390755 Sequence-Specific DNA Detection at 10 Fm By Electromechanical Signal Transduction

Tuesday, November 18, 2014: 10:45 AM
408 (Hilton Atlanta)
Leyla Esfandiari, Bioengineering, Unversity of California Los Angeles, Los Angeles, CA, Jacob Schmidt, Bioengineering, University of California Los Angeles, Los Angeles, CA and Harold G. Monbouquette, Chemical and Biomolecular Engineering, University of California, Los Angeles, Los Angeles, CA

The development of a rapid, sensitive, and cost-effective nucleic acid detection platform is highly desired for a range of diverse applications including in patient screening during epidemics, oncological status assessment during surgery, detection of food contaminants, and biowarfare agent detection. We previously described a simple, PCR-free, optics-free, and potentially low-cost device for sequence-specific nucleic acid (NA) detection. The key element of the system is a peptide nucleic acid (PNA) capture probe conjugated to polymer spheres. PNA oligomers are uncharged analogs to DNA and RNA that share the same base chemistry and hybridize strongly to complementary NA sequences. Since the sphere-PNA conjugates carry little or no charge, they do not exhibit electrophoretic movement in response to a steady, DC field imposed through a pore. However, the substantial negative charge acquired upon capture of a target NA sequence makes the hybridized conjugate electrophoretically mobile. If the pore diameter is smaller than that of the spheres, the charged conjugate carrying the hybridized PNA and target NA would be expected to block the pore and significantly decrease its conductance, thereby causing a very strong, sustained drop in measured current. In such a way, the selective PNA-NA hybridization signal is transduced electromechanically, and the system would be expected to give an essentially binary response signaling the absence or presence of a target NA. In proof of principle work with our device, we detected 20-mer DNA oligomers with a 10 pM detection limit. Here, we demonstrate the testing of our device with longer DNA strands, and we describe the resulting improvement in the limit of detection (LOD). We examined the detection of target DNA oligomers of 110, 235, 419, and 1613 nucleotides at 1 pM–1 fM concentrations, finding that the LOD decreased as DNA length increased, with 419 and 1613 nucleotide oligomers detectable down to 10 fM. In addition, no false positive responses were obtained with non-complementary, control DNA fragments of similar length. Also, we successfully detected target fragments against a DNA background simulating the non-complementary genomic DNA present in real samples. The 10 fM LOD achieved with a ~1600 base analyte suggests our device would be useful clinically for identification of bacteria using similar size, species specific 16S rRNA as the target. Results to date have been acquired with a micro-scale device; we currently are creating and testing nano-scale devices that should provide further performance enhancements.

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