dna based biosensors

Talk Outline Introduction # Principles of DNA based sensors # Applications of DNA based biosensors @ Bioplatform Design & Fabrication Good Food Tutorial In Lt Society Athens Nov...

Talk Outline

Introduction

# Principles of DNA based sensors

# Applications of DNA based biosensors

@ Bioplatform Design & Fabrication

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Technologies

# Contains genetic material for all living organisms

# Double helix structure

@ Made of four different Molecule: @2

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@ Hydrogen bonding between base pairs

@ Stacking interaction between bases along axis

of double-helix

@ Size and base content and sequence

Biosensors are analytical devices which use biological

interactions to provide either qualitative or quantitative results

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Principles of DNA Biosensors : yy `

Working Principle of a DNA Biosensor

# Nucleic acid hybridization

-rennealing b/w the ssDNAs from different sources

Are localised deposition and

attachment of “spots” of DNA

Strands at a passive or active

substrate, e.g., glass or silicon

chip, respectively

These high density DNA spot arrays

(microarrays) can be employed to

monitor the presence and/or activity

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, Applications

@ Gene expression

» Usually Looking for RNA expression

» Differences between cells

= Differences in time

@ Polymorphisms

=» Change in base pairs

=» Single base pair change

#@ Comparative genomic hybridisation

» Compare entire genome

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Design and Fabrication of Bio-platform Ê

@ Considerations

» Substrate Type

¢ Glass, Au, SiO,, Plastic, Metal, nylon, etc

= Porus, planar, etc

„m Probe Immobilisation method

¢ Adsorption, Covalent, Entrapment,

» Patterning on the surface

¢ Lithography, insitu synthesis, printing

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Dependent on immobilisation method

Optimal density Specificity of attachment Probe orientation

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@ Use of biotinylated DNA for complex

formation with a surface-confined avidin or

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Technologies

Spot test Silver-Staining PV scattered

eon ty l a Science, 2000 White light

The probe is attached to the core dendrimer

Surface & TT r8 DY comnplernen(ary oligonucleotide of the

Immobilised dendrimer outer arms

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On-Chip Oligo Synthesis

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Oligo

High density

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‘Technologies

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Design & Fabrication : Ị

Typical DNA Based Biosensors Platforms §&

%, Preparation of DNA from fooc

sample after brief cultivatio

Design & Fabrication : »"

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Example of DNA Bio-platform Fabrication §

Key process steps:

— Substrate selection and clean — Anchor layer formation at surface OLIGO =P

— Amino - DNA probe monolayer ue£° — Amino-terminated

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In-house example using an oxide surface i IP

Printing a Probe DNA Microarray

Oligos Attached tin in:

SpotBot™ microarray spotter with

Typical microarray fabrication process Stealth™ microspotting pins

Oligo Deposition

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In-house example using an oxide surface 7s

Probe Printing & Attachment: Linker 3°NH

terminated terminated terminated terminated

Demonstrated the importance of both linker molecule and amino modified DNA

Following Deposition and Attachment

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(Dye-labelled) (unlabelled) ) hybridised

complement

Bifunctional linker

Oxide substrate / /

(1) VeriÍication of attachment (11) Verification of hybridisation

of oligo probe layer to oligo probe layer

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Microarray Fabrication (a) (a)

1 = Control oligo modified 2= Oligo A

3 = Oligo B

4 = Control non-modified

Hybridisation Cycle ] (b) (b)

1 = Control oligo modified

2 = Oligo A Oligo A’

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@ Each application has its own challenges

# Design and characterisation depends on the