We present the electrochemical response to single adrenal chromaffin vesicles filled with catecholamine hormones as they are adsorbed and rupture on a 33 μm diameter disk-shaped carbon electrode. The vesicles adsorb onto the electrode surface and sequentially spread out over the electrode surface, trapping their contents against the electrode. These contents are then oxidized, and a current (or amperometric) peak results from each vesicle that bursts. A large number of current transients associated with rupture of single vesicles (86%) are observed under the experimental conditions used, allowing us to quantify the vesicular catecholamine content.

No abstract

Acetylcholine is a highly abundant nonelectroactive neurotransmitter in the mammalian central nervous system. Neurochemical release occurs on the millisecond time scale, requiring a fast, sensitive sensor such as an enzymatic amperometric electrode. Typically, the enzyme used for enzymatic electrochemical sensors is applied in excess to maximize signal. Here, in addition to sensitivity, we have also sought to maximize temporal resolution, by designing a sensor that is sensitive enough to work at near monolayer enzyme coverage. Reducing the enzyme layer thickness increases sensor temporal resolution by decreasing the distance and reducing the diffusion time for the enzyme product to travel to the sensor surface for detection. In this instance, the sensor consists of electrodeposited gold nanoparticle modified carbon fiber microelectrodes (CFMEs). Enzymes often are sensitive to curvature upon surface adsorption; thus, it was important to deposit discrete nanoparticles to maintain enzyme activity while depositing as much gold as possible to maximize enzyme coverage. To further enhance sensitivity, the enzymes acetylcholinesterase (AChE) and choline oxidase (ChO) were immobilized onto the gold nanoparticles at the previously determined optimal ratio (1:10 AChE/ChO) for most efficient sequential enzymatic activity. This optimization approach has enabled the rapid detection to temporally resolve single vesicle acetylcholine release from an artificial cell. The sensor described is a significant advancement in that it allows for the recording of acetylcholine release on the order of the time scale for neurochemical release in secretory cells.

We report the lithographic microfabrication of a movable thin film microelectrode array (MEA) probe consisting of 16 platinum band electrodes placed on top of a supporting borosilicate glass substrate. These 1.2 μm wide electrodes were tightly packed and positioned parallel in two opposite rows within a 20 μm × 25 μm square area and with a distance less than 10 μm from the edge of the glass substrate. We demonstrate the ability to control and place the probe in close proximity to the surface of adherent bovine chromaffin cells and to amperometrically record single exocytosis release events with high spatiotemporal resolution. The two-dimensional position of single exocytotic events occurring in the center gap area separating the two rows of MEA band electrodes and that were codetected by electrodes in both rows was determined by analysis of the fractional detection of catecholamine released between electrodes and exploiting random walk simulations. Hence, two-dimensional electrochemical imaging recording of exocytosis release between the electrodes within this area was achieved. Similarly, by modeling the current spikes codetected by parallel adjacent band electrodes positioned in the same electrode row, a one-dimensional imaging of exocytosis with submicrometer resolution was accomplished within the area. The one- and two-dimensional electrochemical imaging using the MEA probe allowed for high spatial resolution of exocytosis activity and revealed heterogeneous release of catecholamine at the chromaffin cell surface.

No abstract