(306a) Sequence-Specific Nucleic Acid Detection at <1 Am

A nucleic acid amplification-free, optics-free platform has been created for the detection of bacterial 16S rRNA at <1 aM (10^-18M). This sequence-specific nucleic acid (NA) detector is based on electromechanical signal transduction using a nanopore-based scheme. Our microfluidics device utilizes peptide nucleic acid (PNA) capture probes conjugated to submicron diameter polystyrene beads. Since PNA is charge neutral, the bead-PNA conjugates may be designed to be “charge neutral” until they hybridize target NA, at which point the complex becomes negatively charged and mobile in an electric field. If the electric field is oriented through a nanopore in a glass membrane that is too small for the bead-PNA conjugate with hybridized target to pass through, it will block the pore thereby causing an easily measured step reduction in ionic current. In this way, the selective, NA hybridization event is electromechanically transduced. An opposing electroosmotic flow through the glass pore provides an active mechanism to reduce false positives. No false positive signals were observed with P. putidaRNA when this device was configured to detect E. coli16S rRNA. In fact, this device has proven capable of detecting E. coli16S rRNA at ~0.1 aM against a 1 pM background of RNA from Pseudomonas putida. Finally, the device readily detected E. coliat 10 CFU/mL in a 1 mL sample, also against a million-fold background of viable P. putida. These results suggest that this new technology may serve as the basis for small, portable, low power, and low-cost systems for rapid detection of specific bacterial species in clinical samples, food, and water.