biosensorsibruce/courses/ee3ba3_2011/ee3ba… · dnafet real world use of this technology used for...

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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