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Project SummaryThis project is focused on feasibility testing of a new device based on electromechanical signal transduction forthe low-cost (~$10 in electronics), optics-free and amplification-free (e.g., no PCR) detection of DNA/RNA atultralow concentration. The device addresses the projected $12.8B disease diagnostics market and is wellsuited for the detection of bacterial pathogens in body fluids, food and water through the presence of specific16S rRNA sequences. A compelling need persists for rapid (minutes), cost effective, point-of-care (POC)nucleic acid (NA) detection devices for infectious disease diagnostics. The investigators recentlydemonstrated detection of Escherichia coli (E. coli) 16S rRNA against a 106-fold background of Pseudomonasputida RNA at <10-18 M (<1 CFU/10 mL). This ultralow detection limit was achieved using a simple RNAextraction and hybridization protocol that is accomplished in <30 mins at the lab bench. It is likely that anintegrated microfluidic device eventually can be produced for pathogen detection in <15 mins. A key feature ofthe proposed device is the use of peptide nucleic acid (PNA) capture probes, which are uncharged polyamideanalogs to NAs that share the same base chemistry. Since the bead-PNA conjugates are designed to becharge neutral, they do not exhibit electrophoretic movement in the presence of a DC electric field. However,the substantial negative charge acquired upon capture of a target NA sequence makes the hybridizedconjugate mobile. Electrophoresis of the bead-PNA conjugate with hybridized target NA to the mouth of asmaller diameter glass pore causes a significant increase in pore resistance, thereby resulting in a strong,sustained drop in measured ionic current. Nonspecifically bound NA is removed from the bead conjugate inthe strong electric field in the pore mouth resulting in no sustained signal. Further, the opposing electroosmoticflow through the glass pore sweeps bead-PNA conjugates without hybridized target away from the pore mouth.In such a way, this simple conductometric device gives a highly selective, binary response signaling thepresence or absence of the target NA (and associated pathogen). This project focuses on 2 Aims: 1)Demonstration of selective detection of E. coli in human urine against a background of normal flora at astatistical performance level competitive or better than existing commercial devices and 2) Development andtesting of the inexpensive, device-associated electronics that are suitable for manufacturing. Successfulachievement of these Aims will substantiate the potential of the novel device for integration into a microfluidicsystem for detection of Neisseria gonorrhoeae, a CDC top-three public health threat, and Chlamydiatrachomatis in human urine. Further, this project will illustrate the potential of the technology for developmentof generally applicable, inexpensive POC devices for bacterial pathogens in body fluids, food and water.

Leclair, Timothy
Electronucleics, Inc.
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