473064 Nanopore-Based Sequence-Specific Nucleic Acid Detection at 1aM
Previously, we introduced a first-generation device based on a drawn pipette tip as the “pore” with a 10 fM limit of detection (LOD). In this system, the bead-PNA conjugates and the nucleic acid-containing sample were placed inside the buffer-filled drawn glass pipette tip and an electric field was applied over the length of the pipette. This sensor could distinguish between complementary and non-complementary NA sequences and no false positives were observed with 1613-base DNA oligomers as targets. [1, 2]
In current work, we replaced the drawn glass pipette tip with a glass nanopore in a thin (1 μm or thinner) glass membrane. The planar device with a nanopore is compatible with microfluidic integration and also makes high throughput manufacturing possible. Smaller bead-PNA conjugates become mobile enough to block the nanopore at lower concentrations of target NAs. This device has proven capable of detecting E. coli 16S rRNA (target) at 1 aM against a 1 pM background of RNA from Pseudomonas putida. In the absence of E. coli rRNA, no false positive signals were obtained in the presence of 1 pM P. putida 16S rRNA. Also, a universal PNA probe complementary to both a portion of the 16S rRNA target from E. coli and rRNA from P. putidawas used as a positive control, and as expected, a response to rRNA from both bacteria was detected using the positive control PNA probe. This detection scheme shows promise for integration into portable, low-cost systems for rapid detection of pathogenic bacteria in food, water and body fluids.
 Esfandiari L, Monbouquette HG, Schmidt JJ. Sequence-specific Nucleic Acid Detection from Binary Pore Conductance measurement. J. Am. Chem. Soc. 2012; 134: 15880-15886.
 Esfandiari L, Lorenzini M, Kocharyan G, Monbouquette HG, Schmidt JJ. Sequence-Specific DNA Detection at 10fM by Electromechanical Signal Transduction. Anal. Chem. 2014; 86: 9638-9643.
See more of this Group/Topical: Food, Pharmaceutical & Bioengineering Division