DNA Based Bio Sensors

8/6/2019 DNA Based Bio Sensors

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Good Food TutorialAthens Nov. 29th

Talk OutlineIntroduction

Principles of DNA based sensors

Applications of DNA based biosensors

Bioplatform Design & FabricationConsiderations

Example of Modifying an Oxide Surface withDNA

Summary


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DNA StructureContains 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

Introduction


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DNA Stability

Hydrogen bonding between base pairs

Stacking interaction between bases along axisof double-helix

Size and base content and sequence

Introduction


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Biosensor Format

What is a biosensor?

Biorecogn

iton

Layer

Transdu

cer

Signal (light, current,frequency)

Sample

Analyte

Biosensors are analytical devices which use biologicalinteractions to provide either qualitative or quantitative results.

Introduction


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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

Introduction


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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 basemismatches

----weak hybridization

Principles of DNA Biosensors


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DNA Biosensor PlatformPrinciples of DNA Biosensors

These high density DNA spot arrays(microarrays) can be employed to

monitor the presence and/or activityof thousands of genessimultaneously.

Examples of DNA based Bio-platforms

Microarrays/ Biochips

Are localised deposition andattachment of spots of DNAstrands at a passive or activesubstrate, e.g., glass or silicon

chip, respectively.


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ApplicationsGene 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

Applications of DNA Arrays


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Design and Fabrication of Bio-platform

Considerations Substrate Type

Glass, Au, SiO2, Plastic, Metal, nylon, etc.

Porus, planar, etc

Probe Immobilisation methodAdsorption, Covalent, Entrapment,

Patterning on the surface Lithography, insitu synthesis, printing

Hybridisation Tm, Wash stringency, etc

Detection Optical, electrochemical, etc.

Design & Fabrication


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Immobilization Chemistries

Thiolated DNA for self assembly onto goldtransducers

Covalent linkage to the gold surface viafunctional alkanethiol-based monolayers

Use of biotinylated DNA for complex

formation with a surface-confined avidin orstrepavidin

Covalent (carbodiimide) coupling to functional

groups on carbon electrodes

Simple adsorption onto carbon surfaces



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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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Problems

PCR Samples Homogenous cell samples

Types RNA quantity

Require amplification

Oligos Missing bases (incorrect sequence)

Limitation in length

Expense



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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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Array Fabrication

Manufacturing

Mechanical

Spotting, Soft lithography PCR & Oligo

low density

Inkjet

Spotting, Oligo Synthesis PCR & Oligo

low & density

Photolithography

Oligonucleotdie Synthesis OligoHigh density



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Biosensor Platform Basics

Hybridisation

Denaturation

Hybridisation Buffer

Wash step



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(1) Electrodes (2) DNA Chips

(3) Crystals

Typical DNA Based Biosensors PlatformsDesign & Fabrication


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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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Example of DNA Bio-platform Fabrication

Silanol groupsat glass surfaceSi Si

OH

Si

OH

Si

OH

C

NH

S

PDITCcross-linker

NH

C

S

O

Si

OCH3H3CO

Aminosilane anchor(CH2)3

NH

Novel attachment methodology developed at NMRC.

700 m

Key process steps:

Substrate selection and clean

Anchor layer formation at surface

Amino - DNA probe monolayerattachment

Hybridisation to DNA probemonolayer

OLIGO

PO

O

O-

NH

(H2C)6

O Amino-terminated

probe DNA

In-house example using an oxide surface


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SpotBot microarray spotter withStealth microspotting pins.

Printing a Probe DNA Microarray

Clean Substrate

Anchor Layer

Oligo Deposition

Oligos Attached

Typical microarray fabrication process.



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Probe Printing & Attachment: Linker 3NH2

PDITC

NH2 -

terminated

PDITC

Non-

terminated

No PDITC

NH2 -

terminated

No PDITC

Non-

terminated

Following Deposition

and Attachment

Following

Wash

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

50m



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(i) Verification of attachment (ii) Verification of hybridisation

of oligo probe layer. to oligo probe layer.

DNA Probe Attachment & Hybridisation

Bifunctional linker

Silane anchor

Oxide substrate

Oligo(unlabelled)

Oligo withhybridised

complement

Oligo(Dye-labelled)



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Microarray Selectivity

Microarray Fabrication (a)1 = Control oligo modified

2 = Oligo A

3 = Oligo B

4 = Control non-modified

Hybridisation Cycle 1 (b)1 = Control oligo modified

2 = Oligo A Oligo A

3 = Oligo B4 = Control non-modified

Hybridisation Cycle 2 (c)1 = Control oligo modified

2 = Oligo A

3 = Oligo B Oligo B

4 = Control non-modified

Demonstration of microarray selectivity and reusability.

1 2 3 4

(a)

(c)

(b)

1 2 3 4

(a)

(c)

(b)

100m



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Summary Overview

Each application has its own challenges

Design and characterisation depends on theapplication

Immobilisation chemistries more commonlyused

How to design and manufacture a DNA arrayplatform

Applications

Conclusion

Documents

DNA Based Bio Sensors