This paper describes electrochemical microsensors for the in vivo measurement of glutamate and ascorbate in the extracellular space of brain tissue. To prepare glutamate microsensors, carbon fiber microelectrodes (10 microns in diameter and 300-400 microns long) were modified with a cross-linked redox polymer film containing enzymes. The microsensors were coated with a thin Nafion film before use. The glutamate microsensors were both selective and sensitive toward glutamate, with detection limits in the low micromolar range. Physiologically relevant concentrations of several electroactive compounds found in brain tissue produced no response at the glutamate microsensors and also did not affect their glutamate response, the only exception being glutamine, for which a small response was observed in the absence, but not in the presence, of glutamate. The ascorbate microsensors were used in conjunction with cyclic voltammetry. They were sensitive and selective toward ascorbate, but did exhibit a small sensitivity toward the dopamine metabolite, dihydroxyphenylacetic acid. The in vivo measurements performed establish the ability of the glutamate microsensors to monitor the component of the basal extracellular glutamate level that is derived from the neuronal activity of brain tissue.

Bacteria, plants, and higher and lower animals have evolved an innate immune system as a first line of defense against microbial invasion. Some of these organisms produce antimicrobial peptides (AMPs) as a part of this chemical immune system. AMPs exert their antimicrobial activity by binding to components of the microbe's surface and disrupting the membrane. The overall goal of this study was to apply the AMP magainin I as a recognition element for Escherichia coli O157:H7 and Salmonella typhimurium detection on an array-based biosensor. We immobilized magainin I on silanized glass slides using biotin-avidin chemistry, as well as through direct covalent attachment. Cy5-labeled, heat-killed cells were used to demonstrate that the immobilized magainin I can bind Salmonella with detection limits similar to analogous antibody-based assays. Detection limits for E. coli were higher than in analogous antibody-based assays, but it is expected that other AMPs may possess higher affinities for this target. The results showed that both specific and nonspecific binding strongly depend on the method used for peptide immobilization. Direct attachment of magainin to the substrate surface not only decreased nonspecific cell binding but also resulted in improved detection limits for both Salmonella and E. coli.

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Interest in the detection of hydrogen peroxide in living brain tissue is growing for several reasons. Peroxide and other reactive oxygen species are implicated in neurodegenerative disorders and appear to have neuromodulatory functions in the brain. Also, there is a need to measure peroxide levels as a companion to measurements with amperometric sensors that rely on enzymes to generate peroxide for the detection of glutamate, choline, and glucose. Herein, we report on measurements performed in the brain of anesthetized rats with carbon fiber amperometric sensors coated with a cross-linked redox polymer film that contains horseradish peroxidase. Prior work with these sensors has established that they are both sensitive and selective toward hydrogen peroxide. When implanted in the striatal region of the rat brain, a biphasic response is observed upon electrical stimulation of the dopaminergic pathway that innervates the striatal tissue. No response is observed at sensors lacking HRP, which are not sensitive to peroxide, suggesting that the biphasic response is due to the production of hydrogen peroxide by two separate mechanisms. Additional measurements of dopamine and oxygen, and the administration of two drugs with well-known effects on the biochemical kinetics of the dopamine neurons, are used to identify those mechanisms. One appears to be the production of peroxide upon the oxidation of dopamine by molecular oxygen. This occurs during the electrical stimulation itself, which elevates both dopamine and oxygen levels in the extracellular space. The other appears to be the production of peroxide as a byproduct in the oxidative metabolic conversion of dopamine to DOPAC by the mitochondrial enzyme, monoamine oxidase. The production of peroxide due to dopamine metabolism is also observed after rats receive a dose of L-DOPA, a drug used in the treatment of Parkinson's disease.