Talk Outline

Introduction Principles of DNA based sensors Applications of DNA based biosensors Bioplatform Design & Fabrication Considerations Example of Modifying an Oxide Surface with DNA Summary

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Introduction

DNA Structure
Contains genetic material for all living organisms Double helix structure Made of four different nucleotides-A,T,C,G Sequences of nucleotides define proteins Each sequence is a gene

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Introduction

DNA Stability
Hydrogen bonding between base pairs

Stacking interaction between bases along axis of double-helix Size and base content and sequence
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Introduction

Biosensor Format What is a biosensor?

Sample
Biorecogniton

Transducer

Signal (light, current,

frequency)

Layer

Analyte

Biosensors are analytical devices which use biological interactions to provide either qualitative or quantitative results.
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Introduction

Biosensor Configuration
Configuration
Can be developed from any basic sensor by adding a biological component. Usually incorporates a biomembrane

Transduction
Electrical Optical Mechanical, mass acoustic Thermal Chemical Magnetic
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Principles of DNA Biosensors

Working Principle of a DNA Biosensor

Nucleic acid hybridization ---rennealing b/w the ssDNAs from different sources
Perfect match ---stable dsDNA, strong hybridization One or more base mismatches ----weak hybridization

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Principles of DNA Biosensors

DNA Biosensor Platform

Examples of DNA based Bio-platforms

Microarrays/ Biochips 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 of thousands of genes simultaneously.
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Applications of DNA Arrays

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 & Fabrication

Design and Fabrication of Bio-platform

Considerations
Substrate Type
Glass, Au, SiO2, Plastic, Metal, nylon, etc.
Porus, planar, etc

Probe Immobilisation method

Adsorption, Covalent, Entrapment,

Patterning on the surface

Lithography, insitu synthesis, printing

Hybridisation
Tm, Wash stringency, etc

Detection
Optical, electrochemical, etc.

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Design & Fabrication

Biosensor Basics Immobilisation

(a) Adsorption (i) SAMs (b) Entrapment (c) Crosslinking (d) Covalent bonding Transduction efficiency Dependent on immobilisation method Optimal density Specificity of attachment Probe orientation

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Design & Fabrication

Immobilization Chemistries
Thiolated DNA for self assembly onto gold transducers Covalent linkage to the gold surface via functional alkanethiol-based monolayers Use of biotinylated DNA for complex formation with a surface-confined avidin or strepavidin Covalent (carbodiimide) coupling to functional groups on carbon electrodes Simple adsorption onto carbon surfaces
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Design & Fabrication

Methods of Improving Sensitivity

Bioconjugated Nanoparticles

DNA Dendrimers
Schematic drawing showing the hybridization detection at the dendrimer. The probe is attached to the core dendrimer by complementary oligonucleotide of the outer arms.
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Design & Fabrication

Problems PCR Samples

Homogenous cell samples Types RNA quantity Require amplification

Oligos
Missing bases (incorrect sequence) Limitation in length

Expense
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Design & Fabrication

On-Chip Oligo Synthesis Process Steps

Deprotection
Chemical removal of DMT

Coupling
Addition of new base to active sites

Oxidation
Stablise the phosphoramidite bond

Capping
protect all the unreacted sites

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Design & Fabrication

Array Fabrication Manufacturing

Mechanical
Spotting, Soft lithography
PCR & Oligo low density

Inkjet
Spotting, Oligo Synthesis
PCR & Oligo low & density

Photolithography
Oligonucleotdie Synthesis
Oligo High density

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Design & Fabrication

Biosensor Platform Basics

Hybridisation
Denaturation Hybridisation Buffer Wash step

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Design & Fabrication

Typical DNA Based Biosensors Platforms

(1) Electrodes (2) DNA Chips

(3) Crystals

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Design & Fabrication

Limitations of Biosensor Technologies Cost

commercial expensive

Reproducibility
Biosensor platform performance can vary between batches

Sensitivity
poor signal to noise ratio

Reusability
single use only
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In-house example using an oxide surface

Example of DNA Bio-platform Fabrication

Key process steps: Substrate selection and clean Anchor layer formation at surface Amino - DNA probe monolayer attachment Hybridisation to DNA probe monolayer PDITC
cross-linker
HN C S NH H3CO O H Si (CH )3 2 Si OCH 3 O Si H O Si O H Si

OLIGO
O O P O (H C) 2 6 S C NH NH O-

Amino-terminated probe DNA

Aminosilane anchor

Silanol groups at glass surface

700 m

Novel attachment methodology developed at NMRC.

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In-house example using an oxide surface

Printing a Probe DNA Microarray

Clean Substrate

Anchor Layer

Oligo Deposition

Oligos Attached Typical microarray fabrication process. SpotBot microarray spotter with Stealth microspotting pins.

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In-house example using an oxide surface

Probe Printing & Attachment: Linker 3NH2

PDITC NH2 terminated PDITC Nonterminated No PDITC No PDITC NonNH2 terminated terminated

Following Deposition and Attachment

Following Wash
50 m

Demonstrated the importance of both linker molecule and amino modified DNA.

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In-house example using an oxide surface

DNA Probe Attachment & Hybridisation

Oligo (Dye-labelled)

Oligo (unlabelled)

Oligo with hybridised complement

Bifunctional linker Silane anchor Oxide substrate (i) Verification of attachment of oligo probe layer. (ii) Verification of hybridisation to oligo probe layer.

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In-house example using an oxide surface

Microarray Selectivity
Microarray Fabrication (a)
1 = Control oligo modified 2 = Oligo A 3 = Oligo B 4 = Control non-modified
1
(a)

Hybridisation Cycle 1 (b)

1 = Control oligo modified 2 = Oligo A Oligo A 3 = Oligo B 4 = Control non-modified

(b)

Hybridisation Cycle 2 (c)

1 = Control oligo modified 2 = Oligo A 3 = Oligo B Oligo B 4 = Control non-modified

(c)

100 m

Demonstration of microarray selectivity and reusability.

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Conclusion

Summary Overview
Each application has its own challenges Design and characterisation depends on the application Immobilisation chemistries more commonly used How to design and manufacture a DNA array platform Applications
Good Food Tutorial Athens Nov. 29th