Biosensors ביו-חיישנים – עקרונות ויישומים מרפואה ביתית ועד לניטור סביבתי

1. Biosensorsביו-חיישנים – עקרונות ויישומים מרפואה ביתית ועד לניטור סביבתי Dr. Ronen Almog מכון טכנולוגי חולון

2. Outline • Biosensors • Detection principles: • Electrochemical • Optical • Mechanical • Lab on a chip/BioMEMS • Examples: • Diabetes - glucose monitoring • Water toxicity detection

3. Elements of a biosensor

4. 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.

5. Biosensors applications and importance • Medical/health monitors • Homeland security • Pharmaceutical industry • Food industry • Simplicity • Miniaturization Biosensors Features • Selectivity • Rapid • Sensitivity

6. Biological recognition elements • Enzymes and their substrates • Antibodies and their antigens • Nucleic acids and their complementary sequences • Whole cell A - T G - C

7. On Size and Scale

8. 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.

9. 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.

10. Antibody structure

11. 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.

12. Transducers - sensing methods in biosensors

13. MEMS - MicroElectroMechanichal sensors Use: Bioassay of prostate-specific antigen (PSA)

14. Optical sensors - Biacore example

15. Reasons for Miniaturization

16. 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)

18. Lab-on-a-chip Burns et al. Science 282, 484, 1998

19. 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

20. Biochip - microfluidics Key Attributes of Biochips 1. Small length scale 2. Small thermal mass 3. Laminar flow 4. High surface-to-volume ratio

21. The Oxygen electrode Clark’s Glucose electrode

22. Biosensors History

23. Technology evolution 6 analyses from a drop of blood in about one minute.

24. 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 http://www.diabetesuffolk.com/Managing%20Diabetes/Meters/LifeScan%20Ultrasmart.asp

25. הינשוף Alcohol Test – Drager Alcotest 7110 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

26. 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

27. Example: Toxichip

28. 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

29. 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.

30. The goal To develop a portable system that can detect the presence of unknown acute toxicity chemicals in drinking water within 20 minutes.

31. 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.

32. E. Coli bacteria Biochip array

33. Bioluminescence The emission of light by a living organism as the result of a chemical reaction. Chemical energy is converted to light energy. Insects Marine organisms Fungee Emission spectrum: Visible - blue-green (490-500 nm) Bacteria

34. Product: Oxyluciferin + light Luciferin (substrate) Luciferase (enzyme) + + O2 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.

35. 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

36. 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

37. Toxins list

38. Example - cell bioluminescence response Bacteria response to mitomycin C - chemotherapeutic agent

39. Effect of toxin concentration:

40. 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.

41. SPADs Outlet Stainless steel Inlet Inlet Outlet Glass cover Glass PMMA base PDMS PMMA Bacteria immobilized in Agar 10mm PDMS Biochip Elastomer, Simple, fast, modular, cheap, reproducible, disposable Single Photon Avalanche photoDiode Microfluidics interface system for the PDMS biochip

42. The biochip layout: 4 strains 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.

43. Bacteria panel 2 toxins: Nitrogen mustard, Potassium cyanide 2 strains (promoters): nhoA, grpE

45. Acknoledgments HUJI - Shimshon Belkin’s group: • Sharon Yagur-kroll • Tal Elad • Sahar Melamed • Prof. Yosi Shacham • Ramiz Daniel • Klimentiy Levkov • Matan Peer • Yaniv Chen • Ragini Raj Singh • Sefi Vernick • Amit Ron • Mordechai Aharonson • Tsvi Shmilovich • Arthur Rabner

46. The end Thank you!

47. E. Coli bacteria Biochip array

48. Optical sensors - Biacore example