430671 Sequence-Specific Nucleic Acid Detection Based on Blockade of a Nanopore in a Thin Glass Membrane

Monday, November 9, 2015: 4:12 PM
Canyon C (Hilton Salt Lake City Center)
Allison M. Yorita1, Bonhye Koo1, Leyla Esfandiari2, Jacob Schmidt3 and Harold G. Monbouquette1, (1)Chemical and Biomolecular Engineering, University of California, Los Angeles, Los Angeles, CA, (2)Biomedical Engineering, University of Cincinnati, Cincinnati, OH, (3)Bioengineering, University of California Los Angeles, Los Angeles, CA

A new platform for sequence-specific nucleic acid (NA) detection with binary response has been demonstrated. The detection of nucleic acid sequences is useful for diagnosing the presence of bacteria, especially those proven to be harmful to public health. A platform for low cost, reliable detection of the presence of pathogenic bacteria has a wide variety of applications, ranging from the diagnosis of bacterial infections to identification of contaminated food or water. Most existing methods are complex and require special reagents, as they rely on polymerase chain reaction (PCR) for target sequence amplification and fluorescence or enzyme labels. In our previous work, we described our first-generation, PCR-free, label-free system based on polystyrene beads conjugated with uncharged peptide nucleic acid (PNA) as sequence-specific probes, and demonstrated a detection limit of 10 fM.[1,2] A drawn glass pipette tip served as the micropore and was placed between two buffer-filled chambers, one of which contained the bead-PNA conjugates. Since the bead-PNA conjugates carry little to no charge, they do not respond to an electric field applied across the pore. However, in the presence of target NA sequence, the bead-PNA conjugates acquire enough negative charge to become mobile by hybridizing the NA sequence of interest. Upon pore blockade by the beads, a change in the pore conductance occurs, which is reflected in a step-decrease in current that signals the presence of the target nucleic acid.[1,2]

At lower concentrations of NA where fewer target NA molecules hybridize per bead, smaller beads are expected to have a higher mobility. Accordingly, by decreasing both the size of the pore and the beads, detection limits below 10 fM may be achieved. Motivated by this analysis, we have fabricated thin (1 μm or thinner) glass membranes in which a nanopore is milled with a focused ion beam (FIB). This device has two benefits: 1) By milling a pore via FIB, the pore size can be reduced significantly compared to drawn glass pipette tips, which lie in the micrometer range; 2) Since the nanopore is milled in a planar substrate, it can be mass-manufactured and can be assembled into devices more easily. To date, the nanomachined devices have proven capable of distinguishing between complementary and non-complementary sequences of ssDNA at various bead sizes. In addition, a promising trend has been observed showing that with decreasing bead size, higher mobility is attained at lower NA concentrations. These results support the expectations that detection limits less than 10 fM may be achieved with the nanomachined device and that this work could give rise to a diagnostic platform for the rapid, sensitive, and cost-effective detection of NA of specific sequence.

[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.

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