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

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.

Microelectrode Biosensors for in vivo Analysis of Brain Interstitial Fluid

“…Microelectrode biosensors can detect choline by using choline oxidase. Acetylcholine can be detected as well if acetylcholine esterase and choline oxidase are mixed and immobilized together at the microelectrode surface . However, it was observed that these dual enzyme biosensors did not detect any substantial levels of acetylcholine within interstitial fluid , probably because this molecule is quickly degraded within the synaptic cleft and it remains at extremely low concentrations within interstitial fluid overall.…”

Mechanism of neurotransmitter release coming into focus

“…However, even higher time resolution will be required to monitor fusion at the speeds occurring in vivo. Novel methods, including for instance amperometric recordings on artificial cells, may be necessary for this purpose. Of course, electrophysiological studies will continue to be essential to test the models emerging from structural and reconstitution studies, as well as to study the functions of the release machinery.…”

Frontiers in electrochemical sensors for neurotransmitter detection: towards measuring neurotransmitters as chemical diagnostics for brain disorders

“…Keighron et al modied carbon ber microdisk electrodes with spatially discrete nanoparticles, onto which acetylcholinesterase and choline oxidase were deposited. 91 The enzyme layer was near monolayer thickness, allowing for rapid (millisecond) response time, the fastest reported for biosensing tools. This technique was used to measure exocytotic ACh release from articial secretory cells.…”