1
1 Introduction
1.1 Protein electrochemistry
The field of protein electrochemistry deals with redox proteins which are able to
exchange electrons with an electrode either with the protein free in solution or
confined on an electrode surface. It can be usually divided into two groups (Figure 1).
In one case an external shuttle molecule is employed as mediator to facilitate the
electron transfer (ET) and it is referred as mediated electron transfer (MET). In any
mediated enzyme catalytic reaction, the mediator must exchange electrons rapidly
with the electrode, since a sustained flow of electrons is required. The electrons are
provided by the electrode via the mediator.
Figure 1. Scheme of electron transfer processes on an electrode surface. (a) Direct Electron Transfer
(DET) and (b) Mediated Electron Transfer (MET) between electrode and protein.
In the other case the electron transfer occurs directly between the protein and the
electrode and is called direct electron transfer (DET). It was thought for a long time to
be virtually impossible. However, from the first publications in the ‘70s (Eddowes et
al., 1977; Yeh et al., 1977) investigations in this field boomed. DET provides rapid
and direct measurements of redox properties and a wide range of electrode
potentials can be applied. Together with the precise redox control afforded by the
2
electrode potential it offers an excellent temporal resolution of the activity assay.
Therefore a precise characterization how activity quickly evolves with time following
an instant change in experimental conditions is possible. Furthermore unspecific side
reactions of the mediator, that may cause erroneous results, are prevented.
1.1.1 Direct protein electrochemistry
Direct protein electrochemistry where a protein is confined on an electrode surface
is a powerful tool for investigating the catalytic properties of redox enzymes. From an
operational perspective, direct protein electrochemistry of surface immobilized
molecules also has a number of other advantages, not at least the very small
amounts of the often “priceless” biological material required, down to pmol cm
-2
, in
comparison with other more classical techniques (Armstrong et al., 1988; Armstrong,
2002; Léger et al., 2008). After immobilization, the same sample can be reused for
the further studies. Precondition for the application of this technique, it is the ability to
connect the active site of the enzyme to the electrode. Basically, two different
strategies can be employed, either protein modification with genetic or chemical
engineering techniques (Campàs et al., 2009; Caruana et al., 2010) or novel
interfacial technologies (Hill et al., 1989; Fedurco, 2000).
The ET is a radiation less electronic rearrangement where an electron moves from
an initial state on an electrode or reductant to a receiving state on another solvated
species or on an electrode of the same energy. The rate is strongly dependent from
the potential difference and the spatial distance between the two redox sites (Marcus
et al., 1985; Marcus, 1993). Direct protein electrochemistry enables to exploit the
naturally high efficiency of biological systems for developing selective biosensors,
energy storage and production systems like biofuel cells, heterogeneous catalysts,
and biomolecular electronic components (Léger et al., 2008).
The most successful electrodes for proteins so far have been noble metals and
carbon due to their elevated conductivity and easy handling. However they often lead
to an irreversible adsorption and denaturation of the proteins onto the electrode
surface and therefore to the impossibility to establish fast ET. A wide used method to
solve this problem is the modification of the electrode by a promoter, which can
prevent the protein denaturation and can lead also to a specific protein-electrode
orientation (Armstrong, 2002). The promoter can reduce the distance between the
active site of the protein and the electrode (Armstrong, 1990). It is not electroactive
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itself and can interact with the enzyme by different kind of forces (electrostatic,
hydrophobic, hydrogen bounds, etc.). By this approach, a DET reaction can be
obtained or in alternative the usage of mediators can solve possible distance
problems and permits the electron shuttling (Figure 1). General promoters are self-
assembly monolayers of amphiphiles or polyelectrolytes (Scheller et al., 2002; Allen
et al., 1984; Fedurco, 2000; Rusling et al., 2008) with several possible functionalities.
A drawback of this approach, however, is that the amount of immobilized protein is
limited to monolayer coverage. Larger amounts of protein, can be obtained by
alternate deposition of proteins and polyelectrolytes (“layer -by-layer” technique)
(Beissenhirtz et al., 2004; Grochol et al., 2007; Spricigo et al., 2009; Dronov et al.,
2007; Ram et al., 2001; Calvo et al., 2004). However this advantage is
counterbalanced by the limited accessibility of the active sites of the proteins in the
inner layers and the low stability.
The advent of nanoscaled materials such as nanotubes, nanoparticles, conductive
and non-conductive metal oxides opens new horizons for the field of bioelectronics
due to the likely deep interactions between the nanomaterials and the proteins
(Armstrong, 2002; Bernhardt, 2006; Chen et al., 2007; Wollenberger et al., 2008).
1.1.2 Protein spectroelectrochemistry
The coupling of electrochemical and optical methods has been used for decades
to study a large range of organic, inorganic and biological redox systems (Kuwana et
al., 1976; Heineman et al., 1984). Althought a large variety of electrochemical
methods are available, they do not render any structural information of the electrode
system besides the detailed knowledge of charge transfer, transport and distribution.
The combination of electrochemical and optical methods to monitor the spectroscopic
variations associated to the potential changes allows a qualified picture of the
chemical structures in electrochemical reactions. The potential of the analyzed
solution may be easily changed by addition of reductants or oxidants. On the other
hand it can be electronically changed by potential imposition at an electrode. A
classical set for such an experiment consist in an optical transparent thin-layer
electrochemical (OTTLE) cell, with a metallic paint ensuring electrical conductivity
and preserving some degree of transparency (Pinkerton et al., 1980; Bowden, et al.,
1982; Heineman et al., 1984; Dai et al., 2011).
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A large variety of spectroscopic methods may be coupled, from UV-Vis, infra-red
(IR) (Arion et al., 2011), resonance Raman (RR) (Kavan et al., 2009) and surface
enhanced resonance Raman (SERR) (Murgida et al., 2006) to electron paramagnetic
resonance (EPR) (Paulsen et al., 1992) and nuclear magnetic resonance (NMR)
(Klod et al., 2009). Nevertheless, there are some spectroscopic methods which are
preferred in spectroelectrochemistry. The choice of the method is often dominated by
not the importance of a spectroscopic method which offers the access of important
structural data of an electron-transfer reaction in experimental studies, but in most
cases, the ease of application. UV-Vis spectroscopy is the most applied method in
spectroelectrochemistry irrespective of the fact that other methods would result in
more detailed structural informations (Dunsch, 2011).
Spectroelectrochemical studies were usually restricted to solution samples, where
a relative concentrated sample is required, attenuated total internal reflection mode
or with reflection cells (Bernad et al., 2006). Only with a signal enhancement, like with
Raman spectroscopy through surface plasmon resonance, the noteworthy reduction
of sample volume and concentration can be obtained.
Absorption UV-Vis spectroelectrochemical investigation of protein boosted in the
last time by the improvements in the field of nanostructured transparent conductive
oxide. The possibility to entrap a large amount of protein in the porous structure of
such materials overcomes the lack of sensitivity (Szamocki et al., 2007). In addition
these materials offer a high transparency in the UV-Vis region and elevated
conductivity over the whole potential range commonly used for proteins investigation
(Topoglidis et al., 2001; Panicco et al., 2008; Renault et al., 2011).
1.1.3 Biosensors
A biosensor is defined as a specific type of chemical sensor comprising a
biological or biologically derived recognition element either integrated within or
intimately associated with a physicochemical transducer. The biological element is
capable of recognizing the presence, activity or concentration of a specific analyte in
solution (Thévenot et al., 1999; Thévenot et al., 2001; Hall, 2002). An analyte is the
compound whose concentration has to be measured. Biosensors basically involve
the quantitative analysis of various substances by converting their biological actions
into measurable signals. Generally the performance of the biosensors is mostly
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dependent on the specificity and sensitivity of the biological reaction, besides the
stability of the biological element.
In general a biosensor comprises three parts: a biological recognition element, a
suitable transducer and an amplification element (Figure 2).
The transducer is an important component of a biosensor through which the
measurement of the target analyte(s) is achieved by selective transformation of a
biomolecule-analyte interaction into a quantifiable output signal. The mode of
transduction may be one of several approaches, including electrochemical, optical,
piezoelectric, magnetic or thermometric transducers.
Every biomolecule from enzyme to an antibody, a nucleic acid, a hormone, an
organelle or whole cell which can selectively interact with other substances, can be
theoretically qualified for biosensor development. Usually, sensors are distinguished
(Thévenot et al., 1999) in sensors using catalytic biorecognition elements (enzymes,
cells, microorganisms) (Gu et al., 2004) and affinity-based recognition elements
(antibodies, antigens, protein receptors, synthetic receptors, nucleic acids) ( ale e ,
2005) (Ge et al., 2008). Catalytic sensors own the concomitant ability to amplify the
signal and regenerate the active site of the biorecognition element (Dryhurst et al.,
1982).
A wide range of enzymes, owing a combination of specificity and amplification
properties, has been successfully used as a recognition element. Since enzymes
allow a wide range of transduction technologies, they have found very wide
applications in the field of biosensors (Schuhmann et al., 2003).
Figure 2. Configuration of a biosensor showing biorecognition, interface, and transduction elements
(Chambers et al., 2008).
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Other kinds of proteins used are for example membrane bound receptors that
change their structural conformation binding specific ligands. This modification
triggers an amplified physiological response, such as ion channel opening or
secreting an enzyme. High affinity and specificity towards the natural targets are the
advantages of such recognition system.
Although the biosensors suffer general problem of stability and complexity
connected to the biological recognition element, they offer many advantage in
respect to classical analytical methods. Biosensors show a very broad range of
detectable analytes, depending on the nature of the recognition element, and high
analyte selectivity with the limitation of interferences in complex samples.
Furthermore, the current tendency in biosensor development is the miniaturization.
This enables and will further expand the integration and parallelization of biosensors
in sophisticated systems.
1.1.3.1 Electrochemical enzyme based biosensors
Biosensors that utilize enzymes as recognition elements represent a wide
extensively studied area, with glucose biosensors dominating the market
(Frost & Sullivan, 2006). Enzymes are favored as recognition elements in biosensors
because they provide a broad range of changes of physical and chemical parameters
during the enzymatic reaction, such as electrons, protons, ions, mass, light and heat.
These changes can be detected using suitable transducer elements. Different
electrochemical methods as potentiometriy, voltammetry and amperometry exist,
where either the potential or the current change depending on the concentration of
the analyte can be measured. Selective and sensitive catalysis of a substrate at
relative low potentials are the great power of enzymes.
Electrochemical enzyme-based sensors are often separated in three different
types or generations. First generation sensors measure the signal via the natural
secondary substrates and products of the enzyme catalyzed reaction. In the second
generation sensors an artificial electron mediator is used instead of the natural
co-substrates. Indeed enzymes in direct electronic contact, based on direct protein
electrochemistry are considered as third generation sensors. Their direct electron
transfer (DET) between the electrode and the protein (Figure 1) may avoid most of
the interferences (Wollenberger, 2005).
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1.2 Electrodes
1.2.1 Nanostructured electrode materials
Nanomaterials have number of features that make them ideally suited for sensor
applications, such as its high surface area, high reactivity, controlled electrode
modification and defined interaction with other partner, e.g. biomolecules. They find
large employ in different fields ranging from biosensors to biofuel cells or more
complex bioelectronic systems. Nanostructured materials include dendrimers,
nanoparticles, nanotubes, nanopores etc (Umasankar et al., 2009).
1.2.1.1 Indium Tin Oxide
Using film of intrinsic stoichiometric materials like metals partial transparency, with
moderate reduction in conductivity, can be obtained. However such materials may
not achieve high transparency and coincidentally elevated conductivity.
A solution is to create electron degeneracy in a material with a wide energy
bandgap (E
g
> 3eV or more for visible radiation) by introduction of non-stoichiometry
and/or appropriate dopants. A large number of non-stoichiometric and doped oxide
films (indium, tin, antimony, cadmium, zinc etc.) meet these conditions and exhibit
high transmittance and nearly metallic conductivity (Chopra et al., 1983).
Tin doped indium oxide or indium tin oxide (ITO), with a mean transmittance of
95% and conductivity as high as 10
4
S
-1
cm
-1
, is among the most popular of these thin
films (Granqvist et al., 2002).
ITO is essentially formed by subsititutional doping of In
2
O
3
with Sn which replaces
the In
3+
atoms from the cubic bixbyte structure of indium oxide (Fan et al., 1977). Tin,
which exists either as SnO or SnO
2
, forms an interstitial bond with oxygen. These two
valency states have a direct influence on the ultimate conductivity of ITO. The lower
valence state (+2) results in a net reduction in carrier concentration since the hole
created acts as a trap and reduces the conductivity. On the other hand,
predominance of the SnO
2
state (+4) acts as a n-type donor releasing electrons to
the conduction band. The high conductivity of ITO films is due to high carrier
concentration and their mobility increases due to enhanced crystallinity of films
deposited at high temperatures (Balasubramanian et al., 1989).
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The high optical transmittance of this material is a direct consequence of being a
semiconductor with a wide bandgap and therefore the absorption region generally
lies in the ultraviolet part of the electromagnetic spectrum and shifts to shorter
wavelengths with increasing carrier concentration (Gupta et al., 1989). The
transmittance of ITO films is also influenced by a number of minor defects which
include surface roughness and optical inhomogeneity. Opaqueness has been
attributed also to unoxidised tin metal grains on the ITO surface as a result of
instability due to the absence of sufficient oxygen during the deposition (Fan et al.,
1977) or external induction by an applied potential (Kraft et al., 1994; Senthilkumar et
al., 2008).
X-ray photoelectron spectroscopy studies of ITO surfaces showed high
concentrations of In(OH)
3
-like and InOOH-like surface species, indicating an excess
of negative surface charge (Milliron et al., 2000).
ITO has found an employment in electronic, opto-electronic and mechanical
applications. Uses of ITO have traditionally ranged from transparent heating
elements of aircraft and car windows, heat reflecting mirrors, antireflection and
antistatic coatings, over electronic instrument display panels and even in high
temperature gas sensors. Early electro-optic devices using ITO include
charge-coupled devices, liquid crystal displays and as transparent electrodes for
various display devices like touchscreens. More recently, ITO has been used as a
transparent contact in advanced optoelectronic devices such as solar cells, light
emitting and photo diodes, photo transistors and lasers.
In parallel to the planar films, ITO with a well-defined mesoporous framework
(mpITO) is of considerable interest. With its unique combination of transparency, high
conductivity, well-defined 3D mesoporosity and high surface area, mpITO allows the
incorporation of a high amount of optoelectroactive species, facilitates electron
transport to these centers, and efficiently harvests the electron-induced optical
response or, vice versa, the photon-induced electron flow.
This material thus could open new pathways to novel, highly efficient solar cells
and optoelectronic systems based on transparent electrodes and sensors.
1.2.1.2 Tin rich indium tin oxide
A general problem in the production of ITO is the limited amount of tin, which,
although the cheaper component, is only slightly soluble in the In
2
O
3
phase, typically
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around 1−10 wt %. In case of organized mesostructured ITO, a second problem
appears from the compromise between the conductivity and regular porosity.
To avoid phase segregation of tin-rich ITO, a low-temperature approach with high
control over the In/Sn molar ratio was reported recently, based on the molecular
single-source precursor indium tin tris-tert-butoxide (ITBO; Scheme 1) containing
indium and tin in the molar ratio of 1:1, which facilitates the formation of tin-rich ITO
with an identical stoichiometry in the final product (Aksu et al., 2009).
Most importantly, the resulting tin-rich ITO shows high conductivity and
transparency even in an amorphous state. Using such approach it was possible to
overcome all of the problems connected with the pore
collapse during the crystallization typical for the
template-assisted approach toward mesoporous ITO
(Fattakhova-Rohlfing et al., 2006). As the ITBO
precursor enables the formation of transparent
conducting films without any crystallization step, the
main cause of pore collapse of mesoporous metal
oxides is excluded in these materials. Thus, ITBO
appeared as highly suitable for the preparation of
mesoporous, tin-rich ITO films with reliable high
electrical conductivity and transparency using different templates (Aksu et al., 2011).
1.2.1.3 Antimony doped tin oxide
The research toward the replacing of the rare and expensive indium in transparent
conducting films is of great interest and may in addition provide a different surface
chemistry and energy-level properties.
The most promising materials are the extrinsically doped tin oxides, such as
fluorine- or antimony-doped tin oxide (ATO) (Batzill et al., 2005). Sb is a common
n-type dopant in SnO
2
. Stjerna (Stjerna et al., 1994) reported a strong increase in the
free electron concentration in the SnO
2
band gap when doped with Sb. Therefore it
was concluded that this band could be a half-filled metallic band and that additional
thermal excitation into the Sn-like bands could increase the conductivity. In recent
years, some communications have been published reporting macro and mesoporous
ATO electrodes (Hou et al., 2009; Wang et al., 2009; Urbanová et al., 2010).
Scheme 1 Structure of indium
tin tris-tert-butoxide (ITBO).
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1.2.1.4 Gold nanoparticles
An interesting way to build up conductive three dimensional structures on
electrode is offered by metal nanoparticles (NPs). The chemical functionalities
associated with nanoparticles enable the assembly of 2D and 3D NP architectures on
surfaces (Shipway et al., 2000). On the basis of the tremendous success in
supramolecular chemistry, NPs functionalized with various molecular and
biomolecular units were assembled into complex hybrid systems. The electronic
triggering of redox proteins by the incorporation of nanoparticles represents a novel
strategy for the electrical contacting of redox enzymes with their macroscopic
environment.
Colloidal gold nanoparticles (AuNPs) have been around for centuries
predominantly in the work of artists and craftsman because of their intensive visible
colors. However, through research on size, shape, surface chemistry, and optical
properties of gold nanoparticles a door to some very unique and exciting capabilities
has been opened. Gold nanoparticle chemistry and physics has emerged as a broad
new subdiscipline in the domain of colloids and surfaces. NPs with fewer than 300
gold atoms can display distinct optical and electronic properties compared to the bulk
metal. These unusual optical properties of small gold particles, their size-dependent
electrochemistry, and their high chemical stability have made them the model system
of choice for exploring a wide range of phenomena including self-assembly,
biolabeling, catalysis, electron-transfer theories, phase transfer, DNA melting,
DNA assays and crystal growth. They found a large application range from photonic
device fabrications, to sensing of organic and biomolecules, to charge storage
systems (Jennings et al., 2007; Sardar et al., 2009).
The convergence of biotechnology and nanotechnology has led to the
development of hybrid nanomaterials that incorporate the highly selective catalytic
and recognition properties of biomaterials with the unique electronic, photonic, and
catalytic features of nanoparticles. A very interesting property of gold nanoparticles is
to provide a suitable microenvironment for biomolecules immobilization retaining their
biological activity. Their ability to facilitate ET between the immobilized proteins and
electrode surfaces, in addition to the light-scattering properties and extremely large
enhancement ability of the local electromagnetic field led to an intensive use of this
nanomaterial for the construction of biosensors (Li et al., 2010) and electrochemical
biosensors (Pingarrón et al., 2008; Bon Saint Côme et al., 2011).
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Their high surface-to-volume ratio, high surface energy, ability to decrease
proteins–metal particles distance, and the functioning as electron-conducting
pathways between prosthetic groups and the electrode surface, are regarded to be
the general characteristics of gold nanoparticles responsible to facilitate electron
transfer between redox proteins and electrode surfaces (Liu et al., 2003).
Gold nanoparticles are prepared with a wide variety of preparative methods.
These methods are mostly based on precursors containing gold complexes with
tetrachloroauric acid (HAuCl
4
), being the precursor most commonly used. Various in
situ reactions, such as chemical, photo-induced, thermal decompositions or
controlled solvent evaporation are used for the reduction process (Rao et al., 2000).
Nanoparticles show a relative stabilization in solution towards aggregation and
other modes of decay due to the acquisition of charges either from surface charged
groups or by specific ion adsorption from the bulk solution. Such charges lead to a
repulsive double-layer force between particles. On the other hand nanoparticle
systems adsorbing a polymeric layer can be sterically stabilized due to a steric barrier
which prevente the particles against collision. A much better stabilization is provided
when the adsorbed polymer is a polyelectrolyte. In this case both types of
stabilization can be combined giving rise to electrosterically stabilized systems (Koetz
et al., 2007).
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