(570d) Nanopore-Based Sequence-Specific Nucleic Acid Detection at 1aM

A PCR-free, optics-free platform has been developed for the detection of E. coli16S rRNA at 1 aM. This sequence-specific nucleic acid (NA) detector is based on electromechanical signal transduction using a nanopore-based scheme. Rapid, sensitive, and cost-effective detection of NAs of specific sequence is in high demand for diagnosing pathogenic diseases, detection of food contaminants, patient screening during epidemics, or oncological status assessment during surgery. In order to detect target NAs, our device utilizes peptide nucleic acid (PNA) capture probes conjugated to polystyrene beads. Since PNA is charge neutral, the bead-PNA conjugates are designed to be charge neutral until they hybridize to target NAs at which point the complex becomes negatively charged and mobile in an electric field. If the electric field is oriented through a pore that is too small for the bead-PNA conjugate with hybridized target to pass through, it will at least partially block the pore thereby causing an easily measured step reduction in ionic current. In this way, the selective, nucleic acid hybridization event is electromechanically transduced.

Previously, we introduced a first-generation device based on a drawn pipette tip as the â??poreâ? 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.

[1] Esfandiari L, Monbouquette HG, Schmidt JJ. Sequence-specific Nucleic Acid Detection from Binary Pore Conductance measurement. J. Am. Chem. Soc. 2012; 134: 15880-15886.

[2] 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.