Tapping mode AFM for real-time analysis of DNA hybridization on microarrays
EMSL Project ID
1692
Abstract
Recent advances in AFM make visualization of surface/DNA and DNA/protein interactions possible. Presently, we are designing nucleic acid based microarrays for the detection of bacterial pathogens and bacteria that are important to bioremediation. The microarray surface is coated with a self-assembled monolayer (SAM) which binds oligonucleotide capture probes either by covalent bonding or electrostatic attraction depending on the organo silane monolayer. The microarray is then hybridized with the DNA of interest and complementary target strands are detected via a fluorescent dye. Uniform deposition and correct orientation of SAM at the surface is needed to promote efficient spatial binding of oligos deposited onto the surface as 150 mnm spots. Arrays will be prepared on silicon wafers with epoxy and amino silane surfaces, both with and without linkers. AFM operated in the tapping mode will enable direct visualization of the surface morphology, enabling a determination of the density and uniformity of oligonucleotide ocverage. We also plan to use AFM flow cell attachment to monitor real-time DNA hybridization onto the oligonucleotide surface. Both low and high resolution AFM imaging of 150 micrometer array elements will be investigated. This will provide information about the effect of steric hindrance and the surface morphology required for DNA hybridization. The microarray hybridization asays are performed at room termperature with no hazardous reagents. Operation of the AFM will require initial training on the use of the equipment, but additional analysis will be perfromed with limited to no direct assistance.
Project Details
Project type
Exploratory Research
Start Date
1999-10-01
End Date
2002-06-01
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members