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, sensitive, specific, and cost-effective tool for detection and identification of the category A pathogen, B. anthracis, and a battery of other pathogens with similar clinical syndromic presentation using a single sample.
In Specific Aim 1, we will develop a rapid, high-sensitivity, high-selectivity sequence-specific test based on the optical, hybridization, and catalytic properties of gold nanoparticle DNA conjugates and Raman-active dyes for the simultaneous detection of multiple target polynucleotide molecules.
In Specific Aim 2, we will design, develop, and fabricate a flexible polynucleotide array-based microfluidic platform with cell and analyte handling capabilities for cost-effective, high-throughput, selective detection of captured target DNA and RNA bound to nanoparticles capable of surface-enhanced Raman scattering (SERS). Lastly, in Specific Aim 3, we will establish proof-of-principle and validate the performance of the array-based multiplex detection of conserved polynucleotides derived from anthrax and other infectious pathogens with a similar respiratory syndromic presentation at the point-of-care.
| Root, Brian E; Agarwal, Abhishek K; Kelso, David M et al. (2011) Purification of HIV RNA from serum using a polymer capture matrix in a microfluidic device. Anal Chem 83:982-8 |
| Kim, Eun-Young; Stanton, Jennifer; Korber, Bette T M et al. (2008) Detection of HIV-1 p24 Gag in plasma by a nanoparticle-based bio-barcode-amplification method. Nanomedicine (Lond) 3:293-303 |
| Root, Brian E; Hammock, Mallory L; Barron, Annelise E (2008) Thermoresponsive N-alkoxyalkylacrylamide polymers as a sieving matrix for high-resolution DNA separations on a microfluidic chip. Electrophoresis 29:4677-83 |
| Kim, Eun-Young; Stanton, Jennifer; Vega, Rafael A et al. (2006) A real-time PCR-based method for determining the surface coverage of thiol-capped oligonucleotides bound onto gold nanoparticles. Nucleic Acids Res 34:e54 |
