Chip Based Electroanalytical Systems For Monitoring Cellular Dynamics

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Chip Based Electroanalytical Systems for Monitoring Cellular Dynamics
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CHIP BASED ELECTROANALYTICAL SYSTEMS
FOR MONITORING CELLULAR DYNAMICS
A. HEISKANEN, M. DUFVA, AND J. EMNÉUS

Department of Micro- and Nanotechnology, Technical University of
Denmark, Ørsteds Plads 345 East, DK-2800 Kgs. Lyngby, Denmark,
Jenny.Emneus@nanotech.dtu.dk
Abstract. Electroanalytical methods are highly compatible with micro- and

nano-machining technology and have the potential of invasive but “non-
destructive” cell analysis. In combination with optical probes and imaging
techniques, electroanalytical methods show great potential for the development
of multi-analyte detection systems to monitor in real-time cellular dynamics.
1. Introduction
In cell biology and pharmacology, the determination of cellular functions

and responses to exogenous effectors is a general part of the scientific quest.
However, assays that, for instance, determine the activity of an enzyme
upon induction of gene expression are customarily conducted after the cells
have been lysed, and the resulting cell extract or a further purified enzyme
fraction is used for the assay [1]. Furthermore, high-throughput screening
(HTS) of compound libraries in drug discovery has strongly relied on assays
conducted using purified or isolated targets, i.e. enzymes, ion channels,
signaling proteins as well as cell surface- and nuclear receptors [2]. The
fundamental question is: How reliable and true are the obtained results that
are based on an isolated fraction of the whole system, i.e. a cell, organ or
organism? In a living cell, the different cellular functions and subcellular
compartments, although to a certain degree autonomous, they are at the
same time strongly dependent on feedback from each other. As examples
can be mentioned the activity of enzymes that transfers electrons to or
from different cellular metabolites, such as glucose, which is the main
energy source of mammalian cells, and the activation of G protein coupled
receptors (GPCRs) that mediate external signals upon binding of a ligand to
the receptor, giving rise to an intracellular cascade of biochemical events,
leading to the execution of a certain function.
The significance of cell-based assays as a source of information that
yields a more holistic view of the cellular dynamics, i.e. the interaction of
S. Kakaç et al. (eds.), Microfluidics Based Microsystems: Fundamentals and Applications, 399
DOI 10.1007/978-90-481-9029-4_19, © Springer Science + Business Media B.V. 2010
400 A. HEISKANEN, M. DUFVA, AND J. EMNÉUS
biological functions in general and the intercompartmental biological effect

of different compounds has been recognized in HTS of compound libraries
in drug discovery. The advantages are associated with the involvement of
the entire cellular environment as the modulator of the monitored responses
whether these are primarily connected to the activation of GPCRs, ion
channels or enzyme activity. If an assay involves binding of a ligand to a
receptor (target), this takes place in the real biological environment of the
target. Additionally, when the target is located in the intracellular environment,
a cell-based assay also gives possibility to screen for secondary cellular
events (multi-parameter monitoring) as well as bioavailability of the used
test compounds [3]. Work featuring methods and techniques for assaying
biological parameters in the context of intact cells comprise e.g. intracellular
[4] and extracellular [5] monitoring of oxygen consumption, monitoring of
enzyme activity and cofactor availability [6–9], cellular adhesion [10] as
well as cellularly released secondary metabolites [11, 12] and G-protein
coupled receptor (GPCR) activation [13].
Although cell-based assays have been strongly implemented in drug
discovery, they are primarily in microtiter plate format, including applications
for even 1,536 well-plates [14] and screening of compound libraries of
100,000 compounds [15]. Implementation of cell-based assays in HTS
suffers, however, from problems caused by the microtiter plate format.
These concern reliability of temperature and CO2 control in the incubator as
well as increased evaporation and difficulties involved in liquid handling
due to the large number of wells comprising an extremely small volume
[15]. The emergence of perfusion based microfluidic cell culture chips
(see Chapter “Perfusion Based Cell Culture Chips” of this book) with the
inherent technological capability to undergo sufficient miniaturization and
parallelization represents a new trend that can both alleviate the drawbacks
of microtiter plate based assays and facilitate real-time monitoring of
cellular dynamics instead of only end-point detection.
2. Detection of Cellular Dynamics
2.1. DETECTION TECHNIQUES
2.1.1. Fluorescence Detection

As consequence of implementation of parallelization in microfluidic cell
culture chips, detection of biologically relevant cellular parameters imposes
further requirements on the development of the applied detection techniques.
Using available motorized microscope stages, time-lapse fluorescence
microscopy is a widely applied technique in monitoring cellular responses.
Alternatively, fluorescent plate readers facilitate real-time monitoring in
highly parallelized systems (readouts for 1,536 well microtiter plate format).
CHIP BASED ELECTROANALYTICAL SYSTEMS 401
Implementation of fluidic functions inside a plate reader is however not a

straightforward task. For short-term detection, the chip depicted in Fig. 10f
[16] in the Chapter “Perfusion Based Cell Culture Chips” of this book could
be amenable for use with a plate reader since its function does not require
any external pump. However, for long- term real-time monitoring, this system
is not suitable due to the need for a CO2 incubator. An alternative approach
could be based on a multichannel pump, suitable for integration with a
polymeric microfluidic cell culture chip independent of a CO2 incubator.
The pump shown in Fig. 10b [17] in the Chapter “Perfusion Based Cell
Culture Chips” of this book could function as the basis for such an
approach.
Fluorescence based monitoring of dynamic cellular processes is not,
however, a complete solution to the need of detection in cell culture
systems. Long-term monitoring causes photobleaching of fluorophores and
photodamage to the cells in the case of autofluorescence detection [18]. To
alleviate the problem with photobleaching [19] and photodamage [20], two-
photon excitation microscopy has emerged as a microscopic technique.
However, the required instrumentation is expensive and the technique itself
does not facilitate easy automation and high-throughput monitoring. Further-
more, fluorescence detection, in general, is not suitable for monitoring of all
relevant parameters of cellular dynamics. For instance, monitoring of calcium
triggered vesicular release (exocytosis) of cellular secondary metabolites,
such as the neurotransmitter dopamine or hormone insulin, is not possible
directly using fluorescence detection. Only indirect detection of the process
has been demonstrated using, for instance, internal reflection fluorescence
microscopy of co-exocytosed fluorescent dye upon loading of cellular
vesicles [21].
2.1.2. Electrochemical Detection

In many cases, monitoring of cellular dynamics involves detection of
molecules that are either released or taken up by the cells. Monitoring of such
parameters comprises (i) nutrients and primary metabolites (e.g. glucose
[22], lactate [23] and oxygen [5]), (ii) secondary metabolites, such as neuro-
transmitters (e.g. dopamine [12] and glutamate [11]) and hormones (e.g. insulin
[24]), and (iii) compounds resulting from xenobiotic1 metabolism and physical
stress (e.g. hydrogen peroxide and superoxide radical [25], glutathione
conjugates [26] quinones [27] and quinols [28]). All the listed compounds
are examples of species that can be detected electrochemically either directly
as electroactive species or by using enzymes as the biorecognition element.
______
1
In xenobiotic metabolism, foreign compounds, such as drugs and toxicants, are
enzymatically detoxified.
Figure 1 illustrates the detection scheme of such cellular factors: Extracellularly

placed electrodes are used to detect compounds that are produced in cellular
metabolism and released by the cells either based on active transport or
diffusion through the plasma membrane. Analogously, compounds used by
the cells in their metabolism are taken up from the extracellular environment
and consequently the decrease in concentration is detected.
Figure 1. A schematic illustration of electrochemical monitoring of cellular dynamics. An

extracellularly placed electrode is used to detect cellular release and uptake of molecules.
Traditionally, extracellular microelectrodes that have been used to

detect, for instance, release of compounds have been placed adjacent to
the cell body using a micromanipulator [29] or scanning electrochemical
microscope (SECM) [27]. Such measurements are normally conducted
using cells that form a part of a population in a culture vessel, such as a
Petri dish. Although the published results, have contributed to a highly
accurate and mechanistic description of the studied biological phenomena
based on single-cell measurements, the approach to use micromanipulated
microelectrodes has some severe drawbacks. Detection cannot be automated
and throughput is limited. Furthermore, the required instrumentation and
operational skills are beyond what normally are needed for electrochemical
detection. A new approach has emerged that applies microchips having
planar microelectrodes on a substrate, most commonly an oxidized surface
of a silicon wafer (for fabricational aspects, see [30]), simultaneously
functioning as the substrate, on which cells either sediment or grow.
The approach to use chip based electroanalytical systems to monitor the
dynamics of processes in living cells facilitate the possibility to integrate the
detection systems to microfluidic cell culture chips. In virtue of the functional
principle of such systems, cells can be cultured on the platform where
detection takes place. Hence, the measurements can be conducted in an
environment that has been tailor-made for proper adaptation to the require-
ments of the cultured cells. Furthermore, such miniaturized systems possess
the capability to achieve operational automation and facilitate measurements
on a small population of cells or even single cells. Aside from single-cell

measurements, the miniaturization of systems, comprising a microfluidic
cell culture chip with an integrated microchip for electrochemical detection,
also enables utilization of small amounts of chemicals that are needed as
cellular effectors to, for instance, trigger certain metabolic responses. This
feature facilitates cell-based assays with a high degree of parallelization
without extensive increase in the incurred expenses.
Electrochemical measurements on cells are primarily conducted using
impedimetric [10], potentiometric [31] and amperometric measurements
[12]. In this chapter, amperometric measurements are described based on
examples comprising monitoring of cellular redox environment and detection
of exocytosis, i.e. Ca2+-triggered release of cellular secondary metabolites,
e.g. the neurotransmitter dopamine [12]. In amperometric measurements,
most commonly, a three-electrode configuration controlled by a potentiostat
is applied: A microelectrode functioning as the working electrode (WE) is
in direct contact with the cells the measurements are to be conducted on
and has a certain poised potential, at which the detected chemical species
is either oxidized2 or reduced. A reference electrode (RE) is used under
potentiostatic control to adjust the applied potential of the WE with respect
to the third electrode, the counter electrode (CE),3 which is the site of the
electrochemical process complementary to the one taking place at the WE.
If oxidation of a chemical species involving the donation of n electrons is
detected at the WE, a corresponding number of electrons are accepted
arbitrarily at the CE by any chemical species in the electrolyte that is used
as the medium of measurements. Hence, the electrochemical processes
taking place at the electrode–electrolyte interface of the WE and CE
together with the potentiostat form a closed electrical circuit that facilitates
movement of electrons that can be registered as a faradaic current. The
faradaic current (I), according to Faraday’s law of electrolysis, is directly
proportional to the number of moles of molecules oxidized or reduced
(Eq. (1)):
Q nFn x
I= = (1)
t t
where Q is the total charge carried by the electrons that are accepted or
donated, t is the time during which the current is recorded, n is the number
______
2
In amperometry, oxidation refers to donation of a number of electrons from a chemical
species to an electrode and reduction refers to acceptance of a number of electrons by a
chemical species from an electrode.
3
In electrochemical literature, counter electrode is oftentimes also called auxiliary
electrode.
of accepted or donated electrons, F is the Faraday constant (96,485 C mol–1)

and nx is the number of moles of the chemical species oxidized or reduced.
In amperometry, the applied potential at the WE is chosen to provide a
sufficient driving force, overpotential, for the desired electrochemical
process, i.e. oxidation or reduction. Each electroactive chemical species is
characterized by a certain reduction potential (E°), at which the oxidized
and reduced form of the species are in equilibrium.4 Since oxidation or
reduction of different chemical species that are detected in biological
systems is also pH dependent due to involvement of proton transfer, the
formal potential (E°′) is used instead of reduction potential. The tabulated
values of formal potential for different chemical species are usually valid at
pH 7. The formal potential also implicitly comprises the contribution of
activity coefficients. A sufficient overpotential for oxidation or reduction is
obtained by poising the WE at a potential that is more positive or negative,
respectively, than the formal potential of the detected species. This means
that at the chosen potential, predominantly either oxidation or reduction
takes place independent of whether both the oxidized and reduced component
of the redox couple are present. For example, in a system containing the
redox couple, ferrocyanide ([Fe(CN)6]4–)/ferricyanide ([Fe(CN)6]3–), which
has the reduction potential 274 mV with respect to a Ag/AgCl5 RE [32], at
an applied potential of 400 mV with respect to a Ag/AgCl RE, [Fe(CN)6]4–
is oxidized whereas [Fe(CN)6]3– is not affected by the electrode process.
Generally, the effect of an applied overpotential at a WE can be presented
using an energy level diagram schematically depicting the energy levels of
the electrons in the electrode material as well as the lowest unoccupied
(LU) and highest occupied (HO) molecular orbital (MO). When the over-
potential is sufficiently positive, to make the energy of electrons in the
electrode material lower than that of the energy of the HOMO of the species
to be oxidized, electrons can be donated to the electrode, resulting in
oxidation (Fig. 2A). In the opposite case, a negative potential rendering the
______
4
At equilibrium, the oxidized and reduced form of an electroactive species, collectively
termed as redox couple, are reduced and oxidized, respectively, at an equal rate.
5
Most often, the tabulated values of formal potential are given with respect to the normal
hydrogen electrode (NHE), which has the defined potential 0 V. However, in practise, a
silver/silver chloride (Ag/AgCl) electrode or a plain metal surface (e.g. Au or Pt) is
commonly used as a RE. An Ag/AgCl RE, having an internal electrolyte of saturated KCl,
has a characteristic potential of 197 mV with respect to the NHE. A plain metal surface, on
the other hand, does not have a characteristic potential that can be expressed in terms of
NHE. Instead, its potential depends on the prevailing conditions, affected by the deposited
species and the electrolyte. E.g., if a Au surface is used as an RE to adjust the potential of,
for instance, another Au surface (WE), both the RE and WE are affected by the same
conditions. The equilibrium potential between such electrodes is ideally 0 V and a poised
potential is directly an overpotential with respect to the equilibrium potential.
electrons in the electrode material with an energy higher than that of the
energy of the LUMO of the species to be reduced, electrons can be donated
by the electrode, resulting in reduction (Fig. 2B).
Figure 2. Energy diagrams schematically illustrating oxidation-reduction (redox) reactions.

(A) An electrode material with a sufficiently positive potential can accept an electron from the
highest occupied molecular orbital (HOMO) of species A, which is oxidized (A → A+ + e−).
(B) An electrode material with a sufficiently negative potential can donate an electron to the
lowest unoccupied molecular orbital (LUMO) of species B, which is reduced (B + e− → B−).
3. Monitoring of Cellular Redox Environment
3.1. CELLULAR REDOX ENVIRONMENT
3.1.1. Cellular Redox Couples

Organisms obtain the necessary energy and building blocks for cellular
functions, such as locomotion, contraction and biosynthesis, from digested
food. The main constituents of food, carbohydrates, fats and proteins,
are digested to the monomers making up the biopolymeric structures.
Carbohydrates consist of different hexoses, such as glucose, fructose and
galactose. Fats are esters of glycerol and fatty acids with different length
of carbon skeleton. Proteins are formed of amino acids through amide
linkages. The resulting hexoses, glycerol, fatty acids and amino acids are
taken up by cells, where they undergo further degradation, i.e. catabolic
processes. Partly, products of the catabolic processes are utilized for synthesis
of new biomolecules needed for building new cellular material to maintain
the cellular structures and sustain the needs of growth, i.e. anabolic processes.
However, these processes require energy, which also comes from the cata-
bolic processes. In order not to release the entire energy contents in one
single process, which would be too exothermic for the cells to bear, the cells
may store the energy in the form of catabolic intermediates, e.g. reduced
cofactors nicotinamide adenine dinucleotide (NADH) and nicotinamide
adenine dinucleotide phosphate (NADPH) as well as acetyl coenzyme A
(Acetyl-CoA), the energy of which can be released in subsequent processes
to synthesize, for instance, adenosine-5′-triphosphate (ATP) for energy
requiring cellular processes. Collectively, NADH and NADPH as well as
the corresponding oxidized forms, NAD+ and NADP+, respectively, are
referred to as cellular redox couples. Examples of other redox couples are
flavin adenine dinucleotide (FAD-FADH2) involved in metabolic processes,
and glutathione (GSSG-GSH) involved in cellular detoxification processes
to alleviate, for instance, oxidative stress. The general functional principle
of cellular redox couples is to participate in enzymatic processes catalyzing
oxidation or reduction of nutrients and other biomolecules. The oxidized form
of a redox couple functions as an electron acceptor, whereas the reduced
form functions as an electron donor.
3.1.2. Definition of Cellular Redox Environment

Each of the cellular redox couples has a characteristic formal potential,
the value of which is valid under equimolar composition of the oxidized
and reduced form. However, the functions of living cells require a non-
equimolar composition. For instance, in the case of the redox couple
NADP+-NADPH, the ratio [NADP+]/[NADPH] << 1, and for NAD+-
NADH, the ratio [NAD+]/[NADH] can approach 1,000. The small value of
[NADP+]/[NADPH] and consequently an excess of NADPH is necessary to
maintain the biosynthetic processes that utilize NADPH. The large value of
[NAD+]/[NADH] indicates the presence of an excess of NAD+, which is
needed to support, for instance, mitochondrial respiration that involves
continuous reduction of NAD+. The actual potential (E) a redox couple has
is dependent on the ratio of the oxidized form to the reduced form according
to the Nernst equation (Eq. (2)):
RT [Ox ]
E = E°' + ln (2)
nF [Red ]
where [Ox] and [Red] are the concentration of the oxidized and reduced
form, respectively, and all the other symbols are as previously described.
Although the prevailing potential of the redox couples, e.g. NADP+/
NADPH and NAD+/NADH, determined by the concentration ratio of the
individual components, indicates the instantaneous direction of cellular
processes, reductive or oxidative, also the actual concentration of the reduced
components are significant in determining the cellular reducing capacity.
The cellular redox environment (CRE) is a combination of the influence of
the potential of different cellular redox couples and their reducing capacity.
Schafer and Buettner have defined CRE as the sum of products of potential
and reducing capacity of each cellular redox couple according to Eq. (3)
[33].
n (redox couple)
CRE = Σ E i × [Red ]i (3)
i =1
3.1.3. The Biological Significance of CRE

CRE has a significant role in controlling different cellular functions,
e.g. signaling and enzyme activation [34], ultimately being responsible
for controlling cellular growth and differentiation. Upon too drastic or
uncontrollable perturbations of CRE, cells may undergo either programmed
cell death (apoptosis) or necrosis. Considering the diversity of cellular
functions involved in the response to and defense against oxidative stress as
well as genetic engineering, a modification of the definition of CRE presented
by Schafer and Buettner [33] is necessary. Aside from only including the
reducing capacity and potential of different cellular redox couples, also the
cellular activity of different redox enzymes, such as cytochrome P450 (cyt
P450), NAD(P)H: Quinone oxidoreductase1 (NQO1)6 and the complexes of
the mitochondrial electron transport chain (ETC), should be taken into
consideration. This would give a more functional definition, which goes
beyond merely observing the redox state of the different redox couples.
Such an approach emphasizes the significance of determining both the
cellular availability of relevant redox couples, such as NAD(P)+/NAD(P)H,
and the activity of certain key enzymes directly in living cells.
Perturbations in CRE may arise as a consequence of cellular functions
as well as environmental factors and pathological disorders. However, no
clear categorization is possible since the causative factors and consequences
may be interrelated. Pathological disorders, such as cancer, may be caused
by perturbations in CRE and when the full pathogenic state has been
______
6
NQO1 is also known as DT-diaphorase.
reached, the disorder itself may cause further perturbations in CRE. On the
other hand, a perturbation in CRE caused by one pathological disorder may
serve as the causative factor for the onset of another disorder. This is, for
instance, valid in the relationship between mitochondrial disorders and
neurodegenerative diseases. An additional cause for perturbations in CRE
has arisen with the emergence of microbial strain engineering. In this case,
the perturbations are desired and capable of improving the strain properties
for a certain application.
Although the normal function of the mitochondrial ETC yields water
upon reduction of O2 by Complex IV through four consecutive one-electron
transfers, according to estimations 1–2% of the O2 taken up by cells results
in formation of H2O2 [35], which originates from superoxide radical (O2–)
generated by the ETC complexes. Through the generation of O2–, mito-
chondria are a major contributor to cellular oxidative stress, which has
been implicated as a causal factor for neurodegenerative diseases, such as
Parkinson’s disease [36], and cancer [37]. The effect of oxidative stress in
the development of different pathological disorders is mediated through
mechanisms involving lipid peroxidation, DNA fragmentation and protein
modification [38]. Cells have different defense mechanisms to counteract
the deleterious effects of oxidative stress. These include, for instance,
enzymatic conversion of O2− into H2O2 and further into water in reactions
involving oxidation of GSH to GSSG. GSSG-GSH is a cellular redox
couple that strongly contributes to the overall CRE. In order to maintain the
reducing capacity of GSH, and hence effective protection against oxidative
stress, cells utilize NADPH-dependent enzymatic reactions for reducing
GSSG. Despite varying functions, the pools of different cellular redox
couples are interconnected. Although the rigorous cellular defense against
oxidative stress is capable of normalizing the perturbations of CRE caused by
normal activity of the ETC, the effect of mitochondrially caused oxidative
stress may be more prominent in pathological disorders that cause abnormal
function of the ETC and deficiency in enzyme activity involved in elimination
of O2−, reduction of GSSG or formation of NADPH.
Organisms are exposed to a myriad of harmful chemicals, such as
quinones, that may induce oxidative stress by increasing the intracellular
concentration of reactive oxygen species (ROS) including O2−. Especially,
the liver cells have enzymes that function as a defense against such external
attack. Two examples of such enzymes are cyt P450 and NQO1. The redox
reactions catalyzed by cyt P450 utilize NADPH as cofactor, whereas those
catalyzed by NQO1 utilize either NADH or NADPH as cofactor. An
additional defense mechanism functioning against the effect of harmful
chemicals is based on the action of GSH, which may either contribute to
scavenging the formed ROS or directly conjugate with the chemicals, after
which they may be expelled from the cells [39]. These examples show a
direct connection between the effect of harmful chemicals and CRE, involving
the pools of different redox couples and ultimately cellular catabolism.
Cyt P450 and NQO1 have a fundamental difference in their function as
defense against harmful chemicals. Cyt P450 catalyzes one-electron reduction
reactions, which, for instance, upon reduction of quinones yield semiquinone
free radicals. These, like free radicals in general, are short lived and tend to
react with biomolecules oxidizing them or with O2 forming of O2−. NQO1,
on the other hand, catalyzes two-electron reductions, which in the case of
quinones yield the fully reduced form, hydroquinone.
Study of the properties of cancer cells has revealed that in certain types
of cancer the expression of the gene encoding for NQO1 is up-regulated in
comparison to normal cells. This has opened the possibility to employ
quinoid substances for chemotherapy, which selectively can affect cancer
cells at the same time minimizing the harmful effects on normal cells [40].
When the enzymatically reduced quinones are auto-oxidized, the resulting
oxidative stress selectively causes apoptosis in cancer cells. In research, the
determination of NQO1 activity in general or screening for NQO1 substrates,
to be used as chemotherapeutic drugs, as well as screening for the inductive
effect of certain compounds on the expression of the gene encoding for
NQO1 in different cell lines [1] has become significant. An additional aspect
concerning cancer cells is that, due to depressed vascularization, and hence
lack of oxygen, they have an up-regulated function of the NADH forming
glycolytic pathway (GP) [41]. This results in increased ATP production
through the Cytosolic substrate level phosphorylation instead of ATP
synthesis in the mitochondria. The other significant consequence is that
NADH from the GP is more abundantly available, indicating that NADH
availability is also significant for the activity of the NQO1.
Especially in microbial strain engineering, metabolic pathways are altered
either by deleting a gene, cloning a gene from another organism, or over-
expressing a naturally existing gene. Applications relying on these approaches
range from fundamental research of cellular functions to industrial exploitation
of microbes. In research towards the elucidation of mechanisms that control
the function of catabolic pathways in Saccharomyces cerevisiae (baker’s
yeast), the gene PGI1 encoding for phosphoglucose isomerase (PGI), the
enzyme that functions as the branching point between the NADH forming GP
and the NADPH forming cytosolic pathway, the pentosephosphate pathway
(PPP), has been deleted. Studies have revealed that as the consequence of
the deletion of PGI1, glucose catabolism is diverted into the PPP, which
very rapidly depletes the NADP+ pool [42]. In an analogous way, fructose is
only catabolized in the GP producing NADH. Hence, the deletion of one
gene strongly influences the reducing capacity of the NADP+-NADPH and
NAD+-NADH redox couple as well as their potential.
During ethanol production from lignocellulosic hydrolysates in S.

cereviciae another problem has been observed; upon acidic pretreatment,
the raw material yields toxic compounds, such as 5-hydroxymethyl furfural
(HMF), which inhibit fermentation [43]. Gene expression analysis of a S.
cerevisiae mutant with increased tolerance to HMF showed that the gene
ADH6, encoding for the NADPH-dependent alcohol dehydrogenase6 (ADH6),
was up-regulated. ADH6 exerts aldehyde reducing activity [44], and is
hence capable of reducing HMF. Over-expression of this gene in a non-
tolerant S. cerevisiae strain made the cells overcome inhibition with the
concomitantly increased consumption of NADPH. Despite the resulting
shift in CRE with respect to the NADP+-NADPH redox couple, the cells are
able to grow in the presence of HMF.
3.2. MEDIATED AMPEROMETRIC REAL-TIME MONITORING

OF CELLULAR REDOX ENVIRONMENT
Coupling between a biologically catalyzed reaction and an electrochemical

reaction, referred to as bioelectrocatalysis, is the constructional principle
for enzyme-based electrochemical biosensors. This means that the flow of
electrons from a donor through the enzyme to an acceptor must reach the
electrode in order for the corresponding current to be detected. In case a
direct electron transfer between the active site of an enzyme and an electrode
is not possible, a small molecular redox active species, e.g. hydrophobic
ferrocene, meldola blue and menadione as well as hydrophilic ferricyanide,
can be used as an electron transfer mediator. This means that the electrons
from the active site of the enzyme reduce the mediator molecule, which, in
turn, can diffuse to the electrode, where it donates the electrons upon
oxidation. When these mediator molecules are employed for coupling of
an enzymatic redox reaction to an electrode at a constant potential, the
resulting application can be referred to as mediated amperometry or mediated
bioelectrocatalysis.
Monitoring of the intracellular redox activity in eukaryotic cells imposes
the requirement that the utilized mediator is capable of readily crossing
the plasma membrane into the intracellular environment to communicate
with the enzyme(s), the activity of which is to be monitored. This strictly
requires the utilization of a lipophilic mediator that can diffuse through the
plasma membrane. Using chip based amperometric detection on S. cerevisiae,
menadione was shown to possess the desired properties [28]. Figure 3
depicts the functional principle of the chip based detection technique to
monitor CRE in eukaryotic cells, which aside from the lipophilic menadione,
also utilizes the hydrophilic mediator, [Fe(CN)6]3−. The lipophilic menadione

(M) diffuses through the plasma membrane into the intracellular environment,
where it is distributed between different subcellular compartments, undergoing
reduction to menadiol (MH2) (Scheme 1) by NAD(P)H-dependent menadione
reducing enzymes (MREs). A short review of MREs and the different
metabolic processes that provide the reduced cofactors for menadione
reduction in S. cerevisiae can be found in the Supplementary Material of
Heiskanen et al. [7]. NAD(P)H originates from the oxidative catabolic
pathways in the cytosol, the PPP and the GP, as well as the mitochondrial
tricarboxylic acid (TCA) cycle that produces NADH. The equally hydrophobic
MH2 diffuses through the plasma membrane into the extracellular environ-
ment, where it can surrender its electrons to [Fe(CN)6]3− upon the concomitant
reduction of [Fe(CN)6]3− to [Fe(CN)6]4− and regeneration of M, which can
continue redox cycling by diffusing back into the intracellular environment.
The bioelectrocatalytic process is completed by oxidation of [Fe(CN)6]4−
at the microelectrode that has been poised at the potential of 400 mV vs. a
Ag/AgCl electrode. For each intracellularly reduced M two electrons are
shuttled to the electrode. Figure 4A shows the obtained current upon
introduction of menadione based on the basal level of NAD(P)H in cells
as well as in response to glucose, which increases the level of NAD(P)H
through the activity of the GP and the PPP. The current-time trace also
illustrates the fact that due to the hydrophilicity of [Fe(CN)6]3−, which
remains in the extracellular environment, no considerable response is obtained.
[Fe(CN)6]3− is used for enhancing the amplitude of the amperometric signal
as well as the kinetics of the response. During real-time measurements, only
2 mM of [Fe(CN)6]3− are needed due to continuous reoxidation of the formed
[Fe(CN)6]4− at the electrode. Figure 4B shows an electrode microchip for
mediated amperometry with S. cerevisiae cells.
Scheme 1. Menadione reduction by MREs to menadiol.

Figure 3. The functional mechanism of the ferricyanide-menadione double mediator system.

The driving force for electron flow from the highly reduced substrates to the mediators and
finally to the utilized electrodes is the increasing reduction potential of the components
involved in the processes along the shown potential gradient.
Figure 4. (A) Current-time trace recorded upon introduction of [Fe(CN)6]3–, menadione and
glucose to S. cerevisiae cells on an electrode microchip. (B) A silicon microchip for
mediated amperometry (upper panel) and a microscope image of S. cerevisiae cells on a
microband electrode (width/length: 25/1,000 µm). (Reprinted with permission from Ref. [8],
© 2009 Elsevier BV.) (Lower panel).
The biological function of the double mediator system is based on the

controlled metabolic perturbation caused by menadione, which serves as an
analytical tool. Menadione reduction competes for the available NAD(P)H
in the entire cellular environment. Hence, depending on what endogenous
processes there are that supply and consume NAD(P)H, the obtained ampero-
metric signal either increases or decreases with respect to a utilized control.
Menadione is also known for its ability to increase oxidative stress but the
utilized concentration, 100 µM, can be regarded as non-destructive [26],
when short-lived measurements are conducted. The technique can answer to
different biological questions depending on how an experiment is designed.
The technique is found useful in screening of changes in NADPH and NADH

availability as the consequence of (i) genetic modifications (genotype), such
as deletion [6, 8] or over expression [9] of a gene, (ii) changes in phenotype
as exemplified by respiratory and fermentative cells [8], (iii) the simultaneous
influence of different subcellular compartments (cytosol vs. mitochondria)
on the cellular response to different chemicals [8]. Upon parallelization
by using multiple electrode arrays, the technique facilitates simultaneous
monitoring of different geno- and phenotypes [8]. Figure 5 depicts the
obtained relative responses to glucose and fructose using a S. cerevisiae
deletion mutant strain lacking the gene PGI1 (EBY44 strain) in comparison
with a non-modified laboratory strain (CEN.PK) [6].
Figure 5. Relative responses to glucose and fructose (left panels in A and B) obtained for S.
cerevisiae cells (A) with and (B) without phosphoglucose isomerase (PGI). (Right panels in
A and B: a schematic presentation of the pentose phosphate pathway (PPP) and glycolytic
pathway (GP); the deletion of PGI1 gene is indicated in B.)
The responses are relative with respect to the baseline current recorded
prior to introducing either glucose or fructose. In the presence of PGI1,
introduction of either glucose or fructose results in an equal availability of
NAD(P)H and hence an equal current response (Fig. 5A). Upon deletion of
PGI1, glucose is predominantly shuttled into the PPP increasing the
availability of NADPH, whereas fructose is catabolized through the GP with a
concomitant increase in the availability of NADH. A considerable difference
can be seen in the obtained current response (Fig. 5B). The result also
demonstrates that NADPH is the preferred cofactor for MREs.
Additionally, the technique is capable of delivering in vivo enzyme kinetics

data as dose response curves, which can be fitted to the four-parameter
logistic equation (Eq. (4)):
Top - Bottom
Δi = Bottom + (4)
(Hill slope )
log (XC50 )
⎛ 10 ⎞
1+ ⎜ ⎟⎟
⎜ log[S]
⎝ 10 ⎠
The response is expressed in either nA or % (relative response), XC50 is
either IC50 or EC50, the Hill slope is the midpoint slope, and the bottom and
top indicate the response for the minimal and maximal curve asymptote,
respectively. Data for enzyme kinetics is obtained through titration with the
utilized substrate or another effector, such as an inhibitor or a competing
substrate. The titration curves in Fig. 6A were obtained by titration with
HMF in studies involving a S. cerevisiae strain overexpressing the ADH6
gene encoding for the NADPH dependent alcohol dehydrogenase6 (ADH6
strain) and the corresponding parental strain with an empty plasmid (control
strain) [9]. The titration curves show the response of MREs, which is
decreasing due to the decreasing NADPH pool as the consequence of
consecutive additions of HMF. The data for the dose response curve is
obtained as the difference between consecutive steady-states; now, however,
the differences are negative and an absolute value is taken. Figure 6B shows
the corresponding dose response curves. As can be seen, the dose response
curves do not show the sigmoidal shape characteristic of the four-parameter
logistic equation. This is due to the fact that the Hill slope is 1 and the
bottom is zero, i.e. initially at 0 µM addition the response (decrease in
current) is 0 nA.
Mathematically, the obtained curves have the same hyperbolic form as
the well known Michaelis–Menten equation. However, the curves are
expressed in the form of current vs. concentration, yielding IMAX instead of
VMAX (Eq. (5)):
× [S]
Δi = Iapp
MAX (5)
K M, cell + [S]
where [S] is the concentration of any substrate and K appM, cell is the apparent
cellular Michaelis–Menten constant. In order to convert these into a more
informative or conventional VMAX with, for instance, unit nmol substrate/min,
Faraday’s law of electrolysis (Eq. (6)) can be used to obtain the number of
moles of substrate corresponding to a given value of current:
Figure 6. (A) Current-time traces for S. cerevisiae cells recorded upon titration with 5-
hydroxymethyl furfural (HMF). (Reprinted with permission from Ref. [9], © 2009 American
Chemical Society.) (B) Dose response curves based on Δi values in (A). (Lower panel: a
schematic presentation of the ADH6 catalyzed NADPH dependent reduction of HMF.)
n 60 × I MAX
V MAX = S = (6)
τ nF
where nS generally refers to the number of moles of a substrate oxidized or

reduced, τ is the time-base of VMAX (e.g. min) and 60 is the conversion
factor between the time-base of VMAX (min) and IMAX (s). The other symbols
are as previously described. Other enzyme kinetic parameters can be
derived from the obtained VMAX and KM by using knowledge regarding the
number of cells involved in the assay [8, 9]. Ultimately, the determined
kinetic parameters should purely reflect the intracellular electron transfer
but they cannot be separated from other contributions, such as mass
transport across the plasma membrane into the cells and out of the cells,
extracellular reaction between menadiol and ferricyanide, mass transfer of
ferrocyanide to the electrode and oxidation of ferrocyanide at the electrode.

Due to the influence of these factors, the determined enzyme kinetic
parameters are referred to as apparent. The extracellular mass transfer steps
are also affected by convection in the utilized fluidic system where electrode
chip is placed. Additionally, the intracellular electron transfer is dependent
on the concentration of the participating species, i.e. the concentration of
the entering menadione and competing substrate etc. as well as NAD(P)H,
the latter of which also determines the potential of the redox couple. When
determining the enzyme kinetic parameters for a competing substrate, such
as HMF, the most significant criterion is that the mass transfer and electron
transfer of menadione reduction is not slower than the corresponding
processes of the studied substrate in order not to become the limiting factor.
4. Monitoring of Exocytosis
4.1. THE BIOLOGICAL SIGNIFICANCE OF EXOCYTOSIS
Signal propagation by neurons takes place through both electrical and

chemical means. Along the axon of a neuron, opening and closing of Na+
and K+ ion channels, increasing the Na+ and K+ conductance, generates and
propagates action potentials. When a propagating action potential in the
form of ionic current reaches the gap between two neurons, the electrical
mode of signal propagation is converted into its chemical counterpart. This
change in the mode of signal propagation takes place in a finite gap between
two neurons (~100 nm wide [45]), the synaptic cleft. The arrival of a
propagating action potential to the synapse results in the opening of a Ca2+
ion channel, increasing the intracellular Ca2+ concentration of the pre-synaptic
neuron from a normally low level (~100 nM) to concentrations 1,000-fold
higher in the microenvironment near the Ca2+ ion channels [45]. Through
a cascade of biochemical processes, the inflow of Ca2+ serves as a trigger
of fast and regulated excretion, exocytosis, of a signaling molecule, neuro-
transmitter, which propagates the neuronal signal across the synaptic cleft.
Secretion of different regulatory substances through a Ca2+ dependent
machinery is not only limited to neurons, but also includes, for instance,
insulin-secreting pancreatic β-cells and catecholamine-secreting chromaffin
pheochromocytoma (PC12) cells of the adrenal gland. The significance lies,
however, in the nature of the secreted compound and the receptors to which
this compound functions as a ligand. Much of the information illustrating
the overall process of exocytotic release of neurotransmitters from their
storage vesicles originates from studies using chromaffin and PC12 cells.
Although these are not neurons and hence the catecholamines in their vesicles
do not function as neurotransmitters, the functional principle of the release
is the same as the release of catecholamines and other neurotransmitters from
pre-synaptic neurons. This process contains four distinct stages: (i) docking,
(ii) priming, (iii) triggering and (iv) fusion/exocytosis. During docking, vesicles
loaded with the neurotransmitter molecules are brought to the vicinity of the
plasma membrane at the site of the synapse (active zone). During priming,
vesicles are bound to the plasma membrane through complex formation
between certain plasma membrane and vesicle membrane proteins. An exo-
cytotic event is triggered as the consequence of Ca2+ ion influx through ion
channels that are opened upon arrival of a propagating action potential. In
exocytosis, the vesicle membrane fuses with the plasma membrane in the
active zone resulting in opening of a fusion pore. Upon electrical, mechanical
and chemical stimulation (e.g. using an elevated K+ ion concentration) of cells
capable of undergoing exocytosis, the neurotransmitters or other signaling
substances are released as packages of one vesicle at a time. This mode is
referred to as quantal release. Hence, in neuronal synapses, the overall post-
synaptic response is a sum of discrete responses corresponding to single
vesicles. In large vesicles, the number of released neurotransmitter molecules
per exocytotic event (quantum) can be several millions, whereas in small
neuronal vesicles the number of neurotransmitter molecules can be as low
as 3,000–30,000 [46].
4.2. DETECTION PRINCIPLE
Amperometry is the most widely utilized electrochemical technique for

monitoring of exocytosis. Its fundamental application was presented by
Wightman and his co-workers [29]. The technique was originally presented
using carbon-fiber microelectrodes (CFME) as WEs and most of the described
applications are based on this approach [47]. The position of CFMEs can
be adjusted under microscopic observation using a micromanipulator. The
electrode surface is placed into a close contact with the plasma membrane
of the monitored cell, the separation being of the order of magnitude of the
diffusion layer [48]. The close proximity of the electrode surface to the
plasma membrane provides a sufficient temporal resolution to monitor
single-vesicle exocytotic events. Aside from good temporal resolution,
amperometry also provides a superior quantitative sensitivity down to 31 zmol7
(~18,700 molecules) of catecholamines [49] and 7.8 zmol (~4,700 molecules)
of serotonin [50], as well as a spatial resolution at best ranging from the
level of 1 µm [51] down to the size of a single vesicle (~100 nm) [52] in
catecholamine detection. Hence, amperometry can yield accurate quantitative
and high-resolution spatio-temporal information regarding single-vesicle
exocytotic events.
______
7
1 zmol ≡ 1·10−21 mol.
15.0
B.
10.0
5.0
0
0 5.0 10.0 15.0
C.
B
A D
C E
F
D
G
Figure 7. (A) A SEM image of an array of well-electrodes capable of accommodating sigle

cells (left panel) and a magnification of a single well-electrode (right panel). (Reprinted with
permission from Ref. [53], © 2003 American Chemical Society.) (B) An AFM image of an
array of microelectrodes in the size regime of a single cell. (Reprinted with permission from
[54], © 2002 IOP Publishing Ltd.) (C) A schematic view of a microfluidic system (left
panel) able to capture single cells in the middle of a ring-electrode and a microscope image
of a PC12 cell on a ring-electrode (right panel). (Reprinted with permission from Ref. [55],
© 2008 The Royal Society of Chemistry.)
Emergence of lithographic techniques in electrode fabrication has opened

new possibilities for monitoring of exocytosis using chip based electro-
analytical systems [30]. Primarily, these systems utilize planar electrodes,
facilitating detection of exocytosis from vesicles at the active zones near the
basal membrane in contact with the electrode surface. However, Chen et al.
have reported on monitoring of exocytosis from single cells utilizing
well-electrodes [53] (Fig. 7A) In this case, detection covers active zones
in a large fraction of a cell. Planar electrodes cannot be positioned to a
certain part of a monitored cell, somewhat decreasing the spatial resolution.
However, with an additional system for guiding and positioning cells, a
single cell can be placed on measuring electrodes to monitor single-vesicle
exocytotic events with good spatial resolution. Figure 7B shows an electrode
microchip having an array of four platinum microelectrodes around a
central area [54, 56]. Simultaneous detection of dopamine exocytosis from
single chromaffin cells accommodated on top of the electrode array could
be used to localize the opening of a fusion pore with a high spatial
resolution. Left and right panel of Fig. 7C show a schematic view of a
microfluidic cell handling system and a ring-shaped microelectrode with a
single rat pheochromocytoma (PC12) cell in the middle of the electrode,
respectively [55]. In virtue of an aperture in the middle of the ring-electrode,
single cells could be captured and held in position to facilitate exocytosis

measurements that provided a high spatial resolution of the position of the
active zone having the dopamine-releasing vesicle.
Neurotransmitter
Vesicle
synaps
HO O
Dopamin CH2CH2NH3+ CH2CH2NH3+
+ 2H+ + 2e
HO O
Oxidation av Dopamin
Elektrod
Figure 8. (A) A schematic view of the proximity of a cell to the surface of a planar electrode
and the reaction of dopamine oxidation.
Figure 9. (A) A train of exocytotic spikes representing single-vesicle exocytotic events, (B)
an enlargement of the spike encircled in (A) showing the different parameters of eocytotic
spikes, and (C) a microscope image of PC12 cells on ring-electrodes. (Reprinted with
permission from Ref. [12], © 2007 Wiley-VCH Verlag GmbH & Co. KGaA.)
The application of chip based electroanalytical systems having planar

electrodes provides good temporal resolution and sensitivity for monitoring
of exocytosis. The distance between a cell and the underlying electrode is of
the same order of magnitude as the distance between a presynaptic and
postsynaptic neuron in vivo. Hence, no specific arrangements are needed to
achieve a certain separation between an electrode and a cell, as is the case
when using CFMEs. Figure 8 shows schematically the placement of a cell
on a planar electrode and the release of dopamine molecules, which are
immediately oxidized at the potential (e.g. 700 mV vs. a Ag/AgCl RE) poised
at the WE and the dopamine oxidation reaction. The proximity of a cell to
the electrode surface ensures that sufficient detection kinetics is achieved to
monitor exocytosis from a single vesicle of a single cell. Since the duration
of an exocytotic event can be below 10 ms, the primary constraint is to have
a potentiostat capable of sampling at the rate of 5,000–10,000 Hz. Figure 9A

shows a train of spikes, each of which represents a single-vesicle exocytotic
event monitored using circular electrodes (Fig. 9C) [12]. Figure 9B shows a
magnification of one of the spikes in Fig. 9A, featuring the different details
and hence the parameters that can be obtained to characterize exocytotic
events, e.g. the peak width at half height (t½), the spike amplitude (imax).
Additionally, the kinetics of an event can be characterized by the rise time
(RT), i.e. the time required for recording 25–75% of the imax. By integrating
the current of each spike, it is possible to determine the total charge (Q)
associated with the oxidation of the released molecules from one vesicle.
Application of Faraday’s law of electrolysis (Eq. (1)) can then yield the
total number of released molecules. The proximity also facilitates high
sensitivity since the released molecules can be detected before they are able
to diffuse into the surrounding space. Figure 9B illustrates the ultimate
capability of chip based electroanalytical systems showing a foot signal
that is the consequence of initial leakage of molecules through an opening
fusion pore. The number of molecules contributing to a foot signal can be
below 30,000 [12] and the duration of such a signal only a fraction of the
total duration of an exocytotic event.
Aside from single-cell exocytosis measurements, chip based electro-
chemical systems also facilitate applications where exocytosis is monitored
from a population of growing [57–61] or differentiating adherent cells [61].
This approach provides the possibility to conduct exocytosis measurements
in systems where cells are cultured, making cells fully adapted to the
environment and eliminating the necessity of trypsinization as a part of
the preparation of cells for measurements. When utilizing systems with
cultured cells, parallelization in terms of multiple electrodes can be also
effectively applied to conduct simultaneous measurements providing a
sufficient statistical control. Furthermore, automation of measurements can be
implemented to achieve capability for high-throughput screening, for instance,
to study drug effects on neurotransmitter release. Figure 10 shows a micro-
fluidic system with an integrated electrode ship having a microelectrode
array for monitoring exocytosis [60]. Detection from a cell population
increases the duration of an exocytotic event to seconds since each recorded
event is an averaged sum of many single-vesicle events from several cells.
Figure 11A shows current-time traces for recorded exocytotic events from
populations of growing PC12 cells (traces 1–3) [61]. The cell population
corresponding to the measurements are shown in Fig. 11B (panels 1–3).
The inset of Fig. 11A shows a current-time trace for an exocytotic event
from a population of differentiated PC12 cells and the corresponding
population is shown in Fig. 11B (panel 4). This current-time trace shows a
distinct characteristic compared to traces 1–3, i.e. the rising and descending
portions have a clearly steeper slope, which indicates a faster process. This
Figure 10. (A) A microfluidic system with integrated microelectrode arrays to monitor
exocytosis from cultured cells. (Reprinted with permission from Ref. [60], © 2008 American
Chemical Society.)
Figure 11. (A) Current-time traces for exocytotic events recorded from populations of
growing (traces 1–3) and differentiating (trace 4) PC12 cells. (B) PC12 cell populations used
for recording traces 1–4. (Reprinted with permission from [61], © 2008 The Chemical and
Biological Microsystems Society.)
was attributed to mainly two factors: (i) a closer proximity of the adherent
differentiated cells compared to the more roundish albeit spread non-
differentiated cells and (ii) the presence of only distinctly distributed active
zones in differentiated cells in comparison with non-differentiated cells
where vesicles are located throughout the cell body. The first characteristic
of the differentiated cells gives rise to faster diffusion of the released
dopamine to the electrode surface and the second characteristic makes the
overall duration of the monitored exocytosis shorter than those monitored
from non-differentiated cells. The longer duration of exocytotic events that
are recorded from a population of cells also offers the possibility to widen
the spectrum of detectable compounds from electroactive catecholamines,
serotonin and histamine to, for instance, glutamate [11], the detection of
which requires enzyme-based biosensors having a response time at best in
the regime of seconds.
5. Conclusions
Despite the fact that amperometry on planar electrodes decreases the spatial
resolution in comparison with systems, where microelectrodes are positioned
adjacent to a cell using a micromanipulator or SECM instrument, it at the
same time possess the greatest possibilities for developing monitoring
systems with a sufficient degree of fabricational freedom in order to realize
goals that also require handling, culturing and differentiation of cells in
applications, e.g. characterization of differentiating neuronal stem cells and
their integration into brain tissue. Moreover, further miniaturization to nano-
sized electrode structures could provide the possibility to address only a
very small section of a differentiated cell, e.g. ideally the junction between a
pre- and postsynaptic neuron. Furthermore, no matter whether the goal is
analysis of single cells or cell populations, only chip based systems are
capable of providing a sufficient throughput and the automation necessary
for screening in drug discovery, decreasing the need of continuous intervention
by an operator.
Acknowledgments
The EU FP7 NMP project EXCELL is kindly acknowledged for financial

support.
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Chip Based Electroanalytical Systems for Monitoring Cellular Dynamics

Arto Heiskanen Martin Dufva

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A. HEISKANEN, M. DUFVA, AND J. EMNÉUS

Abstract. Electroanalytical methods are highly compatible with micro- and

In cell biology and pharmacology, the determination of cellular functions

biological functions in general and the intercompartmental biological effect

2. Detection of Cellular Dynamics

2.1. DETECTION TECHNIQUES

2.1.1. Fluorescence Detection

Implementation of fluidic functions inside a plate reader is however not a

2.1.2. Electrochemical Detection

Figure 1 illustrates the detection scheme of such cellular factors: Extracellularly

Figure 1. A schematic illustration of electrochemical monitoring of cellular dynamics. An

Traditionally, extracellular microelectrodes that have been used to

on a small population of cells or even single cells. Aside from single-cell

of accepted or donated electrons, F is the Faraday constant (96,485 C mol–1)

Figure 2. Energy diagrams schematically illustrating oxidation-reduction (redox) reactions.

3. Monitoring of Cellular Redox Environment

3.1. CELLULAR REDOX ENVIRONMENT

3.1.1. Cellular Redox Couples

3.1.2. Definition of Cellular Redox Environment

3.1.3. The Biological Significance of CRE

During ethanol production from lignocellulosic hydrolysates in S.

3.2. MEDIATED AMPEROMETRIC REAL-TIME MONITORING

Coupling between a biologically catalyzed reaction and an electrochemical

also utilizes the hydrophilic mediator, [Fe(CN)6]3−. The lipophilic menadione

Scheme 1. Menadione reduction by MREs to menadiol.

Figure 3. The functional mechanism of the ferricyanide-menadione double mediator system.

The biological function of the double mediator system is based on the

The technique is found useful in screening of changes in NADPH and NADH

Additionally, the technique is capable of delivering in vivo enzyme kinetics

where nS generally refers to the number of moles of a substrate oxidized or

ferrocyanide to the electrode and oxidation of ferrocyanide at the electrode.

4.1. THE BIOLOGICAL SIGNIFICANCE OF EXOCYTOSIS

Signal propagation by neurons takes place through both electrical and

4.2. DETECTION PRINCIPLE

Amperometry is the most widely utilized electrochemical technique for

Figure 7. (A) A SEM image of an array of well-electrodes capable of accommodating sigle

Emergence of lithographic techniques in electrode fabrication has opened

single cells could be captured and held in position to facilitate exocytosis

The application of chip based electroanalytical systems having planar

a potentiostat capable of sampling at the rate of 5,000–10,000 Hz. Figure 9A

The EU FP7 NMP project EXCELL is kindly acknowledged for financial

1. A. Begleiter and J. Fourie, Induction of nqo1 in cancer cells, Methods in

7. A. Heiskanen, C. Spegel, N. Kostesha, S. Lindahl, T. Ruzgas and J. Emneus,

22. N. Pereira Rodrigues, Y. Sakai and T. Fujii, Cell-based microfluidic biochip

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