Evaluating the Diffusion Coefficient of Dopamine at the Cell Surface During Amperometric Detection: Disk vs Ring Microelectrodes

During exocytosis, small quantities of neurotransmitters are released by the cell. These neurotransmitters can be detected quantitatively using electrochemical methods, principally with disk carbon fiber microelectrode amperometry. An exocytotic event then results in the recording of a current peak whose characteristic features are directly related to the mechanisms of exocytosis. We have compared two exocytotic peak populations obtained from PC12 cells with a disk carbon fiber microelectrode and with a pyrolyzed carbon ring microelectrode array, with a 500 nm ring thickness. The specific shape of the ring electrode allows for precise analysis of diffusion processes at the vicinity of the cell membrane. Peaks obtained with a ring microelectrode array show a distorted average shape, owing to increased diffusion pathways. This result has been used to evaluate the diffusion coefficient of dopamine at the surface of a cell, which is up to an order of magnitude smaller than that measured in free buffer. The lower rate of diffusion is discussed as resulting from interactions with the glycocalyx

Amperometric Detection of Single Vesicle Acetylcholine Release Events from an Artificial Cell

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

Evaluating the Diffusion Coefficient of Dopamine at the Cell Surface During Amperometric Detection: Disk vs Ring Microelectrodes

During exocytosis, small quantities of neurotransmitters are released by the cell. These neurotransmitters can be detected quantitatively using electrochemical methods, principally with disk carbon fiber micro-electrode amperometry. An exocytotic event then results in the recording of a current peak whose characteristic features are directly related to the mechanisms of exocytosis. We have compared two exocytotic peak populations obtained from PC12 cells with a disk carbon fiber microelectrode and with a pyrolyzed carbon ring microelectrode array, with a 500 nm ring thickness. The specific shape of the ring electrode allows for precise analysis of diffusion processes at the vicinity of the cell membrane. Peaks obtained with a ring microelectrode array show a distorted average shape, owing to increased diffusion pathways. This result has been used to evaluate the diffusion coefficient of dopamine at the surface of a cell, which is up to an order of magnitude smaller than that measured in free buffer. The lower rate of diffusion is discussed as resulting from interactions with the glycocalyx