Optical interference based microarray imaging for label-free multi-analyte detection

Knowing the binding affinities of biomolecular interactions is crucial for understanding both cell physiology and disease progression. Microarrays are promising tools since they provide high-throughput capability for measuring affinities of thousands of different interactions simultaneously. A considerable amount of effort is expended to improve the technology further to allow a broader utilization. Label-free detection schemes generated a great excitement as they not only simplify the assay process by eliminating the complications of using fluorescent molecules, but they also allow for determining the kinetics of the interactions. Current technologies that provide high-throughput and label-free detection either suffer from low sensitivity or they involve complex procedures limiting their applicability.;In this dissertation, we describe a simple interferometric technique in which optical phase difference as a result of accumulation of biological mass at the binding sites is monitored. A silicon substrate with a thick thermally grown oxide on the top is used as a solid support for spotted probe molecules. The reflectivity of the substrate depends on the wavelength of the light and the exact oxide + biomolecular layer thickness, because of the interference signature. Reflectance spectrum is recorded for the entire surface simultaneously by illuminating the substrate with a tunable laser and capturing the intensity images of reflectance at different wavelengths.;We proved that our technique is quantitative by directly relating the measured optical thickness of the biolayer to the absolute amount of molecules on the surface and verified that the measurement has a linear dynamic range of 4 orders of magnitude. Proof-of-concept experiments demonstrated that this technique can be used for protein-protein, protein-DNA and DNA-DNA interactions. In a set of independent experiments: (i) specific binding of antigens and antibodies were monitored dynamically for hundreds of independent spots of ligands. (ii) the hybridization of single-stranded DNA was detected and the ability to distinguish mismatched strands (corresponding to single nucleotide polymorphism) from complete complementary was shown. (iii) the application of the technique to transcription factor discovery studies was demonstrated. Sensitivity of the current system is ∼ 10 pg/mm2 per spot for an array of 1000 spots, comparable to far more complex state-of-the-art technologies.