Microsoft PowerPoint - DNA Tutorial Athens_public version

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 Good Food Tutorial Athens Nov. 29th 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 Good Food Tutorial Athens Nov. 29th Introduction DNA Stability Hydrogen bonding between base pairs Stacking interaction between bases along axis of double-helix Size and base content and sequence Good Food Tutorial Athens Nov. 29th 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. Good Food Tutorial Athens Nov. 29th 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 Good Food Tutorial Athens Nov. 29th 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 Good Food Tutorial Athens Nov. 29th 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. Good Food Tutorial Athens Nov. 29th 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 Good Food Tutorial Athens Nov. 29th 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. Good Food Tutorial Athens Nov. 29th Design & Fabrication Biosensor Basics Immobilisation (a) Adsorption (i) SAM’s (b) Entrapment (c) Crosslinking (d) Covalent bonding Transduction efficiency Dependent on immobilisation method Optimal density Specificity of attachment Probe orientation Good Food Tutorial Athens Nov. 29th 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 Good Food Tutorial Athens Nov. 29th 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. Good Food Tutorial Athens Nov. 29th Design & Fabrication Problems PCR Samples Homogenous cell samples Types RNA quantity Require amplification Oligos Missing bases (incorrect sequence) Limitation in length Expense Good Food Tutorial Athens Nov. 29th 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 Good Food Tutorial Athens Nov. 29th 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 Good Food Tutorial Athens Nov. 29th Design & Fabrication Biosensor Platform Basics Hybridisation Denaturation Hybridisation Buffer Wash step Good Food Tutorial Athens Nov. 29th Design & Fabrication Typical DNA Based Biosensors Platforms (1) Electrodes (2) DNA Chips (3) Crystals Good Food Tutorial Athens Nov. 29th 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 Good Food Tutorial Athens Nov. 29th 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 µm Novel attachment methodology developed at NMRC. Good Food Tutorial Athens Nov. 29th 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. Good Food Tutorial Athens Nov. 29th In-house example using an oxide surface Probe Printing & Attachment: Linker 3’NH2 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. Good Food Tutorial Athens Nov. 29th 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. Good Food Tutorial Athens Nov. 29th 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) 2 3 4 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. Good Food Tutorial Athens Nov. 29th 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