Full Paper

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 dont 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 its unique properties, such as favorable surface kinetics

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 Huntingtons 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
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 pKa = 4.10) is negatively
charged whereas DA (pKa = 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].

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In recent years, carbon nanotubes (CNTs) have been
extensively used for the fabrication of electrochemical
sensors and biosensors due to their unique structural, me-
chanical, chemical and electronic properties. Usually
found in two categories, single wall (SWCNTs) and multi-
wall (MWCNTs), CNTs provide high surface-to-volume
ratios, promote electron transfer reactions, decrease over-
potentials of various electroactive compounds, increase
sensitivity and reduce fouling [7,15–17].
To overcome the insolubility of CNTs in most solvents,
Nafion can be used to make relatively stable suspensions
via ultrasonication procedures [18]. In particular, the use
of Nafion/CNT nanocomposite films offer an excellent
matrix due to the chemical inertness, thermal stability,
mechanical strength, antifouling property and good bio-
compatibility [10,19,20]. By using MWCNT modified mi-
croelectrodes [21] and carbon nanotube fiber microelectr-
odes [22], DA has been measured in the presence of AA.
More recently, a polyaniline/polyacrylic/MWCNT modi-
fied Pt macrodisc electrode has been reported for measur-
ing AA in the presence of DA and uric acid [23]. Further-
more, the simultaneous determination of AA, DA and
uric acid by using a screen-printed graphene electrode
[24] and chitosan-graphene modified electrodes has been
reported [25].
Electrochemical methods in vivo require the use of fast
techniques, such as constant potential amperometry, chro-
noamperometry or fast cyclic voltammetry (FCV), high
sensitivity and selectivity and the use of ultrasmall sensors
[8]. To date, most of the studies reported in the literature
concerning the measurement of AA in biological media
or tissues did not fulfill these criteria. The in vivo voltam-
metric measurement of AA in the striatum of rat brain
has been reported by using CFMs coated with a DMF/
MWCNT film, but the use of a slow scanning technique
such as differential pulse voltammetry (DPV) did not
allow the measurement of AA in a real-time fashion [26].
In the present work, we have developed CFMs modi-
fied with Nafion and CNTs composite films for in vivo
measurement of AA in the rat brain. The electrochemical
characterization was studied, namely the electrocatalytic
oxidation of AA and DA, the analytical performance in
terms of sensitivity, LOD, selectivity and response time
was also assessed. Furthermore, the modified microelectr-
odes were stereotaxically inserted in the rat hippocampus
for selective measures of both basal and glutamate-stimu-
lated AA release in the extracellular space in real-time
and with high spatiotemporal resolution.
2 Experimental
2.1 Chemicals and Solutions
All compounds were analytical grade and were used as
received. AA, DA and DOPAC were obtained from
Fluka. l-Glutamic acid and NA were purchased from
Sigma and Nafion (5 wt.% solution in a mixture of ali-
phatic alcohols and water) was purchased from Aldrich.
1758 www.electroanalysis.wiley-vch.de  2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
N. R. Ferreira et al.
Sodium nitrite was obtained from Riedel de Haen and
AAOx was obtained from AppliChem. Chemicals used
for buffer solutions were obtained from Panreac.
MWCNT and SWCNT were purchased from Nanolab
(USA). Solutions were prepared in ultra-pure deionized
water (Milli-Q, Millipore Company, Bedford, MA, USA)
with a resistivity  18.2 MW cm. Phosphate-buffered
saline (PBS lite) solution (0.05 M), pH 7.4 was prepared
with the following composition (mM): 100 NaCl, 10
NaH2PO4 and 40 Na2HPO4. AAOx solutions were pre-
pared by dissolving 1 mg (288 U) of the enzyme in 1.0 mL
of 0.9 % NaCl solution.
2.2 Apparatus
A scanning electron microscope JEOL (JSM-5310) was
used to visualize the surface morphology of the micro-
electrodes. AA and DA oxidation potential was deter-
mined by square wave voltammetry (SWV), using an
IVIUM Compactstat (Ivium Technologies, The Nether-
lands). Calibration and in vivo experiments were per-
formed by amperometry, (hold potential: + 0.05 V vs. Ag/
AgCl) using a FAST-16 MK III high-speed electrochemi-
cal system (Quanteon, L.L.C., KY, USA) in a two-elec-
trode configuration mode.
2.2.1 Microelectrode Modification
Carbon fiber microelectrodes were fabricated as previ-
ously described [9,27] and were used with a tip length of
150–250 mm. SWCNTs and MWCNTs were used for coat-
ing the tip surface of CFMs. Both types of CNTs were
suspended in 0.5 % Nafion for a final concentration of
100 mg/mL. A homogeneous suspension was obtained by
sonication during at least 1/2 hour. A single drop of the
suspension was applied onto a glassy plate, and the tip
was immersed into the droplet for 30 s and then dried at
170 8C for 5 minutes.
2.3 Procedures
2.3.1 Calibration
Calibration was performed by successive additions of an
AA standard solution to the beaker followed by the inter-
ferents, DA, NA, DOPAC and nitrite (NO2 ). The sensi-
tivity, detection limit (LOD), linearity and selectivity
were calculated from the data. The LOD was estimated
according to the equation LOD = 3 SD/m, where m rep-
resents the slope of the calibration curve obtained by
linear regression and SD the standard deviation of the
baseline noise. The selectivity ratio refers to the ratio of
the sensitivity for AA over interferents (AA:interfer-
ents). The response time expressed as the half-maximal
response time (t50 %), was calculated as the time to reach
50 % of the maximum current response to an injection of
100 mM AA. The sensitivity and selectivity of the modi-
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Real Time In Vivo Measurement of Ascorbate in the Brain

fied-microelectrodes were determined before and after in

vivo experiments.

2.4 In Vivo Electrochemistry

In vivo studies were conducted in the hippocampal CA1
region of adult male Wistar rats (8–12 weeks; weight
250–350 g) maintained in a 12 : 12 light:dark cycle with
food and water available ad libitum. Experiments were
carried out in accordance with the European Community
Council Directive for the Care and Use of Laboratory
Animals (86/609/ECC) and were approved by the local
institutional animal care committee. Rats were anesthe-
tized with urethane (1.25–1.50 g/kg, i.p.) and placed in
a stereotaxic frame (Stoelting, USA) analogous to previ-
ous studies [9,28]. After exposing the surface, a hole was
drilled in the skull overlying the brain area of interest. A
small hole was drilled in a site remote from the recording
area for insertion of an Ag/AgCl reference electrode
(200 mm diameter). After removing the dura mater, a mi-
croelectrode/micropipette array (tip separation of ca.
200 mm) was inserted into the hippocampus according to
the coordinates calculated based on the rat brain atlas of
Paxinos and Watson (2007). After the insertion of the
array into the hippocampus, AA release was stimulated
by pressure ejection of l-glutamate from the micropipette
using a Picospritzer III (Parker Hannifin, USA).

2.5 Data Analysis
Data were analyzed for statistical significance defined at
p < 0.05 and were expressed as the mean  standard devi-
ation (SD). One-way analysis of variance (ANOVA) was
performed to make comparisons between multiple groups
followed by Dunnett pos-test. For each group of micro-
electrodes the confidence intervals (CI) at 95 % were also
calculated. Statistical analyses were performed using
Origin Pro 7.5 and GraphPad Prism 5. In vivo recordings
were averaged and represented as black line (mean) with
gray area (standard error).
3 Results and Discussion
3.1 Scanning Electron Microscopy of Microelectrode
Surface
Figure 1 illustrates SEM images of typical bare CFM tip
surface (A) and after modification with SWCNT (100 mg/
mL) in Nafion composite film (B) (herein referred as
CFM/Nafion/SWCNT). At low magnification, a very
smooth surface was observed for bare CFM whereas
a rugged surface was clearly seen at the CFM tip modi-
fied with the composite film. These surface morphologies
obtained with SWCNT composite films are very similar
to that obtained by MWCNTs (not shown). The rough
surface of the CNT-modified microelectrodes suggests an
increase of the surface area of the active tip when com-
pared with bare CFMs.
Fig. 1. Typical SEM images of the surface tip of bare carbon
fiber microelectrodes (A) and modified with a Nafion/SWCNT
composite film (B) with the same magnification (1500 ).
3.2 Electrocatalytic Oxidation of Ascorbate at Modified-
Microelectrodes
It is well described that oxidation of AA at bare electro-
des is totally irreversible and requires high overpotentials
because of the slow electron transfer kinetics and fouling
of the electrode surface by the AA oxidation products.
At physiological pH, the oxidation of AA involves a loss
of a single proton and two electrons, yielding dehydroas-
corbate [29,30]. As shown in Figure 2A the anodic peak
potential of AA determined by SWV was + 0.243 V vs.
Ag/AgCl when using bare CFMs but, meaningfully, the
peak was shifted negatively to 0.044 V in the case of
Nafion/SWCNT-modified CFMs, indicating a strong elec-
trocatalytic effect of CNTs. This observation is in agree-
ment with previous works describing the electrocatalytic
effect on the AA oxidation of MWCNTs-IL-Gel deposit-
ed on glassy carbon electrode [31], of MWCNTs/Nafion
modified carbon fiber electrode [21] and of MWCNTs/
Nafion layer over platinum disc electrode on AA oxida-
tion [23]. Noticeably, the electrocatalytic oxidation of DA
at CFM/Nafion/SWCNT suffered a less pronounced nega-
tive shift than AA. The average oxidation potential for
DA changed from + 0.224 V for bare CFM to + 0.196 V
in CFM/Nafion/SWCNT (Figure 2B). Thus, the peak reso-

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Full Paper
Fig. 2. (A) Ascorbate peak oxidation potential of bare (black
dashed line; 1 mM; Ep = + 0.243 V) and CNT-modified CFMs
(solid line; 0.5 mM; Ep = –0.044 V). (B) Dopamine peak oxida-
tion potential of bare (black dashed line; 20 mM; Ep = + 0.224 V)
and CNT-modified CFMs (solid line; 20 mM; Ep = + 0.196 V).
(C) Ascorbate (400 mM) and dopamine (20 mM) peak oxidation
potential in CNT-modified CFM (Ep = 0.028 V and Ep = +
0.178 V, respectively). Gray dashed lines represent baseline in
each experiment. Square wave voltammetry parameters: f =
10 Hz, Es = 2 mV, Esw = 10 mV.
lution of both compounds was clearly visible by SWV as
illustrated in Figure 2C.
Table 1 summarizes data of oxidation peak potential
obtained by SWV for bare and surface-modified CFMs.
When compared with bare CFM, both nanocomposite
films evaluated exhibited an electrocatalytic effect on the
AA oxidation corresponding to a negative shift of ca.
0.3 V. Data analysis by One-way ANOVA followed by
Dunnett post-test, indicate that the differences are statis-
tically significant (p < 0.001) between bare (control) and
modified-microelectrodes but not between Nafion/CNT
modified microelectrodes (p > 0.05).
1760 www.electroanalysis.wiley-vch.de  2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
N. R. Ferreira et al.
Table 1. Ascorbate peak oxidation potential of carbon fiber mi-
croelectrodes. The data are represented as mean  SD. The
number of microelectrodes is given in parenthesis.
CFM Oxidation potential (V)
Bare + 0.243  0.050 (n = 12)
Nafion/SWCNT 0.044  0.033 (n = 10)
Nafion/MWCNT 0.020  0.006 (n = 11)
3.3 Characterization of Microelectrode Response to
Ascorbate
3.3.1 Linearity, Sensitivity and Detection Limit
Figure 3 shows a typical amperometric recording of the
CFM/Nafion/SWCNT modified microelectrode at holding
potential of + 0.05 V vs. Ag/AgCl upon addition of
50 mM, 100 mM, 200 mM, 400 mM, and 800 mM AA to
a PBS buffer solution. At the beginning of the recording,
DA, NA, DOPAC and NO2 were added to determine
the corresponding selectivity ratios of the modified micro-
electrodes. Response time (t50 %) was also calculated from
the recordings and, typically, the modified microelectrode
reached a steady-state in less than 0.5 s.
The modified microelectrodes displayed a good linear
response to AA in the range from 50 mM up to 800 mM
(R2 = 0.998) as shown in Figure 3 Inset. Calibration sensi-
tivity was obtained from the linear regression of current
peak height vs. concentration of AA, giving a slope of
42.6 pA/mM and an intercept of 965 pA with a R2 = 0.998
for the recording presented.
Data in Table 2 supports that the sensitivity of the
modified microelectrodes is ca. 10 fold higher when com-
pared with bare microelectrodes. Data analysis by One-
way ANOVA followed by Dunnett post-test, indicates
that the differences between bare (control) and modified-
microelectrodes are statistically significant (p < 0.05).
Likewise, the differences on LOD are also statistically
significant (p < 0.05). These results emphasize the electro-
catalytic properties of the Nafion/CNT composite in
a way that allows a dramatic increase in sensitivity when
working at + 0.05 V. These sensitivity and LOD values
compare favorably with previous reports [26,32], despite
the use of different electrochemical techniques, experi-
mental conditions and CFMs tip dimensions.
Although sensitivity is of great importance in the char-
acterization of the microelectrode response to AA, its
loss during in vivo exposure is always a major concern.
Upon in vivo implantation of CFM/Nafion/SWCNT mi-
croelectrodes the decrease of sensitivity observed in a typ-
ically experiment lasting for 3–4 hours, was 82 % (n = 7)
of their initial value. These values are in agreement with
those obtained by others with electrochemical pretreated
microelectrodes (84 %) [13]. A 66 % decrease in sensitivi-
ty of a CFM coated with dimethylformamide and
MWCNTs inserted in brain tissue during 10 minutes
when assessed by DPV has been also reported [26].
The loss of sensitivity in vivo may be due to implanta-
tion and electrode fouling by interaction with the sur-
Electroanalysis 2013, 25, No. 7, 1757 – 1763
Real Time In Vivo Measurement of Ascorbate in the Brain

Fig. 3. Amperometric current at + 0.05 V vs. Ag/AgCl of a CFM/Nafion/SWCNT to successive additions of 50, 100, 200, 400 and
800 mM of AA and to the addition of potential interferents present in the hippocampus. Inset: Calibration curve.

3.4 Selectivity
rounding environment. Nevertheless, such loss of sensitiv-
ity is observed mainly during the first 20 minutes after im- The oxidation potential of AA at most common electrode
plantation needed to allow stabilization of the back- materials (e.g. carbon, platinum) is very close to that of
ground before starting the experiments. Afterwards and catecholamines (e.g. DA) and their metabolites (e.g.
throughout the remaining time course of the in vivo re- DOPAC), and by this reason the overlapping of the oxi-
cordings the sensitivity loss is much lower. In fact, follow- dation currents is a major problem.
ing the periodic ejection of AA the signals remained con- Direct measurements of AA by electrochemical meth-
stant (data not shown), indicating that measurements ods in complex biological media such as brain tissue re-
based on post-calibration results are reliable ruling out quires the use of microelectrodes with high selectivity
a gradual loss of sensitivity, a fact that could bias the ob- against major compounds found in the extracellular
tained results. space. Taking into consideration the relatively poor reso-
lution power of the voltammetric techniques, one of the
Table 2. Ascorbate calibration parameters obtained for carbon best experimental strategies that have been followed to
fiber microelectrodes modified with carbon nanotubes composite improve selectivity is the modification of the microelec-
films at + 0.05 V vs. Ag/AgCl. The data are represented as trode surface properties. This is an approach of utmost
mean  SD. The confidence interval (CI) for sensitivity, limit of importance when the microelectrodes are associated with
detection (LOD) and response time is given in square brackets. amperometry.
The number of microelectrodes tested is given in parenthesis. The CNT-modified CFMs, by shifting the oxidation po-
Calibration CFM Bare CFM/Nafion/ CFM/Nafion/ tential of AA negatively, allowed the measurement of
parameter SWCNT MWCNT anodic currents at very low potential (+ 0.05 V in this
Sensitivity 1.3  1.5 37.5  34.9 41.2  30.6 work), a value ca. 100 mV higher than the oxidation peak
(pA/mM) potential for AA (Epa = 0.05 V vs. Ag/AgCl). In addi-
[0.5–2.1] [22.0–52.9] [20.7–61.8] tion, the electrocatalytic effect of the CNT-modified
(n=16) (n=22) (n=11) CFMs to DA was much less pronounced when compared
LOD (mM) 18.1  28.7 2.6  3.1 1.6  1.7
with AA. Therefore, a very high selectivity was expected
[2.9–33.5] [1.2–4.0] [0.5–2.7]
(n=16) (n=22) (n=11) against the majority of electroactive compounds present
Linearity 0.995  0.002 0.990  0.013 0.992  0.012 in the brain extracellular space. In fact, the recording de-
(R2) [0.991–0.998] [0.985–0.996] [0.983–1.000] picted in Figure 3 demonstrates that the interferents DA,
(n=16) (n=22) (n=11) NA, DOPAC, NO2 added sequentially did not induce
Response 0.35  0.25 0.30  0.10 0.50  0.25 measurable current changes, confirming the high selectivi-
time (s) [0.20–0.55] [0.25–0.40] [0.25–0.70]
ty of these modified-microelectrodes for AA measure-
(n=12) (n=14) (n=6)
ments.

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Full Paper N. R. Ferreira et al.

Fig. 4. Average amperometric recording of AA dynamics in rat hippocampus in vivo using CFM/Nafion/SWCNT following stimula-
tion with glutamate 20 mM. Square wave voltammograms were carried out for each phase of the response (inset). The amperometric
current was recorded at + 0.05 V vs. Ag/AgCl. SWV parameters: f = 50 Hz, Es = 2 mV, Esw = 10 mV.

3.5 Real Time Measurements of Brain Extracellular

Ascorbate
The CFM/Nafion/SWCNT coupled to amperometry were
used for measuring rapid changes of extracellular AA in
the rat brain evoked by local stimulation with the excita-
tory neurotransmitter glutamate. The identity of the re-
leased compound was also assessed by SWV. Figure 4
shows a representative in vivo amperometric recording
(hold potential: + 0.05 V vs. Ag/AgCl) using a CFM/
Nafion/SWCNT microelectrode following local pressure
ejection of 20 mM Glutamate to induce the release of
AA in the hippocampal CA1 region of the rat hippocam-
pus. Glutamate stimulation induced a rapid increase in
the oxidation current corresponding to an AA concentra-
tion change of 400 mM according to the post-calibration.
The AA concentration dynamics evoked by glutamate
induced a biphasic signal, an observation that was not de-
scribed in previous studies carried out in hippocampus,
cortex and striatum [13,32,33]. In order to demonstrate
that the current signal was derived from AA oxidation,
SWV has been performed before and after glutamate
stimulation as depicted in Figure 4 Insets. On basis of
pharmacological studies, the mechanism underlying gluta-
mate evoked AA efflux into brain extracellular space
likely involves the glutamate/ascorbate heteroexchange
[2,33].
Considering that extracellular AA has been shown to
be homeostatically regulated at the expense of intracellu-
lar stores [2,34] a further strategy to provide a biologically
relevant AA measurement rely on the biochemical oxida-
tion of AA with AAOx. Thus, basal AA concentration in
the CA1 region of the hippocampus was also assessed by
measuring the decrease in current with CFM/Nafion/
SWCNT microelectrodes following pressure ejection of
ca. 50 nL of AAOx (0.1 mg/mL) as shown in Figure 5.
The current change was converted to AA concentration
according to post-calibration, corresponding on average
to 290  130 mM (n = 12), which is consistent with the
value reported previously in rat hippocampus by using
electrochemically pre-treated CFMs [13]. Local ejection
of glutamate (20 mM) following AAOx did not induce
any measurable oxidation current (Figure 5).
4 Conclusions
Since the beginning of in vivo voltammetry in the brain
tissue, AA has gained recognition as a neuromodulator
linked to extracellular glutamate and other monoamine
neurotransmitters. The fast and selective measurement of
AA with CFMs became challenging in the brain extracel-
lular space mainly because of the interference of DA and

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Real Time In Vivo Measurement of Ascorbate in the Brain
Fig. 5. Average amperometric current recording at + 0.05 V of
basal AA in the CA1 region of the rat hippocampus following
ejection of AAOx (0.1 mg/mL).
its metabolite DOPAC. By using CFMs and a nanocompo-
site film based on the CNTs and Nafion we have devel-
oped a novel AA microelectrode that fulfils the criteria
of size, speed, selectivity and sensitivity required for di-
rectly probing in vivo the temporal and spatial dynamics
of brain chemicals. Due to the electrocatalytic properties
of CNTs, the modified-CFMs revealed a very high selec-
tivity against major interfering compounds present in the
brain extracellular space, as well as appropriate sensitivity
and limit of detection for the measurement of AA. Re-
gardless of the observed decrease in sensitivity during in
vivo implantation, the nanocomposite film prepared from
Nafion provides excellent biocompatibility which is essen-
tial for long in vivo recordings in brain tissue.
This study demonstrates that when combined with am-
perometry the modified CFMs allow not only the measure-
ment of basal extracellular AA in the rat hippocampus but
also the real-time changes following stimulation with gluta-
mate with high selectivity, sensitivity and spatio-temporal
resolution. Hence, the modified-microelectrode described
in the present work could be of interest for assessing the
role of AA in brain physiology and pathophysiology such
as, for instance, in Huntingtons disease.
Acknowledgements
Financial support from Fundażo para a CiÞncia e Tecno-
logia (FCT) Portugal, Grant PTDC/SAU-BEB/103228/
2008, PTDC/SAU-NEU/103538/2008 and PEst-C/SAU/
LA0001/2013-2014. NF and RS acknowledge doctoral fel-
lowship SFRH/BD/65396/2009 and postdoctoral fellow-
ship SFRH/BPD/82220/2011, respectively from FCT.
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