Ex Vivo Electrochemical Monitoring of Cholinergic Signaling in the Mouse Colon Using an Enzyme-Based Biosensor.

Cholinergic signaling, i.e., neurotransmission mediated by acetylcholine, is involved in a host of physiological processes, including learning and memory. Cholinergic dysfunction is commonly associated with neurodegenerative diseases, including Alzheimer's disease. In the gut, acetylcholine acts as an excitatory neuromuscular signaler to mediate smooth muscle contraction, which facilitates peristaltic propulsion. Gastrointestinal dysfunction has also been associated with Alzheimer's disease. This research focuses on the preparation of an electrochemical enzyme-based biosensor to monitor cholinergic signaling in the gut and its application for measuring electrically stimulated acetylcholine release in the mouse colon ex vivo. The biosensors were prepared by platinizing Pt microelectrodes through potential cycling in a potassium hexachloroplatinate (IV) solution to roughen the electrode surface and improve adhesion of the multienzyme film. These electrodes were then modified with a permselective poly(m-phenylenediamine) polymer film, which blocks electroactive interferents from reaching the underlying substrate while remaining permeable to small molecules like H2O2. A multienzyme film containing choline oxidase and acetylcholinesterase was then drop-cast on these modified electrodes. The sensor responds to acetylcholine and choline through the enzymatic production of H2O2, which is electrochemically oxidized to produce an increase in current with increasing acetylcholine or choline concentration. Important figures of merit include a sensitivity of 190 ± 10 mA mol-1 L cm-2, a limit of detection of 0.8 µmol L-1, and a batch reproducibility of 6.1% relative standard deviation at room temperature. These sensors were used to detect electrically stimulated acetylcholine release from mouse myenteric ganglia in the presence and absence of tetrodotoxin and neostigmine, an acetylcholinesterase inhibitor.

Electrokinetic Enrichment and Label-Free Electrochemical Detection of Nucleic Acids by Conduction of Ions along the Surface of Bioconjugated Beads.

In this paper, we report a method to integrate the electrokinetic pre-enrichment of nucleic acids within a bed of probe-modified microbeads with their label-free electrochemical detection. In this detection scheme, hybridization of locally enriched target nucleic acids to the beads modulates the conduction of ions along the bead surfaces. This is a fundamental advancement in that this mechanism is similar to that observed in nanopore sensors, yet occurs in a bed of microbeads with microscale interstices. In application, this approach has several distinct advantages. First, electrokinetic enrichment requires only a simple DC power supply, and in combination with nonoptical detection, it makes this method amenable to point-of-care applications. Second, the sensor is easy to fabricate and comprises a packed bed of commercially available microbeads, which can be readily modified with a wide range of probe types, thereby making this a versatile platform. Finally, the sensor is highly sensitive (picomolar) despite the modest 100-fold pre-enrichment we employ here by faradaic ion concentration polarization (fICP). Further gains are anticipated under conditions for fICP focusing that are known to yield higher enrichment factors (up to 100,000-fold enrichment). Here, we demonstrate the detection of 3.7 pM single-stranded DNA complementary to the bead-bound oligoprobe, following a 30 min single step of enrichment and hybridization. Our results indicate that a shift in the slope of a current-voltage curve occurs upon hybridization and that this shift is proportional to the logarithm of the concentration of target DNA. Finally, we investigate the proposed mechanism of sensing by developing a numerical simulation that shows an increase in ion flux through the bed of insulating beads, given the changes in surface charge and zeta potential, consistent with our experimental conditions.