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Microfabricated Devices for the Detection of Neurotransmitters and Nucleic Acids

  • Author(s): Yorita, Allison Mika
  • Advisor(s): Monbouquette, Harold G
  • et al.
Abstract

Studying neurological disorders on a chemical level requires sensitive and fast techniques in order to monitor the release of neurotransmitters in near-real time. When creating probes for the purpose of sensing these neurotransmitters, these sensors must demonstrate high selectivity for the neurotransmitter of interest, high sensitivity towards the neurotransmitter, and a low detection limit such that small releases of the neurotransmitter can still be detected. Creating platinum-based electrodes, onto which various permselective polymers are deposited, has been demonstrated to be an effective means by which sensors can be created for use in vivo in behavioral studies. The first example is the creation of a platinum wire-based sensor, on which polymers and the enzyme glutamate oxidase were deposited to create a glutamate sensor. The benefit to using a platinum wire its small physical footprint, making it effective for in vivo studies where tissue damage is a big concern, such as when studying brain slices. The second example is the fabrication of a multielectrode microprobe, with which numerous neurotransmitters can be detected simultaneously. Using these sensors, a dopamine-sensing site was modified and optimized to decrease the limit of detection to 10 μM, which is comparable to the lowest concentration that needs to be detected in the brain. Ultimately, this dopamine sensor will be incorporated as one site on the multielectrode microprobe, such that dopamine and glutamate can be detected simultaneously on one sensor in vivo. This dual sensor could be used to study how imbalances in both neurotransmitters affect neurological disorders, in hopes of elucidating the link between neurotransmitter release and behavioral response.

There is a great need for sensors that detect specific sequences of nucleic acids quickly and at a low cost. The applications behind a nucleic acid sequence sensor are broad, ranging from detection of food or water contamination to quick diagnosis of the presence of harmful bacteria. Current nucleic acid sensors rely on methods such as polymerase chain reaction, making it less portable and requiring more complex electronics for detection. We have created a sensor made of a thin (1 μm or thinner) glass membrane in which a single nanopore is milled. An electric potential is applied and the resulting current flowing through the pore is measured. On one side of this nanopore, microspheres conjugated with a peptide nucleic acid (PNA) sequence complementary to the target sequence are injected. These microspheres are neutrally charged, such that when a potential is applied across the glass membrane, they will not respond to the electric field. However, in the presence of the target nucleic acid sequence, the beads’ PNA probe sequence hybridizes to the target sequence, thus imparting negative charge onto the bead. As a result, these beads will move towards the nanopore upon application of an electric field. The pore is designed to be smaller than the beads, such that when the bead blocks the pore, a discernible decrease in current is detected which confirms the presence of the target nucleic acid. To date, this platform has demonstrated the ability to distinguish between complementary and non-complementary nucleic acid sequences, down to a detection limit of 1 aM of 16S ribosomal RNA extracted from Escherichia coli. These results indicate that this sensor can selectively detect low concentrations of pathogenic bacteria, even in the presence of other organisms.

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