Real Time In Vivo Measurement of Ascorbate in the Brain Using Carbon Nanotube-Modified Microelectrodes Nuno R. Ferreira, a Ricardo M. Santos , a Jo¼o Laranjinha, a, b Rui M. Barbosa* a, b a Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal b Faculty of Pharmacy, University of Coimbra, 3000-548 Coimbra, Portugal tel: + 351239488463 *e-mail: rbarbosa@ff.uc.pt Received: January 30, 2013 Accepted: April 26, 2013 Published online: June 14, 2013 Abstract A carbon fiber microelectrode modified with a composite film of carbon nanotubes and Nafion was developed for in vivo ascorbate (AA) measurements in brain tissue. The modified-microelectrodes exhibit an electrocatalytic ac- tivity for AA oxidation by shifting the peak potential negatively to 0.040 V, showing a sensitivity of 37 pA/mM, a detection limit of 2.5 mM, a response time of 0.3 s and dont respond to several electroactive compounds found in the brain extracellular space. In the rat hippocampus, the basal concentration of AA was 290 mM, and glutamate- evoked changes in AA were biphasic comprising fast and slow components. Keywords: Ascorbate, Carbon fiber microelectrodes, Carbon nanotubes, In vivo electrochemistry DOI: 10.1002/elan.201300053 1 Introduction Ascorbate (AA) plays a modulator role in the brain, namely at the information processing and behavior func- tions linked to glutamate and dopamine-mediated neuro- transmission, beyond its well known function as an anti- oxidant and enzyme co-factor [1–4]. In addition, AA defi- ciency seems to be implicated in behavioral deficit in a transgenic mouse model of Huntingtons disease [5,6]. Although the brain contains the highest concentration of AA in the body, its regional asymmetric distribution within different areas and the concentration change in re- sponse to neural activity, raise the need for its dynamic measurement upon neurotransmitter stimulation [3]. Par- adoxically, because of its high concentration in the brain extracellular space, (up to 500 mM), AA is usually viewed as an interferent molecule when major neurotransmitters (e.g. dopamine (DA), noradrenaline (NA) and seroto- nine) are measured by electrochemical methods in combi- nation with microelectrodes [4]. A number of electrochemical methods have been re- ported in the literature for measurement of AA in biolog- ical fluids but most of them still suffer from major draw- backs. A major issue is that most of these methods do not allow the detection of AA in brain tissue microenviron- ments in a real-time mode. Carbon fiber microelectrodes (CFMs) have been extensively used to study neurotrans- mitter release and uptake in association with fast electro- chemical techniques such as amperometry, chronoamper- ometry and fast cyclic voltammetry (FCV). By virtue of its unique properties, such as favorable surface kinetics and biocompatibility, measurements can be performed with high spatial and temporal resolution inducing mini- mal damage to brain tissue [7–9]. At physiological pH, AA (first pK a = 4.10) is negatively charged whereas DA (pK a = 8.87) is positively charged. So far the most used approach to improve the selectivity of carbon fiber microelectrodes for measuring catechola- mine neurotransmitters in vivo is by coating the electrode surface with Nafion. This perfluorosulfonated polymer acts as an ion-exchange membrane highly permeable to cations but almost impermeable to anions [10]. Converse- ly, the use of polymers to improve the selectivity of CFMs for measuring AA anion is very limited. Therefore, different approaches have been proposed which are based on the anodization treatment of CFMs in associa- tion with scanning electrochemical techniques such as dif- ferential pulse voltammetry [11,12]. Despite the fact that the anodization treatment allows the selective measure- ment of AA, the sensitivity diminishes significantly after in vivo implantation of CFMs in the brain tissue. This problem has been partially solved by coating the surface with a thin polymer film [13]. A further strategy to im- prove selectivity for AA has been achieved by incorporat- ing biochemical approaches with the electrochemical measurements. For instance, regional differences in extra- cellular AA levels have also been reported by using a combination of FCV and ascorbate oxidase (AAOx) [14]. Electroanalysis 2013, 25, No. 7, 1757 – 1763  2013 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim 1757 Full Paper