DNA & Protein Biosensors Vishwindera S. Kailey Vivek Govardhanam

Transcription

1 BIOSENSORS DNA & Protein Biosensors Vishwindera S. Kailey Vivek Govardhanam 1

2 OUTLINE OF PRESENTATION Basic Information on DNA. Introduction to Biosensors DNA & Protein biosensors DNA- DNAFET & Southern Blotting Protein- ELISA 2

3 DNA -101 Deoxyribonucleic Acid Contains genetic information for the organism. Structure of DNA Super coiling Reading the DNA- Transcription 3

4 DNA- 101 Biological function of DNA Mutations in DNA Engineering Applications with DNA Genetic Engineering, Forensics, Bioinformatics. Biosensors. 4

5 INTRODUCTION TO BIOSENSOR Definition of biosensors Basic components of general biosensors. 5

6 BIOSENSORS History of biosensors Development in technology. Mechanism and working of biosensors Applications in different industries. 6

7 REAL WORLD BIOSENSORS Efficient : Time and Money Minimal maintenance Meet the needs of potential market in terms of standards and production Continuous Research and Development of new applications in different and new fields. 7

8 TYPES OF BIOSENSORS Biosensors are characterized based on type of property used to analyze a reaction. A bio-sensing element is closely integrated within a transducing microsystem. Some types are: Thermometric : heat detection Piezoelectric : detects change of Mass (Acoustic) Optical : light detection Amperometric : analysis of current flow Many other types 8

9 DETAILED ANALYSIS OF BIOSENSORS DNA Biosensors DNAFET (MOSFET based) Southern Blotting Protein Biosensors : ELISA 9

10 DNA BIOSENSORS Generally speaking it is simply based on nucleic acid recognition (actually not that simple!!) Mechanism and working of DNA biosensors Detection of DNA damage and interactions 10

11 DNAFET DNA- MOSFET Some Background information of MOSFETs PLEASE READ THE CHAPTER 5!!!!! 11

12 MOSFET Metal Oxide Semiconductor Field Effect Transistor. Composition of MOSFET NMOS and PMOS 12

13 MOSFET Modes of Operation Saturation or Active mode: V GS > V th and V DS > ( V GS - V th ) Triode or Linear mode: V GS > V th and V DS < ( V GS - V th ) 13

14 DNAFET It is a specialized FET that acts as biosensor that uses the field effect due to partial charges of DNA The structure is different from MOSFET as the gate structure is replaced by layer of immobilized ssdna ( single strand DNA)molecules acting as surface receptors. (DNA) 14

15 DNAFET Mechanism hybridization of DNA strands leads to current transport through semiconductor transducer In common MOSFET DNA chip, the DNA with negative charge will vary the gate potential of MOSFET and thus can be considered as a response 15

16 DNAFET Real world use of this technology Used for DNA detection Detection of Single nucleotide polymorphism Detecting cancer marker DNA (CK20) The main reason DNAFET is better than the previously used optical detection methods as it doesn t require labeling of molecules Disadvantages includes: Debye length increases in physiological conditions, availability of the DNAFET. 16

17 DNAFET Future of DNAFET The research and development is continuously going on to make them (near) real-time More efficient 17

18 WHAT IS BLOTTING? 18

19 TYPES OF BLOTTING TECHNIQUES Blotting Techniques Southern Blotting DNA Northern Blotting mrna Western Blotting Protein 19

20 SOUTHERN BLOTTING Sir Edwin Southern 20

21 METHOD Step 1: Endonuclease treatment of DNA 21

22 E.COLI, K-12 STRAIN 22

23 METHOD Step 2: Gel electrophoresis 23

24 AGROSE GEL ELECTROPHORESIS 24

25 METHOD Step 3: Transfer of DNA to a nitrocellose membrane 25

26 METHOD Step 4: Hybridization of ssdna 26

27 SUMMARY 27 faculty.ksu.edu.sa

28 APPLICATIONS AND FUTURE A very valuable tool for recognition of specific DNA sequences Early detection of genetic disorders Regularly used in forensic laboratories 28

29 ANGELMAN SYNDROME (AS) AND PRADER- WILLI SYNDROME (PWS) 29

30 WHAT IS AN ASSAY? analysis to determine the presence and/or quantity of one or more components in a solution. ( 30

31 BEFORE ELISA RIA- Radioactive Immunoassay 31

32 WHAT WAS RIA USED FOR? To measure the concentration of a component biological fluid such as serum/urine using the reaction of an antibody to its antigen Practical Example - Measuring the Insulin concentration in Blood. 32

33 PROBLEMS WITH RIA Building special laboratory facilities Safety of the lab personnel Radioactive Waste Expensive counting device 33

34 ELISA - WHAT IS IT? First of all it is an assay, i.e. used for determining presence or to quantify a component It has components which are linked to an enzyme thereby, Enzyme Linked Uses the binding of an antigen or antibody to a solid support as a technique 34

35 ELISA - HOW IT ALL BEGAN Two scientists, Engvall E. and Perlmann P. Described ELISA in their paper, Enzyme-Linked immunosorbent assay(elisa). Quantitative assay of immunoglobulin G. 35

36 WHAT DID THEY DO? Engvall and Perlman demonstrated quantitative measurement of IgG (Antibody) in rabbit serum with alakaline phosphatase as the reporter label 36

37 PRINCIPLE BEHIND THE ASSAY! 37

38 ANTIGEN-ANTIBODY COMPLEX An antibody has an affinity to its specific antigen. QuickTim e and a decom pressor are needed to see this picture. 38

39 IMPORTANT COMPONENTS OF ELISA Microtiter Plates Also known as microplates A flat plate containing small wells Analogous to small test tubes Antigen or antibodies are bound to the solid phase support QuickTime and a decompressor are needed to see this picture. 39

40 ELISA- TYPES There are two kinds of ELISA techniques Direct & Indirect In direct technique the antigen is bound to the solid-phase support In indirect technique the antibody is bound to the solid-phase state 40

41 IMPORTANT COMPONENTS OF ELISA CONT. Antibodies (Primary) Enzyme Linked Antibodies which recognize Primary Antibodies Enzyme Substrate 41

42 WHAT HAPPENS IN ELISA 42

43 WHAT HAPPENS IN ELISA 43

44 WHAT HAPPENS IN ELISA 44

45 WHAT HAPPENS IN ELISA 45

46 WHAT HAPPENS IN ELISA 46

47 WHAT HAPPENS IN ELISA 47

48 WHAT HAPPENS IN ELISA 48

49 WHAT HAPPENS IN ELISA 49

50 WHAT HAPPENS IN ELISA 50

51 WHAT HAPPENS IN ELISA 51

52 WHAT HAPPENS IN ELISA 52

53 WHAT HAPPENS IN ELISA 53

54 WHAT HAPPENS IN ELISA 54

55 WHAT HAPPENS IN ELISA 55

56 WHAT HAPPENS IN ELISA 56

57 ELISA CAN BE USED FOR Parasitology -- Malaria, trichinosis Biochemistry -- fibronectin, laminin, integrins, muscular dystrophies Reproductive endocrinology -- total estrogen and human placental lactogen 57

58 HOW IS ELISA USEFUL IN THE RESEARCH ARTICLE?(APPLICATION) Hug C, Lodish HF (2005). "The role of the adipocyte hormone adiponectin in cardiovascular disease". Current opinion in pharmacology 5 (2):

59 BEFORE WE BEGIN Bariatric Surgery Surgical procedure used to cure obesity GI tract is modified so that the intake of nutrients is reduced 59

60 BEFORE WE BEGIN.. Adiponectin An adipocyte-specific protein Insulin-sensitizing Anti-infamatory Suppresses the metabolic derangement that result in Type 2 diabetes and Obesity 60

61 IN THE PAPER The researchers were checking to see if the surgery affected the levels of adeponectin 61

62 IN THE PAPER How did they detect the presence of adeponectin? Using a Multemeric Adiponectin ELISA kit 62

63 THEY CONCLUDED After the bariatric surgery, the levels of adiponectin rose. Reference (2) 63

64 END OF PRESENTATION Thank You Questions!! 64

65 REFERENCES Lecture notes 3Ba3, Dr Ian Bruce Microelectronics, Sedra and Smith 6e