Hydrogen peroxide is a reactive oxygen species that is implicated in a number of neurological disease states and that serves a critical role in normal cell function. It is commonly exploited as a reporter molecule enabling the electrochemical detection of non-electroactive molecules at electrodes modified with substrate-specific oxidative enzymes. We present the first voltammetric characterization of rapid hydrogen peroxide fluctuations at an uncoated carbon fiber microelectrode, demonstrating unprecedented chemical and spatial resolution. The carbon surface was electrochemically conditioned on the anodic scan and the irreversible oxidation of peroxide was detected on the cathodic scan. The oxidation potential was dependent on scan rate, occurring at +1.2 V vs. Ag/AgCl at a scan rate of 400 V·sec -1 . The relationship between peak oxidation current and concentration was linear across the physiological range tested, with deviation from linearity above 2 mM and a detection limit of 2 μM. Peroxide was distinguished from multiple interferents, both in vitro and in brain slices. The enzymatic degradation of peroxide was monitored, as was peroxide evolution in response to glucose at a glucose oxidase modified carbon fiber electrode. This novel approach provides the requisite sensitivity, selectivity, spatial and temporal resolution to study dynamic peroxide fluctuations in discrete biological locations.

Rapid changes in extracellular dopamine concentrations in freely moving or anesthetized rats can be detected using fast-scan cyclic voltammetry (FSCV). Background-subtracted FSCV is a real-time electrochemical technique that can monitor neurochemical transmission in the brain on a subsecond timescale, while providing chemical information on the analyte. Also, this voltammetric approach allows for the investigation of the kinetics of release and uptake of molecules in the brain. This chapter describes, completely, how to make these measurements and the properties of FSCV that make it uniquely suitable for performing chemical measurements of dopaminergic neurotransmission in vivo.

Neurotransmission occurs on a millisecond timescale, but conventional methods for monitoring non-electroactive neurochemicals are limited by slow sampling rates. Despite a significant global market, a sensor capable of measuring the dynamics of rapidly fluctuating, non-electroactive molecules at a single recording site with high sensitivity, electrochemical selectivity, and a subsecond response time is still lacking. To address this need, we have enabled the real-time detection of dynamic glucose fluctuations in live brain tissue using background-subtracted, fast-scan cyclic voltammetry. The novel microbiosensor consists of a simple carbon fiber surface modified with an electrodeposited chitosan hydrogel encapsulating glucose oxidase. The selectivity afforded by voltammetry enables quantitative and qualitative measurements of enzymatically-generated H2O2 without the need for additional strategies to eliminate interferents. The microbiosensors possess a sensitivity and limit of detection for glucose of 19.4 ± 0.2 nA mM−1 and 13.9 ± 0.7 μM, respectively. They are stable, even under deviations from physiological normoxic conditions, and show minimal interference from endogenous electroactive substances. Using this approach, we have quantitatively and selectively monitored pharmacologically, evoked glucose fluctuations with unprecedented chemical and spatial resolution. Furthermore, this novel biosensing strategy is widely applicable to the immobilization of any H2O2 producing enzyme, enabling rapid monitoring of many non-electroactive enzyme substrates.

Electrochemical monitoring of non-electroactive species requires a biosensor that is stable and selective, with sensitivity to physiological concentrations of targeted analytes. We have combined glucose oxidase-modified carbon-fiber microelectrodes with fast-scan cyclic voltammetry for real-time measurements of glucose fluctuations in brain tissue. Work presented herein quantitatively compares three approaches to enzyme immobilization on the microelectrode surface-physical adsorption, hydrogel entrapment, and entrapment in electrospun nanofibers. The data suggest that each of these methods can be used to create functional microbiosensors. Immobilization of glucose oxidase by physical adsorption generates a biosensor with poor sensitivity to glucose and unstable performance. Entrapment of glucose oxidase in poly(vinyl alcohol) nanofibers generates microbiosensors that are effective for glucose measurements over a large linear range, and that may be particularly useful when targeting glucose concentrations in excess of 3 mm, such as in blood. Hydrogel entrapment is the most effective in terms of sensitivity and stability. These microbiosensors can be used for simultaneous monitoring of glucose and dopamine in real time. The findings outlined herein should be applicable to other oxidase enzymes, and thus they are broadly important for the development of new tools for real-time measurements of fluctuating molecules that are not inherently electroactive.