Univerza v Ljubljani
Fakulteta za matematiko in fiziko
Sensing with electrochemical cell
Miha Devetak
Supervisor: Prof. Dr. Dragan Mihailović
Abstract
Electrochemical cell can be used for various purposes and one of them is sensing of specific
types of species, for instance nitric oxide (NO). For such specific tasks specially designed
microelectrodes are used that consist of carbon fibre, thermally sharpened, and coated with
polymers in order to apply selectivity to the sensor. It is important to understand physical
processes that enable selective detection or even manipulation in vitro or in situ.
Ljubljana, April 2006
1
Index
1
Introduction
Page 3
2
Electrochemical Cell
2.1
Potentials in Electroanalytical Cells
2.2
The Thermodynamics of Cell Potential
2.3
Effect of Activity on Electrode Potential
2.4
Currents in Electrochemical Cells
2.5
Ohmic potential; IR Drop
2.6
Polarization
2.7
Ideal Polarized and Nonpolarized Electrodes and Cells
2.8
Sources of Polarization
2.9
Overvoltage
2.10 Concentration polarization
2.11 Potentiostat
Page 5
Page 6
Page 6
Page 7
Page 8
Page 8
Page 8
Page 9
Page 10
Page 11
Page 11
Page 12
3
Carbon Fibre Based Microelectrode
3.1
Microelectrode Surface Modification
3.2
Selectivity
3.3
Response Time
3.4
NO Detection in Brain Hippocampal Slices
Conclusion
Page 13
Page 14
Page 15
Page 16
Page 16
Page 17
4
References
Page 18
2
1. Introduction
Sometimes it is important to measure specific types of molecules, like nitric oxide (NO) to
monitor and understand reactions and processes that are occurring inside of a cell in vitro or in
even in situ. One way of achieving this is with a sensor that is based on an electrochemical cell.
[1] Understanding physics behind this method can help us research different possibilities this
method has to offer. Robert F. Furchgott, Luis J. Ignarro and Ferid Murad have received the
Nobel Prize for discovery of nitric oxide as signaling molecule in cardiovascular system in 1998.
Figure 1: Two microelectrodes next to a bovine adrenal medullary cell along with a small pipette.
[1]
Nitric Oxide
Nitric oxide is an important bioregulatory molecule, being responsible, for example, for activity
of endothelium-derived relaxing factor (EDRF). Acute hypertensia, diabetes, ischemia and
atherosclerosis are associated with abnormalities of EDRF. Although most extracellular signals
are hydrophilic molecules that bind to receptors on the surface of the target cell, some signal
molecules are hydrophobic enough and/or small enough to pass readily across the target-cell
plasma membrane. Once inside, they directly regulate the activity of a specific intracellular
protein. An important and remarkable example is the gas nitric oxide (NO), which acts as a signal
molecule in both animals and plants. [2] In mammals, one of its functions is to regulate smooth
muscle contractions. Acetycholine, for example, is released by autonomic nerves in the walls of
blood vessels, and it causes smooth muscles cells to relax. The acetycholine acts indirectly by
inducing the nearby endothelial cells to make and release NO, which then signals the underlying
smooth muscle cells to relax. This effect of NO on blood vessels provides an explanation for the
mechanism of action on nitroglycerine, which has been used for about 100 years to treat patients
with angina (pain resulting from inadequate blood flow to the heart muscle). The nitroglycerine is
3
converted to NO, which relaxes blood vessels. This reduces the workload on the heart and, as
consequence; it reduces the oxygen requirement on the heart muscle.
Many types of nerves cells use NO gas to signal to their neighbors. The NO released by
autonomic nerves in the penis, for example causes the local blood vessel dilation that is
responsible for penile erection. NO is also produced as a local mediator by activated
macrophages and neurophilis to help them kill invading microorganisms. In plants, NO is
involved in the defensive responses to injury or infection.
Figure 2: The role of nitric oxide (NO) in smooth muscle relaxation in a blood vessel wall.
Acetylcholine released by nerve terminals in the blood vessel wall activates NO synthase in
endothelia cells lining the blood vessel, causing the endothelia cells to produce NO. The NO
diffuses out of the endothelia cells and into the underlying smooth muscle cells, where it binds to
and activates guanylyl cyclase to produce cyclic GMP. The cyclic GMP triggers a response that
causes the smooth muscle cells to relax, enhancing blood flow through the blood vessels. [2]
NO gas is made by the deamination of the amino acid arginine catalyzed by the enzyme NO
synthase. Because it passes readily across membranes, dissolved NO rapidly diffuses out of the
cell where it is produced and into neighboring cells. It acts only locally because it has a short
half-life – about 5-10 seconds – in extracellular space before it is converted to nitrates and nitrites
by oxygen and water. In many target cells including endothelia cells, no binds to iron in the
active of the enzyme guanylyl cyclase, stimulating this enzyme to produce the small intracellular
mediator cyclic GMP (Figure 2). The effects of NO can occur within seconds because the normal
rate of turnover of cyclic GMP is high: a rapid degradation by phosphodiestarase constantly
balances the production of cyclic GMP from GTP by guanilyl cytalase. The drug Viagra inhibits
this cyclic GMP phosphodiesterase in the penis, thereby increasing the amount of time that cyclic
GMP levels remain elevated after NO production is induced by local nerve terminals. The cyclic
GMP, in turn, keeps blood vessels relaxed and the penis erect. [2]
Carbon monoxide (CO) is another gas that is used as intracellular signal. It can act in the same
way as NO, by stimulating guanylyl cyclase. These gases are not the only signal molecules that
can pass directly across target-cell plasma membrane. A group of smaller, hydrophobic,
nongaseous hormones and local mediators also enter target cells in this way. But instead of
binding enzymes, they bind to intracellular receptors proteins that directly regulate gene
transcription. [2]
4
2. Electrochemical cell
The sensor is actually an electrochemical cell.
Figure 3: A galvanic electrochemical cell with a salt bridge [1]
A DC electrochemical cell consists of two electrical conductors called electrodes, each immersed
in a suitable electrolyte solution. For a current to develop in a cell, it is necessary that the
electrodes be connected by means of a metal conductor, that the two electrolyte solutions be in
contact and to permit movement of ions from one to another, and that an electron transfer
reaction can occur at each of the two electrodes. Figure 3 shows an example of a simple
electrochemical cell. It consists of a zinc electrode immersed in a solution of zinc sulphate and a
copper electrode in a solution of copper sulphate. The two solutions are joined by a salt bridge,
which consists of a tube filled with solution that is saturated with potassium chloride, or
sometimes, some other electrolyte. The two ends of the tube are equipped with porous plugs that
permit the movement of ions across them but prevent siphoning of liquid from one electrolyte
solution to the other. The purpose of the bridge is to isolate contents of the two halves of the cell
while maintaining electrical contact between them. Isolation is necessary to prevent direct
reaction between copper ions and the zinc electrode. The cell in Figure 3 contains two so-called
liquid junctions one being the interface between the zinc sulphate solution and the salt bridge; the
second is at the other end of the salt bridge where the electrolyte solution of the bridge contacts
the copper sulphate solution. [1]
It is important to realize that electrochemical measurement involve heterogeneous measurements
systems because an electrode can only donate or accept electrons from species that is present in a
layer of solution that is immediately adjacent to the electrode. Thus, this layer may have a
composition that differs significantly from that of the bulk of solution. [1]
5
Figure 4: Electrical double layer formed at electrode surface as a result of an applied potential.
[1]
For example, let us consider the structure of the solution immediately adjacent to an electrode
when a positive potential is first applied to electrode. Immediately after impressing the potential,
there will be a momentary surge of current, which rapidly decays to zero if no reactive species is
present at the surface of the electrode. This current is charging current that creates an excess (or a
deficiency) of negative charge at the surface of the two electrodes. As a consequence of ionic
mobility, however, the layers of solution immediately adjacent to the electrodes acquire an
opposing charge. This effect is illustrated in Figure 4. The surface of the metal electrode is shown
as having an excess of positive charge as a consequence of an applied positive potential. The
charged solution consists of two parts: (1) a compact inner layer (d0 to d1), in which the potential
decreases linearly with distance from electrode surface and (2) a diffuse layer (d1 to d2), in which
the potential decrease is exponential on a few 100 nm scale (see Figure 4). This assemblage of
charge at the electrode surface and in the solution adjacent to the surface is termed an electrical
double layer. [1]
2.1 Potentials in Electroanalytical Cells
Electroanalytical methods may be based on measurement of either (1) the current in an
electrochemical cell at a fixed potential or (2) the potential of a cell while the current is fixed at
some constant level. In general, however, in an electrochemical experiment the experimenter can
only control the potential of the cell at a desired level and measure the current that results, or vice
versa. Choosing to control one variable precludes any independent control of the other. [1]
2.2 The Thermodynamics of Cell Potential
It is important to understand that the potential of an electrochemical cell is related to the activities
of the reactants and products of the cell reaction and indirectly to their molar concentrations.
Often we shall make approximations that these activities are equal to molar concentrations, but it
should always be borne in mind that this assumption may produce errors in calculated
potentials.[1]
Recall that activity ax of the species X is given by ax =γx[X]
6
Here, γx is the activity coefficient of solute X and the bracket term is the molar concentration of
X.
For instance:
aA+bB ↔ cC+dD
The equilibrium constant for this reaction is given by:
α a xα b
K = Ac B d
α C xα D
where α-s are the activities of the various species indicated by the subscripts.
If some of the activities have the same value and cancel each other, for instance if α B= αC we can
write
K=
α Aa
αDd
It is convenient to define second quantity Q such that
αA a
Q=
αD d
i
i
Here the subscript i indicates that the terms in the parentheses are instantaneous activities and not
equilibrium activities. The quantity Q, therefore, is not a constant but changes continuously until
equilibrium is reached; at that point Q becomes equal to K and the i subscripts are deleted.[1]
From thermodynamics, it can be shown that the change in free energy ∆G for a cell reaction (that
is, the maximum work obtainable at constant temperature and pressure) is given by
∆G=RT ln(Q)-RT ln(K)=RT ln(Q/K)
where R is the gas constant and T is the temperature in kelvins; This relationship implies that the
magnitude of the free energy for the system is from the equilibrium state. It can also be shown
that the cell potential Φcell is related to the free energy of the reaction by the relationship
∆G=-nF Φcell
where F is the faraday (96,485 coulombs per mole of electrons) and n is the number of moles of
electrons associated with the oxidation/reduction process.
Φcell =-(RT/nF) ln(Q)+(RT/nF) ln(K)
The last term in this equation is a constant, which is called the standard electrode potential, Φcell0
for the cell. That is
Φcell0 =(RT/nF) ln(K)
So
Φcell = Φcell0-(RT/nF) ln(Q)
This is a form of a Nernst equation. [1]
2.3 Effect of Activity on Electrode Potential
Let us consider the half reaction
pP+qQ+…+ne-↔rR+sS…
where the capital letters represent formulas of reacting species (whether charged or uncharged), erepresents the electron, and the lower-case italic letters indicate the number of moles of each
species (including electrons) participating in the half-cell reaction.
7
RT  α r R xα s S x... 

ln
nF  α p P xα q Q x... 
At room temperature (289K), the collection of constants in front of the logarithm has units of
joules per coulomb or volt. Therefore,
RT/nF = (8.316Jmol-1K-1298K)/(n 96487 C mol-1) = (2.568x10-2 /n) V
This equation is a general statement of the Nernst equation, which can be applied both to half-cell
reactions or cell reactions. [1]
Φ = Φ0 −
2.4 Currents in Electrochemical Cells
Only one general type of electrochemical method is based upon measurements that are made in
the absence of appreciable current, namely potentiometric methods. The remaining several
methods all involve currents and current measurement. Thus, we need to consider the behavior of
cells when significant currents are present.
Electricity is carried within a cell by the movement of ions. With small currents, Ohm’s law is
usually obeyed and we may write Φ=IR where Φ is potential difference in volts, responsible for
movement of the ions, I is current in amperes and R is resistance in ohm of the electrolyte to the
current. The resistance depends upon the kinds and concentration of ions in solution.
When DC electricity is carried through an electrochemical cell, the measured cell potential
normally departs from the derived thermodynamic calculations. This departure can be traced to a
number of phenomena, including ohmic resistance and several polarization effects, such as
charge-transfer overvoltage, reaction overvoltage, diffusion overvoltage, and crystallization
overvoltage. Generally, these phenomena have the effects of reducing the potential of a galvanic
cell or increase the potential needed to develop a current in an electrolytic cell. [1]
2.5 Ohmic Potential; IR Drop
To develop a current in either a galvanic or an electrolytic cell, a driving force in the form of a
potential is required to overcome the resistance of the ions to movement toward the anode and the
cathode. Just as in metallic conduction, this force follows Ohm’s law and is equal to the product
of the current in amperes and the resistance of the cell in ohms. The force is generally referred to
as ohmic potential, or the IR drop.
The net effect of IR drop is to increase the potential required to operate an electrolytic cell and to
decrease the measured potential of a galvanic cell. Therefore the IR drop is always subtracted
from the theoretical cell potential. That is,
Φcell = Φcathode - Φanode - IR (Equation 1)
2.6 Polarization
Several important electroanalytical methods are based upon current-voltage curves, which are
obtained by measuring the variation in current in a cell as a function of its potential. Equation
(Equation 1) predicts that at constant electrode potentials, a linear relationship should exist
between the cell voltage and the current. In fact, departures from linearity are often encountered;
under these circumstances, the cell is said to be polarized, Polarization may arise at one or both
electrodes.
8
As an introduction to this discussion, it is worthwhile considering current-voltage curves for an
ideal polarized and an ideal nonpolarized electrode. Polarization at a single electrode can be
studied by coupling it with an electrode that is not readily polarized. Such an electrode is
characterized by being large in an area and being based on a half cell reaction that is rapid and
reversible. [1]
2.7 Ideal Polarized and Nonpolarized Electrodes and Cells
Figure 5: Current-voltage curves for an ideal (a) polarized and (b) nonpolarized electrode.
Dashed lines show departure from ideal behavior by real electrodes.
The ideal polarized is one in which current remains constant and independent of potential over a
considerable range. Figure 5a shows a current-voltage curve for an electrode that behaves ideally
in the region between A and B. Here the potential is independent of current.
Figure 6 is a current–voltage curve for a cell having electrodes that exhibit ideal nonpolarized
behavior between points A and B. Because of the internal resistance of the cell, the currentvoltage curve has a finite slope equal to R (Equation 1) rather than the infinite slope for the ideal
nonpolarized electrode shown in Figure 5b. Typical values for the slope, R, are order of
magnitude of a few Ohms. Beyond points A and B, polarization occurs at one or both electrodes,
resulting in departures from the ideal straight line. The upper half of the curve gives the currentvoltage relationship when the cell is operating as an electrolytic cell. Note that when polarization
arises in an electrolytic cell, a higher potential is required to achieve a given current. Similarly,
polarization of a galvanic cell produces a potential that is lower than expected.
9
Figure 6: Current-voltage curve for a cell showing ideal nonpolarized behavior between A
and B (solid line) and polarized behavior (dashed line). (left) [1] An actual measurement:
The growth patterns for poly-TMHPP-Ni, by continuous scan cyclic voltammetry from
-0.2V to 1.2 on a carbon fibre microelectrode. Peaks Ia, Ic correspond to oxidation of
Ni(II) to Ni(III) and reduction of Ni(III) to NI(II) in the film (right) [4]
2.8 Sources of Polarization
Figure 7 depicts three regions of a half-cell where polarization can occur. These regions include
the electrode itself, a surface film of solution. For this half-cell, the overall electrode reaction is
Ox + ne- ↔Red
Anyone of the several intermediate steps shown in the figure may, however, limit the rate at
which this overall reaction occurs and thus the magnitude of the current. One of these steps in the
reaction, called mass transfer, involves movement of Ox from the bulk of the solution to the
surface film. When this step (or the reverse mass transfer of Red to the bulk) limits the rate of
overall reaction and thus the current, concentration polarization is said to exist. Some half-cell
reactions proceed an immediate chemical reaction in which the species such as Ox’ or Red’ form;
this intermediate is then the actual participant in the electron transfer process. If the rate of
formation or decomposition of such an intermediate limits the current, reaction polarization is
said to be present. In some instances, the rate of physical process such as adsorption, desorption,
or crystallization is current limiting. Here adsorption, desorption, or crystallization polarization is
occurring. Finally charge-transfer polarization is encountered, where current limitation arises
from the slow rate of electron transfer from electrode to the oxidized species in the surface of the
film or from the reduced species to the electrode. It is not unusual to encounter half-cells in
which several types of polarization are occurring simultaneous. [1]
10
Figure 7: Steps in the reaction [1]
2.9 Overvoltage
The degree of polarization of an electrode is measured by overvoltage or overpotential η, which
is the difference between the actual electrode potential Φ and the thermodynamic or equilibrium
potential Φeq. That is,
η =Φ-Φeq
where Φ <Φeq. It is important to realize that polarization always reduces the electrode potential
for a system. Thus as we indicated, Φ is always smaller than Φeq, and η is always negative and is
of order of magnitude up to -1 V. [1]
2.10 Concentration Polarization
Concentration polarization arises when the rate of transport of reactive species to the electrode
surface is insufficient to maintain the current demanded by (Equation 1). With onset of
concentration polarization, a diffusion overvoltage develops. For example, consider a cell made
up of an ideal nonpolarized anode and a polarizable cathode consisting of a small cadmium
electrode immersed in a solution of cadmium ions. The reduction of cadmium ion is a rapid and
reversible process so that when potential is applied to this electrode, the surface layer of the
solution comes to equilibrium with the electrode essentially instantaneously. That is, a brief
current is generated that reduces the surface concentration of cadmium ions to equilibrium
concentration, c0 given by
1
0.0591V
φ = φ 0 Cd −
log  (Equation 2)
2
 c0 
If no mechanism existed for transport of cadmium ions from the bulk of the solution to the
surface film, the current would rapidly decrease to zero as the concentration of the film
approaches c0. As we shall see, however, several mechanisms do indeed exist that bring cadmium
ions from the bulk of the solution into the surface layer at a constant rate. As a consequence, the
large initial current decreases rapidly to a constant level that is determined by the rate of ion
transport.
It is important to appreciate that for a rapid and reversible electrode reaction, the concentration of
the surface layer may always be considered to be equilibrium concentration, which is determined
11
by the instantaneous electrode potential (Equation 2). It is also important to realize that the
surface concentration c0 is often far different from that of the bulk of the solution because while
the surface equilibrium is essentially instantaneous achieved, attainment of equilibrium between
the electrode and the bulk of the solution often requires minutes or even hours.
For a current of the magnitude required by (Equation 1) to be maintained, it is necessary that
reactant be brought from the bulk of the solution to the surface layer at the rate dc/dt that is given
by
I = dQ/dt = nFdc/dt
where dQ/dt is the rate of flow of electrons in the electrode (or the current I), n is the number of
the electrons appearing in the half reaction and F is the faraday. The rate of concentration change
can be written as
dc/dt = AJ
where A is the surface area of the electrode in square meters (m2) and J is the flux in mol s-1m-2.
The two equations can be combined to give
I = nFAJ
When this demand for reactant cannot be met by the mass transport process, the IR drop in
(Equation 1) becomes smaller than theoretical, and a diffusion overvoltage appears that just
offsets the decrease in IR. Thus, with appearance of the concentration polarization, (Equation 1)
becomes
Ecell = Ecathode - Eanode +η cathode + η anode - IR
where η anode is the anodic overvoltage. Note that the overvoltage associated with each electrode
always carries a negative sign and has the effect of reducing the overall potential of the cell. [1]
2.11 Potentiostat
A potentiostat is an electronic device that maintains the potential of a working electrode at a
constant level relative to a reference electrode. [1] In order to understand how this circuit works,
consider the equivalent circuit shown in Figure 8a. The two resistances in this diagram
correspond to resistances in two parts of the electrochemical cell shown in Figure 8b. Here Rs is
the cell resistance between the counter electrode and the tip P of reference electrode, and Ru is the
so-called uncompensated cell resistance, which is the cell resistance between P and the working
electrode. Because of the extremely high resistance of the inputs to the operational amplifier,
there is no current in the feedback loop to the inverting input, but the potential difference between
P and the inverting input of the operational amplifier is simply the reference electrode potential
ΦSCE. [1]
Figure 8 Schematic of a system for potentiostaitc coulometry. (a) Equivalent circuit.
(b) Resistance within the cell [1]
12
Recall that in noninverting configuration, the operational amplifier works to keep Φ1 and Φ2 equal
and that the current IC is supplied by the operational amplifier to maintain this condition. If we
consider the path between the inverting input and the circuit common at the output, we see that:
Φ 2 = Φ1 = Φ SCE + I C RU = Φ SCE + Φ C
where ΦC, the cathode potential, is essentially equal to the potential difference between P and the
working cathode (see Figure 8b). Since Φ1 and ΦSCE are constant, ICRU must also be constant. If
RU or RS change in any way during electrolysis, the operational amplifier output voltage changes
in such a way to maintain ΦC= ICRU at a constant level. If RU increases as result of an increase in
the cell resistance or concentration polarization, the output voltage of the operational amplifier
decreases, which leads to decrease in IC; if RU decreases, the operational amplifier output voltage
increases correspondingly to maintain EC constant. [1]
3. Carbon Fibre Based Microelectrode
Now, when we discussed the mechanism of an electrochemical cell, let us have a look of a
practical use in sensing concentration of nitric oxide in vitro. Microsensor used was a p-type
semiconducting polymeric porphyrin and a cationic exchanger (Nafion) deposited on a thermally
sharpened carbon fibre with a tip diameter of ≈ 0.5 µm. The microsensor, which could be
operated I neither the amperometric or voltametric mode, is characterized by a linear response up
to 300µM and a detection limit of 10 nM. Nitric oxide at the level of 10-20mols can be detected in
a single cell. [4]
Figure 9 a) Microscopic photograph of a carbon-fibre microsensor b) electron scanning
micrograph of the part of the isolating wax-rosin mixture c) electron scanning micrograph of a
thermally sharpened tip covered with TMHPP-Ni [4]
Micosensors were produced by threading carbon fibre (7 µm) through the pulled end of a
capillary tube with ≈1cm left protruding. Nonconductive epoxy was put at the glass/fibre
interface. When the epoxy drawn into the tip of the capillary has dried, the carbon fibre was
sealed in place. The carbon fibre was sharpened by gradual burning (propane air microburner
1300-1400oC) as described. The sharpened fibre was immersed in melted wax-rosin (5:1) at a
controlled temperature for 5-15s and, after cooling, was sharpened again. The flame temperature
and the distance of the fibre form the flame needs to be carefully controlled. The resulting
electrode is a slim cylinder with a small diameter rather than a short taper, a geometry that aids
13
implantation and increase the active surface area. Electron scanning microscope shows that the
wax is burned roughly to the top of the sharpened tip. The tip, (length 2-6µm) is the only part of
the carbon fibre, where electrochemical processes can occur. For the sensor to be implanted into a
cell, this length must be less than the cell thickness. The unsharpened end of the fibre was
attached to a copper wire lead with silver epoxy. [1]
A variety of methods for NO measurement are available involving spectrometry,
chemiluminescence, mass spectrometry and electron paramagnetic resonance. Most of the
approaches relay on measurement of secondary and reaction products (e.g., nitrites and nitrates),
and formation of stable adducts (e.g., NO-iron complexes) that require sample processing. They
do not allow real time measurement of endogenous NO production in situ and provide
complementary information that it is difficult to translate into the NO concentration dynamics in
tissues [3]
Carbon fibres were used, because they are mechanically strong and can be controllably
miniaturized for single cell applications (Figure 9). The use of microelectrodes for chemical
detection of NO in tissues makes real time measurements possible, due to high temporal
resolution. The small size of this electrodes allow a high spatial resolution and makes them
excellent sensors for direct placement into biological preparations without causing extensive
damage to tissue. Electrodes coated with polymer modifiers are one of the options. The
combination of the deposited polymer film and a support constitutes a polymer-modified
electrode, and these have better analytical properties, such as selectivity, sensitivity or stability.
In fact, most of the described microelectrodes are based on a conventional two or three-polymer
films design with each film performing specific tasks.
The microelectrode consists of single carbon fibre electrodes coated with nafion acting as an
anionic fibre and electropolimerized with o-phenylenediamine (o-PD), a polymer that works as a
molecular filter, limiting the access of large molecules to the microelectrode surface. The main
goal was to develop a microelectrode that could be sensitive to low nanomolar NO concentrations
and would be selective [3]
3.1 Microelectrode Surface Modification
The microelectrodes were firstly coated with Nafion by dipping the carbon fibres into a Nafion
solution (5% in aliphatic alcohols) at room temperature for 30s and drying for 10 min at 170oC in
a drying oven. Nafion is a sulfonated tetrafluorethylene copolymer. It is the first of a class of
synthetic polymers with ionic properties which are called ionomers. [5] It has been shown that
drying Nafion at high temperatures produces microelectrodes with improved recording
properties. The o-PD film was electrodeposited. Briefly, a 4 mM o-PD solution in PBS
supplemented with 100 µM AA was made fresh and electropolimerized on the carbon fibre
surface by amperometry at a constant potential of +0.9V versus Ag/AgCl during 15 minutes. [3]
The microelectrode tip was placed in CA 1 region of hippocampus, 200 µM deep into the slice of
the Wistar rat brain, with the aid of a micromanipulator and a dissection microscope. Once a
stable background was obtained; slices were challenged by perfusion during 2 min with either a
10µM NMDA solution or a 5mM L-glutamate solution.
In the study the rate of NO release from DETA/NO solution was determined. Figure 10 shows the
time course of spontaneous NO release after addition of 100µM DETA/NO to a deoxygenated
PBS (pH 7.4) solution at 22oC. As can be observed, the current increased continuously and
14
reached a steady-state value NO concentration of 1µM after 1h. [3] The ISO-NOP sensor was
previously calibrated following the chemical procedure to determine accurately the NO
concentration.
Figure 10: Amperometric measurement of NO released upon addition of 10µM
DETA/NO(arrow), a NO-generating compound to a deoxygenated PBS solution. The current
values, measured with ISO-NOP sensor were converted to NO concentrations, according to the
calibration curve. [3]
3.2 Selectivity
The parameter is crucial for reliable NO measurements in tissues due to its high oxidation
potential. Thus, in the amperometric mode, we must be careful with electroactive species present
in the brain, such as catechols, indoles and ascorbate, which oxidize well below the potential of
NO (between +0.1 and +0.5 V).
Using amperometry (+0.9 V versus Ag/AgCl), the selectivity of microelectrodes against a series
of potential interfering compounds was assessed. Table 1 shows the selectivity ratios NO;
interferent, calculated on a molar basis for the tested interferents that produced a measurable
current. [3] The data suggests that one layer of Nafion conferred highly selective versus anions.
However it was not so selective with cations.
Table 1 [3]
NO selectivity ratios for the Nafion/o-PD microelectrodes against various interfering compounds
15
3.3 Response Time
To evaluate the influence of diffusion rate f crossing the coatings, a bare microelectrode was used
as a control. The average half-maximum response time at a bare carbon fibre microelectrode after
the injection of 26 µM DETA/NO solution was 0.95± 0.15 s (n=4). On average microelectrodes
coated with Nafion/o-PD exhibited half-maximal response times of 1.90±0.1 s (n=9), which is ca.
1 s higher when compared with bare microelectrodes. Thus, the response time of the polymermodified microelectrode to t50% is 1 s. [3]
3.4 NO Detection in Brain Hippocampal Slices
Figure 11A shows a typical in situ amperometrical recording of NO production in hippocampal
slices using the polymer-modified carbon fibre microelectrodes. The perfusion of 5 mM Lglutamate for 2 min evoked a transient increase in NO current, measured with the microelectrode
placed in CA1 subregion. [3] The maximum current was reached in 5 min, whereas the decay to
baseline was characterized by a time constant of 384 s, as calculated by a non linear regression
analysis of the signal decay using an exponential first order equation. The total charge of the
pulse calculated by integration of current/time profile was 94nC. [3]
Figure 11: L-glutamate-induced efflux (current/time profile) of NO recorded in the CA1 region of
the hippocampal slice following a 2 min perfusion of 5mM L-glutamate (A) and 10 µM NMDA
(B). [3]
16
In another set of experiments, a L-glutamate stimulus was replaced by perfusion with 10µM
NMDA, during 2min (Figure 11B). Under these stimulation conditions, the NO production was
characterized by a total charge of 128 nC. The signal increase reached the maximum current in
4.2 min, and an exponential decay with time constant of 340 s. It is apparent that stimulation with
10 µM, NMDA promotes a higher overall production of NO than 5mM L-glutamate.
Nevertheless when decay constants are determined, differences are not statistically significant
(P>0.05), as measured by a Student’s t-test. [3]
Thus, on basis of decay time constants, and as would be expected for a selective measurement of
NO, the concentration dynamics of NO produced either by stimulation with L-glutamate or
NMDA, follows a similar pattern. [3]
4. Conclusion
Nafion/o-PD modified carbon fibre microelectrodes, are capable of sensitive id and reproducible
measures of NO. NO2- is not a major interferent for detection of NO. The in vitro brain slice
studies support the notion that the microelectrodes can be used to study NO signaling in brain
slices. Advantages of sensing with microelectrodes using the principle of electrochemical cell are
obvious. Selectiveness, high resolution and real time measurements are three of many attributes
that make such a device a first class sensor. Physics and chemistry behind the mechanism of
microelectrode based electrochemical cell are complex but offer many possibilities in further
development of such devices. The applicability of selective microelectrodes is as in measurement
of a specific species as in electronic manipulation of species which is the next logical step that
can be used for treatment and research in biotechnology, medicine, chemistry and physics.
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References:
[1] Skoog D. A., Holler F.J., Nieman T. A., Principles of Instrumental Analysis, 1998, Harcourt
Brace & Company, USA
[2] Alberts B., Johnson A., Lewis J., Raff M., Roberts K., Walter P., Molecular Biology of the
Cell, 2002, New York
[3] Ferreira N. R., Ledo A., Frade J. G., Gerhardt G. A., Laranjinha J., Barbosa R. M. Analytica
Chemica Acta, 2005, 535, 1-7
[4] Malinski T., Taha Z., Letters To Nature, 1992, Vol 458, 676-678, 20 August
[5] http://en.wikipedia.org/wiki/Nafion (9.4.2006)
[6] Wightman M. R., Science, 2006, Vol 331, 1570-1574, 17 March
[7] http://www.esainc.com/products/HPLC/EC_Detectors/esa_ECcell_designs.html (11.4.2006)
[8] http://www.scientifica.uk.com/a_productdetails.php?id=120 (11.4.2006)
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