Detection of nitroaromatic compounds is important for environmental monitoring given their widespread use in agro and warfare chemicals. The nitroreductase
(NR) from Enterobacter cloacae catalyzes two-electron reductions of a variety of
nitroaromatic compounds, including trinitrotoluene (TNT). The enzyme contains an
FMN cofactor that binds in deep pockets located at the interface of its homodimeric
structure (Fig. 10h) [87]. Naal et al. described a mediated voltammetric biosensor
for TNT based on this NR fused to a maltose-binding protein (MBP) [218]. The
fusion protein was immobilized on N-(3-pyrrol-1-ylpropyl)-4,4′-bipyridine modified electrodes through the MBP domain, which is thought to establish strong
electrostatic interactions with the conducting polymer film and to contribute to the
maintenance of the immobilized enzyme’s activity [218].
Cytochrome c reductase, CcR (bc 1 complex), is an oligomeric membrane protein
complex that catalyzes the oxidation of ubihydroquinone and the reduction of
cytochrome c in photosynthetic and respiratory chains. The catalytic core of the
enzyme is composed of three subunits, which carry a heme b, a heme c 1 , and a
[2Fe-2S] cluster (Rieske type) (Fig. 10i) [256]. Recently, CcR has been employed
in biosensors for the detection of cytochrome c [88, 219] and nitrite [257].
Nanostructured electrodes based on CNTs and GNPs incorporated in PPy films
were shown to improve ET to the deeply embedded enzyme cofactors [88]. Still,
hydroquinone has been used as a redox mediator. The biosensors have been used
for quantification of cytochrome c in apoptotic cardiomyocytes and cytosolic
fractions of human lung carcinoma cells (Table 5) [88, 219] and nitrite in hypoxic
H9c2 cardiac cells [257].
5 Hydrolases
The majority of amperometric/voltammetric devices featuring hydrolases (Fig. 12)
as the biorecognition element are targeted towards organophosphate compounds
monitoring [258]. These compounds (also known as phosphotriesters) are a class of
highly neurotoxic synthetic compounds that are widely used as agricultural insecticides, petroleum additives, plasticizers, refrigerants, dyes, and chemical warfare
agents. In vivo, these compounds inhibit irreversibly acetylcholinesterase, which
hydrolyzes the neurotransmitter acetylcholine into choline and acetic acid at the
cholinergic synapses and neuromuscular junctions, thus serving as a regulator of
neurotransmission [259, 260].
The detection principle of hydrolase-based amperometric/voltammetric biosensors relies on the catalytic activity of the enzyme to produce an electroactive
product that is detectable on the electrode’s surface (much like H 2 O 2 in
oxidase-based devices, Fig. 2). The analyte itself could be the enzyme’s substrate;
alternatively, it could inhibit the bioreceptor, in which case detection is based on the
level of activity inhibition [261, 262].
Selective Enzymes at the Core of Advanced Electroanalytical …
341
(NR) from Enterobacter cloacae catalyzes two-electron reductions of a variety of
nitroaromatic compounds, including trinitrotoluene (TNT). The enzyme contains an
FMN cofactor that binds in deep pockets located at the interface of its homodimeric
structure (Fig. 10h) [87]. Naal et al. described a mediated voltammetric biosensor
for TNT based on this NR fused to a maltose-binding protein (MBP) [218]. The
fusion protein was immobilized on N-(3-pyrrol-1-ylpropyl)-4,4′-bipyridine modified electrodes through the MBP domain, which is thought to establish strong
electrostatic interactions with the conducting polymer film and to contribute to the
maintenance of the immobilized enzyme’s activity [218].
Cytochrome c reductase, CcR (bc 1 complex), is an oligomeric membrane protein
complex that catalyzes the oxidation of ubihydroquinone and the reduction of
cytochrome c in photosynthetic and respiratory chains. The catalytic core of the
enzyme is composed of three subunits, which carry a heme b, a heme c 1 , and a
[2Fe-2S] cluster (Rieske type) (Fig. 10i) [256]. Recently, CcR has been employed
in biosensors for the detection of cytochrome c [88, 219] and nitrite [257].
Nanostructured electrodes based on CNTs and GNPs incorporated in PPy films
were shown to improve ET to the deeply embedded enzyme cofactors [88]. Still,
hydroquinone has been used as a redox mediator. The biosensors have been used
for quantification of cytochrome c in apoptotic cardiomyocytes and cytosolic
fractions of human lung carcinoma cells (Table 5) [88, 219] and nitrite in hypoxic
H9c2 cardiac cells [257].
5 Hydrolases
The majority of amperometric/voltammetric devices featuring hydrolases (Fig. 12)
as the biorecognition element are targeted towards organophosphate compounds
monitoring [258]. These compounds (also known as phosphotriesters) are a class of
highly neurotoxic synthetic compounds that are widely used as agricultural insecticides, petroleum additives, plasticizers, refrigerants, dyes, and chemical warfare
agents. In vivo, these compounds inhibit irreversibly acetylcholinesterase, which
hydrolyzes the neurotransmitter acetylcholine into choline and acetic acid at the
cholinergic synapses and neuromuscular junctions, thus serving as a regulator of
neurotransmission [259, 260].
The detection principle of hydrolase-based amperometric/voltammetric biosensors relies on the catalytic activity of the enzyme to produce an electroactive
product that is detectable on the electrode’s surface (much like H 2 O 2 in
oxidase-based devices, Fig. 2). The analyte itself could be the enzyme’s substrate;
alternatively, it could inhibit the bioreceptor, in which case detection is based on the
level of activity inhibition [261, 262].
Selective Enzymes at the Core of Advanced Electroanalytical …
341