 nm) Bacteria R. Almog Bioluminescence At least two chemicals are required: The one which produces the light is generically called a "luciferin“. The one that drives or catalyzes the reaction is called a "luciferase." Each organism has its own luciferin and luciferase compounds. Luciferin (substrate) + Luciferase (enzyme) + O2 Product: Oxyluciferin + light R. Almog Bacteria engineering for toxins detection Two types of biochemical response to toxins : Type I Normally doesn’t emit light. Toxin exposure induces light emission. Sensitive to low concentration of toxins with a dose-dependent signal. Type II Constitutive test Normally emits light. In the presence of a toxin, the signal intensity decreases. Respond to high concentration of toxins R. Almog Type I - bacteria engineering for toxins detection The fusion of two genetic elements inside a host E. coli bacteria: Sensing element: A promoter of a gene involved in the response to the desired target. Reporting element: A Bioluminescent gene. – generates the Luciferin and Luciferase when experssed Reporting element gene promoter Sensing element luxCDABE Light R. Almog Toxins list Chemical Description Acute Toxicity Species LD50 1 Sodium arsenite Heavy metal, carcinogen. Use as :Herbicide, Insecticide, Rodenticide, Fungicide. Rat (oral) 41 mg/kg, 11.2 ppm 2 Cadmium chloride Heavy metal, Used as stabilizer and pigment in plastics Rat (oral) 88 mg/kg 3 Parathion Cholinesterase Inhibitor.Very potent insecticide and acaricide used on cotton, rice and fruit trees Rat (oral) 3.6-13 mg/kg 4 Paraquat Rat (oral) Quaternary ammonium herbicide (non selective). superoxide generating compound 5 It is the most poisonous naturally occurring substance in the world.A Botulinum Toxin A muscle-nerve paralyzing bacterial toxin, used in minute doses both to treat painful muscle spasms, and as a cosmetic treatment. 6 Clostridium difficile An enterotoxin. Responsible with toxin B for the Pseudomembranous colitis (diarrhea and inflammation)- severe gastrointestinal disease. Toxin A 100 mg/kg משמש בחקלאות קוטל עשבים Rat 1-0.03 ng/kg טרור ביולוגי Mice 500 ng/kg טרור ביולוגי R. Almog Example - cell bioluminescence response Bacteria response to mitomycin C - chemotherapeutic agent R. Almog Effect of toxin concentration: R. Almog •We use Nalidixic Acid (NA) as the model toxin Measurement of the bacterial bioluminescence response to different NA concentrations under static condition (no flow). Here the bacteria were suspended in LB. 30000 Luminescence (RLU) Bacteria + 10 ppm NA Bacteria + 5 ppm NA 25000 Bacteria + 0 ppm NA 20000 No Bacteria + 10 ppm NA 15000 10000 5000 0 0 20 40 60 80 100 120 140 160 180 200 220 Time (min.) R. Almog PDMS Biochip Elastomer, Simple, fast, modular, cheap, reproducible, disposable Single Photon Avalanche photoDiode Microfluidics interface system for the PDMS biochip Inlet Stainless steel Glass cover Inlet SPADs Outlet Outlet PMMA base Glass PDMS PMMA 10mm Bacteria immobilized in Agar R. Almog The biochip layout: Bacteria wells Sample 4 strains 4 channels Positive Negative Constitutive 4 main channels: sample, positive, negative and constitutive. The sample, positive and negative channels consist of four different bacteria strains immobilized in agar. The constitutive channel consists of “Normally On” bacteria. R. Almog Bacteria panel 2 toxins: Nitrogen mustard, Potassium cyanide 2 strains (promoters): nhoA, grpE R. Almog R. Almog Acknoledgments • • • • • • • • • • • Prof. Yosi Shacham Ramiz Daniel Klimentiy Levkov Matan Peer Yaniv Chen Ragini Raj Singh Sefi Vernick Amit Ron Mordechai Aharonson Tsvi Shmilovich Arthur Rabner HUJI - Shimshon Belkin’s group: • Sharon Yagur-kroll • Tal Elad • Sahar Melamed R. Almog R. Almog Biochip array E. Coli bacteria R. Almog Optical sensors - Biacore example R. Almog