Detection of Category A Pathogens by Gold Nanoparticles

DESCRIPTION (provided by applicant): Recent deliberate exposure of civilian population of the United States populace to Bacillus anthracis spores uncovered an unmet need for tests to rapidly diagnose disease caused by this bacterium. A major challenge for rapid and accurate pathogen detection is the development of methods that do not rely on the polymerase chain reaction (PCR) or comparable target- or signal-amplification systems to minimize instrumentation and reagent needs and enable point-of-care or field testing. We propose to develop a highly selective sequence-specific, chip-based method for detection of DNA from B. anthracis by exploiting the unique properties of DNA-gold nanoparticle conjugates. The proposed methodology promises to be far simpler than current methods and obviates the need for large-scale analytical instrumentation and trained personnel. Preliminary data show that detection assays based on these novel nanostructures are substantially more sensitive and selective than conventional assays based on fluorophore-labeled DNA probes. Ultimately, the assay will be as accurate and sensitive as PCR-based assays. By combining gold nanoparticles with Raman spectroscopic fingerprints, we can effectively distinguish dissimilar polynucleotide sequences of multiple infectious pathogens in the same sample without target sequence amplification. In contrast to fluorescence-based hybridization, gold nanoparticle probes labeled with oligonucleotide and Raman-active dyes offer several advantages, including higher potential for multiplexing and orders-of-magnitude higher sensitivity and selectivity. We propose to implement this assay in a simple microfluidic device with integrated capacity for specimen processing, DNA/RNA purification, separation, hybridization, and signal detection, thus offering the possibility for integrated, high-throughput screening in a "lab-on-a-chip," with easily-read outputs. The overall goal of this proposal is to create a rapid, sensitiv