A new platform for sequence-specific nucleic acid (NA) detection based on blockade of a nanopore has been demonstrated. Rapid and sensitive detection of nucleic acid of specific sequence is useful for health-related applications such as diagnosing the presence of pathogenic bacteria, and screening for genetic diseases. Many of the existing platforms require polymerase chain reaction (PCR) amplification, fluorescent or enzymatic labels, and expensive instrumentation. Recently, PCR-independent NA sequence sensing based on measurement of resistive pulses across nanopores have been introduced. In this type of sensor, the electrical resistance is monitored as analyte passes through a buffer-filled pore. Previously, we introduced our first-generation device with a detection limit of 10 fM based on peptide nucleic acid (PNA) as sequence-specific probes.[1,2] This device does not require PCR and or other labels in addition to a PNA-bead conjugate. In the presence of a target NA complementary to the PNA probe, the bead-PNA conjugates acquire negative charges through hybridization with the target NA, and become electrophoretically mobile. This causes the beads to block the drawn glass pipette pore, thereby increasing the resistance of the pore. As a result, the device shows a step-decrease in current.[1,2] In current work, we have fabricated a glass nanopore in a thin (1 μm or thinner) glass membrane by thinning from a planar glass substrate. This scale down to the nano-regime is motivated by the analysis that smaller beads are expected to have a higher mobility at lower concentrations of NA and glass nanopores have lower noise than silicon-based nanopores. In comparison to our previous device, 1) the pore size is significantly reduced and 2) it can be batch-processed and can be assembled into devices more easily due to a planar substrate. This device has been proven to distinguish target DNA from non-complementary DNA at various bead sizes, and work is underway to achieve the lower detection limits expected. [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.

Sequence-specific nucleic acid detection based on ionic current measurement through a glass nanopore

251st National Meeting (2016)

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