We propose to use a new luminescent material, called quantum dots, to extend the detection sensitivity of gene expression microarrays by almost 3 orders of magnitude. Fluorescence from single quantum dots can easily be detected using simple instrumentation. By using quantum dot labels in expression assays, we aim to detect the presence of individual hybridization events at extremely low sample concentrations. At the end of phase 2, we will have a fully functional assay system, including reagents, protocols and an optical reader, capable of detecting bound target on the single molecule level. The goal of phase 1 is to demonstrate the feasibility of single copy detection on microarrays. We will analyze and eliminate the sources of autofluorescence and optimize our detection system to maximize the detection of single quantum dots. We will then perform a simple, non-hybridization assay between streptavidin:quantum dot bioconjugates and biotinylated DNA bound to a microarray surface. The concentration of biotin within each array spot will be varied across a wide range, including concentrations where bound quantum dots can be directly counted, one at a time. Two different quantum dot colors will be independently developed and tested. Results from these experiments will allow us to optimize procedures for bioconjugation, nonspecific binding and detection of quantum dots in phase 2, where we propose to use these techniques to perform actual multiplexed hybridization assays with single hybridization sensitivity. The development of a single molecule counting assay will have significant medical and commercial value since it has the capability of extending both the sensitivity and dynamic range of expression microarrays and other biological assays. PROPOSED COMMERCIAL APPLICATIONS: With the Human Genome project, gene expression assays are becoming of significant commercial value. At the moment, however, many important genes are expressed at levels below our current detection limit. Single molecule detection represents the ultimate limit of detection sensitivity. By developing a product capable of extending the sensitivity of expression assays into the regime where single bound target molecules can be detected, we will be providing a significant technological breakthrough for laboratories and institutions involved in gene expression analysis.
