Barcoded Nanowires for Multiplexed Clinical Diagnostics

We propose to develop rapid multiplexed assays for the diagnosis of respiratory pathogens in clinical samples. Many viral and bacterial infections cause respiratory illnesses with very similar symptoms, thus, timely and accurate pathogen identification is important for patient recovery and public health monitoring. Current methods include direct immunofluorescence and cell culture, both of which currently require individual tests for each pathogen. We will use barcoded metallic nanowires as encoded supports for two types of multiplexed diagnostic assays, which will be tested on clinical samples and ultimately transitioned into a clinical setting during the course of the proposed work. Aim 1. Self quenching nanowire beacons. We will take advantage of the electromagnetic effects near metal surfaces (quenching, enhancement, and alteration of fluorescence lifetimes), to produce nanowire beacon probes that enable many assays to be conducted simultaneously in the same sample. The resulting multiplexible, "closed tube" nucleic acid detection assays will require no target labeling or washing, nor any special instrumentation beyond a conventional fluorescence optical microscope, and are therefore amenable to point-of-contact assays such as clinical diagnostics. We propose here to develop a 17-plexed assay for respiratory pathogens. Aim 2. Simultaneous multiplexed amplification and immobilization for pathogen detection. For viruses shed at very low levels, amplification (by growing in culture or by PCR) is necessary for detection. We will overcome current limitations of viral detection by existing methods by introducing multiplexed amplification and immobilization onto barcoded nanowires (MAIN). To accomplish this we will develop robust surface attachment chemistry that can withstand thermocycling and will provide for minimal steric hindrance of hybridization and extension, and will then use these advances to simultaneously direct all PCR products of interest onto different, visually identifiable nanowire supports for sensitive pathogen detection.