dna based biosensors

Good Food Tutorial Athens Nov. 29 th 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. 29 th 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 Introduction Good Food Tutorial Athens Nov. 29 th DNA Stability Hydrogen bonding between base pairs Stacking interaction between bases along axis of double-helix Size and base content and sequence Introduction Good Food Tutorial Athens Nov. 29 th Biosensor Format What is a biosensor? Biorecogniton Layer Transducer Signal (light, current, frequency) Sample Analyte Biosensors are analytical devices which use biological interactions to provide either qualitative or quantitative results. Introduction Good Food Tutorial Athens Nov. 29 th 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 Good Food Tutorial Athens Nov. 29 th 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 Principles of DNA Biosensors Good Food Tutorial Athens Nov. 29 th DNA Biosensor Platform Principles of DNA Biosensors These high density DNA spot arrays (microarrays) can be employed to monitor the presence and/or activity of thousands of genes simultaneously. 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. Good Food Tutorial Athens Nov. 29 th 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 Applications of DNA Arrays Good Food Tutorial Athens Nov. 29 th Design and Fabrication of Bio-platform Considerations  Substrate Type  Glass, Au, SiO 2 , 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. Design & Fabrication Good Food Tutorial Athens Nov. 29 th 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 Design & Fabrication [...]... 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 (3) Crystals Good Food Tutorial Athens Nov 29th (2) DNA Chips Design & Fabrication Limitations of Biosensor Technologies Cost commercial expensive Reproducibility Biosensor platform performance can vary between... 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 OLIGO O O P O- O Amino-terminated probe DNA (H C) 2 6 S NH C NH cross-linker HN C S (CH )3 2 NH Aminosilane anchor Si H3CO...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... 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)... O H Si Si Si Si OCH 3 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... 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... 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 . 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. base mismatches weak hybridization Principles of DNA Biosensors Good Food Tutorial Athens Nov. 29 th DNA Biosensor Platform Principles of DNA Biosensors These high density DNA spot arrays (microarrays) can. step Design & Fabrication Good Food Tutorial Athens Nov. 29 th (1) Electrodes (2) DNA Chips (3) Crystals Typical DNA Based Biosensors Platforms Design & Fabrication Good Food Tutorial Athens Nov.