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PPT - BIOSENSORS DNA _ Protein Biosensors

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					BIOSENSORS
   DNA & Protein Biosensors




                 Vishwindera S. Kailey
                 Vivek Govardhanam
                                         1
OUTLINE OF PRESENTATION
 Basic Information on DNA.
 Introduction to Biosensors

 DNA & Protein biosensors

 DNA- DNAFET & Southern Blotting

 Protein- ELISA




                                    2
 DNA -101

 Deoxyribonucleic Acid
 Contains genetic information for the organism.

 Structure of DNA

 Super coiling

 Reading the DNA- Transcription




                                                   3
    DNA- 101
 Biological function of DNA
 Mutations in DNA

 Engineering Applications with

  DNA – Genetic Engineering,
  Forensics, Bioinformatics.
 Biosensors.




                                  4
    INTRODUCTION TO BIOSENSOR

 Definition of biosensors
 Basic components of general biosensors.




                                            5
    BIOSENSORS
 History of biosensors
 Development in technology.

 Mechanism and working of biosensors

 Applications in different industries

.




                                         6
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
 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
DETAILED ANALYSIS OF BIOSENSORS

            DNA Biosensors

DNAFET                       Southern Blotting
(MOSFET based)

Protein Biosensors : ELISA



                                             9
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
DNAFET

 DNA- MOSFET
 Some Background information of MOSFETs

 PLEASE READ THE CHAPTER 5 !!!!!




                                           11
MOSFET

 Metal Oxide Semiconductor Field Effect
  Transistor.
 Composition of MOSFET

 NMOS and PMOS




                                           12
MOSFET
   Modes of Operation

   Saturation or Active mode:
    VGS > Vth and VDS > ( VGS - Vth )

   Triode or Linear mode:
    VGS > Vth and VDS < ( VGS - Vth )


                                        13
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
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
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
DNAFET

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




                                                 17
WHAT IS BLOTTING ?




                     18
TYPES OF BLOTTING TECHNIQUES


                      Blotting Techniques




  Southern Blotting     Northern Blotting   Western Blotting
        DNA                  mRNA               Protein




                                                               19
SOUTHERN BLOTTING

   Sir Edwin Southern




                         http://www.oxfordtoday.ox.ac.uk/2005-06/v18n2/01.shtml
                                                                                  20
METHOD

   Step 1: Endonuclease treatment of DNA




                                            21
E.COLI, K-12 STRAIN




                      22
METHOD

   Step 2: Gel electrophoresis




                                  23
AGROSE GEL ELECTROPHORESIS




                             24
METHOD

   Step 3: Transfer of DNA to a nitrocellose
            membrane




                                                25
METHOD

   Step 4: Hybridization of ssDNA




                                     26
SUMMARY




               27
          faculty.ksu.edu.sa
APPLICATIONS AND FUTURE

   A very valuable tool for recognition of specific
    DNA sequences

   Early detection of genetic disorders

   Regularly used in forensic laboratories



                                                       28
ANGELMAN SYNDROME (AS) AND PRADER-
WILLI SYNDROME (PWS)




                                     29
WHAT IS AN ASSAY?



      analysis to determine the presence and/or
       quantity of one or more components in a
       solution.
               (http://www.merriam-webster.com/dictionary/assay)




                                                                   30
BEFORE ELISA…………



    RIA- Radioactive Immunoassay




                                    31
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
PROBLEMS WITH RIA

      Building special laboratory facilities

      Safety of the lab personnel

      Radioactive Waste

      Expensive counting device

                                                33
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
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
WHAT DID THEY DO?

   Engvall and Perlman demonstrated quantitative
    measurement of IgG (Antibody) in rabbit serum
    with alakaline phosphatase as the reporter
    label




                 http://www.karstenfaehnrich.de/Immunosensors/Immunoglobulins.jpg   36
PRINCIPLE BEHIND THE ASSAY!




                              37
ANTIGEN-ANTIBODY COMPLEX

   An antibody has an affinity to its specific
    antigen.



                              QuickTim e™ and a
                               decom pres s or
                       are needed to s ee this picture.




                                                          http://www.drsanderson.org/immunity.htm



                                                                                                    38
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.




                                         http://en.wikipedia.org/wiki/Image:Microtiter_plate.JPG


                                                                                                   39
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
IMPORTANT COMPONENTS OF ELISA CONT.

   Antibodies (Primary)

   Enzyme Linked Antibodies which recognize
    Primary Antibodies

   Enzyme Substrate



                                               41
WHAT HAPPENS IN ELISA




                        42
WHAT HAPPENS IN ELISA




                        43
WHAT HAPPENS IN ELISA




                        44
WHAT HAPPENS IN ELISA




                        45
WHAT HAPPENS IN ELISA




                        46
WHAT HAPPENS IN ELISA




                        47
WHAT HAPPENS IN ELISA




                        48
WHAT HAPPENS IN ELISA




                        49
WHAT HAPPENS IN ELISA




                        50
WHAT HAPPENS IN ELISA




                        51
WHAT HAPPENS IN ELISA




                        52
WHAT HAPPENS IN ELISA




                        53
WHAT HAPPENS IN ELISA




                        54
WHAT HAPPENS IN ELISA




                        55
WHAT HAPPENS IN ELISA




                        56
ELISA CAN BE USED FOR…
   Parasitology -- Malaria, trichinosis

   Biochemistry -- fibronectin, laminin, integrins,
    muscular dystrophies

   Reproductive endocrinology -- total estrogen
    and human placental lactogen


                                                       57
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): 129.




                                                                                                58
BEFORE WE BEGIN…

   Bariatric Surgery
     Surgical procedure used to cure obesity
     GI tract is modified so that the intake of nutrients
     is reduced




                                                             59
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
IN THE PAPER

   The researchers were checking to see if the
    surgery affected the levels of adeponectin




                                                  61
IN THE PAPER
       How did they detect the presence of
        adeponectin?
         Using   a Multemeric Adiponectin ELISA kit




                                                                                            62
                                  http://newenglandbiolabs.de/de/images/stories/cst_elisa_gross.jpg
THEY CONCLUDED…
   After the bariatric surgery, the levels of
    adiponectin rose.




                          Reference (2)




                                                 63
END OF PRESENTATION




          Thank You

            Questions!!
                          64
REFERENCES
   Lecture notes 3Ba3, Dr Ian Bruce
   Microelectronics, Sedra and Smith 6e
   http://www.lsbu.ac.uk/biology/enztech/biosensors.html
   http://www.wikispot.info/2010/08/biosensors-types-uses-applications.html
   http://www.prisner.de/Activities/Seminars/OLD/fresenius1.pdf
   http://nar.oxfordjournals.org/content/28/16/3011.full
   http://en.wikipedia.org/wiki/DNA_field-effect_transistor
   http://en.wikipedia.org/wiki/Biosensor




                                                                               65

				
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