BIOSENSORSDNA & Protein Biosensors
Vishwindera S. Kailey
Vivek Govardhanam
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OUTLINE OF PRESENTATION
Basic Information on DNA.
Introduction to Biosensors
DNA & Protein biosensors
DNA- DNAFET & Southern Blotting
Protein- ELISA
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DNA -101
Deoxyribonucleic Acid
Contains genetic information for the organism.
Structure of DNA
Super coiling
Reading the DNA- Transcription
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DNA- 101
Biological function of DNA
Mutations in DNA
Engineering Applications with
DNA – Genetic Engineering,
Forensics, Bioinformatics.
Biosensors.
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INTRODUCTION TO BIOSENSOR
Definition of biosensors
Basic components of general biosensors.
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BIOSENSORS
History of biosensors
Development in technology.
Mechanism and working of biosensors
Applications in different industries
.
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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.
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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 types8
DETAILED ANALYSIS OF BIOSENSORS
DNA Biosensors
DNAFET Southern Blotting
(MOSFET based)
Protein Biosensors : ELISA
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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
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DNAFET
DNA- MOSFET
Some Background information of MOSFETs
PLEASE READ THE CHAPTER 5 !!!!!
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MOSFET
Metal Oxide Semiconductor Field Effect
Transistor.
Composition of MOSFET
NMOS and PMOS
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MOSFET
Modes of Operation
Saturation or Active mode:
VGS > Vth and VDS > ( VGS - Vth )
Triode or Linear mode:
VGS > Vth and VDS < ( VGS - Vth )
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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)
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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
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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.
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DNAFET
Future of DNAFET
The research and development is continuously
going on to make them (near) real-time
More efficient
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WHAT IS BLOTTING ?
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TYPES OF BLOTTING TECHNIQUES
Blotting Techniques
Southern Blotting
DNA
Northern Blotting
mRNAWestern Blotting
Protein
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SOUTHERN BLOTTING
Sir Edwin Southern
http://www.oxfordtoday.ox.ac.uk/2005-06/v18n2/01.shtml
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METHOD
Step 1: Endonuclease treatment of DNA
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E.COLI, K-12 STRAIN
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METHOD
Step 2: Gel electrophoresis
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AGROSE GEL ELECTROPHORESIS
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METHOD
Step 3: Transfer of DNA to a nitrocellose
membrane
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METHOD
Step 4: Hybridization of ssDNA
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SUMMARY
faculty.ksu.edu.sa 27
APPLICATIONS AND FUTURE
A very valuable tool for recognition of specific
DNA sequences
Early detection of genetic disorders
Regularly used in forensic laboratories
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ANGELMAN SYNDROME (AS) AND PRADER-
WILLI SYNDROME (PWS)
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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)
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BEFORE ELISA…………
RIA- Radioactive Immunoassay
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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.
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PROBLEMS WITH RIA
Building special laboratory facilities
Safety of the lab personnel
Radioactive Waste
Expensive counting device
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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
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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.
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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!
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ANTIGEN-ANTIBODY COMPLEX
An antibody has an affinity to its specific
antigen.
QuickTime™ and a decompressor
are needed to see this picture.
http://www.drsanderson.org/immunity.htm
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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
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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
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IMPORTANT COMPONENTS OF ELISA CONT.
Antibodies (Primary)
Enzyme Linked Antibodies which recognize
Primary Antibodies
Enzyme Substrate
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WHAT HAPPENS IN ELISA
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WHAT HAPPENS IN ELISA
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WHAT HAPPENS IN ELISA
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WHAT HAPPENS IN ELISA
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WHAT HAPPENS IN ELISA
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WHAT HAPPENS IN ELISA
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WHAT HAPPENS IN ELISA
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WHAT HAPPENS IN ELISA
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WHAT HAPPENS IN ELISA
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WHAT HAPPENS IN ELISA
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WHAT HAPPENS IN ELISA
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WHAT HAPPENS IN ELISA
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WHAT HAPPENS IN ELISA
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WHAT HAPPENS IN ELISA
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WHAT HAPPENS IN ELISA
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ELISA CAN BE USED FOR…
Parasitology -- Malaria, trichinosis�
Biochemistry -- fibronectin, laminin, integrins,
muscular dystrophies
Reproductive endocrinology -- total estrogen
and human placental lactogen
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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.
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BEFORE WE BEGIN…
Bariatric Surgery
Surgical procedure used to cure obesity
GI tract is modified so that the intake of nutrients
is reduced
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BEFORE WE BEGIN..
Adiponectin
An adipocyte-specific protein
Insulin-sensitizing
Anti-infamatory
Suppresses the metabolic derangement that result
in Type 2 diabetes and Obesity
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IN THE PAPER
The researchers were checking to see if the
surgery affected the levels of adeponectin
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IN THE PAPER
How did they detect the presence of
adeponectin?
Using a Multemeric Adiponectin ELISA kit
http://newenglandbiolabs.de/de/images/stories/cst_elisa_gross.jpg62
THEY CONCLUDED…
After the bariatric surgery, the levels of adiponectin rose.
Reference (2)
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END OF PRESENTATION
Thank You
Questions!!
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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
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