This work presents several high throughput imaging and analysis techniques performed by fluorescence detection and surface plasmon resonance biosensing. The microarray fabrication methods introduced in this thesis, as well as the DNA functionalization on planar and nanoparticle surfaces, enable and facilitate the real-time study of adsorption events via DNA- DNA hybridization and protein-DNA interaction.
Silica deposited on polyolefin film serves as the base for the development of DNA microarray. Fabrication process involves poly(L-glutamic acid) monolayer to react with amine functional groups functionalized on both the silica surface and the probe DNA strands. Covalently surface-bound probes then hybridize with fluorophore-labeled target DNA. Shrinking the microarray with heat significantly enhances the sensitivity of detection via fluorescence signal amplification.
Polydopamine, a biomimetic polymer, easily and rapidly grows an adhesive layer to link amine-terminated, single-stranded DNA to gold surface. Self-polymerization of polydopamine simply requires mild reagents and reaction condition. The microarray formed with polydopamine attachment technique reserves its stability and specificity to detect DNA and protein targets by surface plasmon resonance imaging. This one-step microarray fabrication approach, when coupled with signal amplification from DNA-modified gold nanoparticles, empowers the detection limit capable of seeing down to 10 pM target concentration.
Electrochemical deposition of polydopamine demonstrates strong control over film localization and thickness. This meticulous polymerization on gold surface at the microscale proves the high toposelectivity of this deposition technique. Amine-modified DNA bound on this polydopamine thin flim hybridizes with fluorophore-tagged DNA to generate fluorescent DNA micropatterns. This electordeposition also forms DNA microarrays used in surface plasmon resonance imaging for DNA-DNA hybridization and DNA-coated gold nanoparticle adsorption measurements.
A 814 nm, near-infrared surface plasmon resonance microscope exemplifies a novel, objective-coupled sensing and imaging system at single-nanoparticle scale. This microscope visualizes individual binding occurrence of gold nanoparticles and polystyrene beads. Adsorption analysis presents the calculated surface plasmon resonance signal (Δ%R) as it varies with particle size. For initial biosensing measurements, the surface plasmon resonance microscopy examines each hybridization-adsorption of DNA-decorated gold nanoparticle on gold surface functionalized with either completely or partially complementary DNA monolayer.