oligosaccharides (e.g, cellobiose, lactose, and cellodextrins) to the corresponding
lactones and the two electrons extracted can be transferred directly to several
two-electron acceptors (e.g., quinones or dichloroindophenol) or, alternatively, to
one-electron acceptors while the second electron is firstly shuttled to the heme iron
through internal ET, and then to one-electron acceptors (e.g., cytochrome c and
ferricyanide) or solid electrodes (e.g., graphite, gold), thus allowing a direct electrochemical response. As mentioned above, for this to happen, the protein should be
correctly oriented on the electrode surface but the simple deposition of CDHs
directly on graphite or gold surfaces leads to a random orientation, hindering an
efficient unmediated ET [149, 187].
Therefore, the electrode modification strategy has played a fundamental role in
the construction of CDHs based third generation biosensors. Gorton’s research
group has done extensive work in this field [170, 182]. In one of their first studies
[188], it was proved that gold electrodes modified with self-assembled monolayers
promote the correct protein orientation, so one could observe both the direct ET
with the heme cofactor and a good catalytic response in the presence of cellobiose.
The latter was dependent on pH, showing a close agreement with the fact that
internal ET between the two cofactors is switched off at high pH values [189, 190].
Similar catalytic behavior was found in many other heme-containing
PQQ-dehydrogenases where the substrate oxidation takes place at PQQ site and
electrons are subsequently transferred to the electrode via the heme domain. This is
the case of FDH, which has been largely used in the development of several third
generation fructose biosensors (see examples in Table 4), though in a less extensive
way than CDH, most likely due to the little relevance of fructose analysis [149, 191,
192]. Other examples are alcohol dehydrogenase [186, 193], pyruvate dehydrogenase [194] or lactate dehydrogenase [195].
4 Reductases
This section is focused on biosensors based on reductase enzymes. The emphasis
was put on enzymes alone, therefore, the reductase activities of ET proteins, such as
chromate reduction by cytochrome c 3 [204], or of O 2 binding proteins, e.g., nitrite
reduction by hemoglobin and myoglobin [205], are not discussed.
A small but diverse group of reductase enzymes has been explored, most of
which are metalloproteins containing iron or molybdenum in their active sites
(Table 1). The majority of works concerns nitrogen oxide reductases, namely
nitrate and nitrite reductases (NaRs and NiRs, respectively), or glutathione reductase [206–209]. The remaining cases include three molybdenum dependent
enzymes (dimethyl sulfoxide (DMSO) reductase, trimethylamine N-oxide (TMAO)
reductase (TorA) and perchlorate reductase), nitroreductase (NR), and cytochrome
c reductase (CcR), for which very few biosensor proposals have been described.
Herein, representative biosensing applications of these enzymes are discussed
alongside with transduction methods, immobilization strategies, and target samples.
Selective Enzymes at the Core of Advanced Electroanalytical …
331
lactones and the two electrons extracted can be transferred directly to several
two-electron acceptors (e.g., quinones or dichloroindophenol) or, alternatively, to
one-electron acceptors while the second electron is firstly shuttled to the heme iron
through internal ET, and then to one-electron acceptors (e.g., cytochrome c and
ferricyanide) or solid electrodes (e.g., graphite, gold), thus allowing a direct electrochemical response. As mentioned above, for this to happen, the protein should be
correctly oriented on the electrode surface but the simple deposition of CDHs
directly on graphite or gold surfaces leads to a random orientation, hindering an
efficient unmediated ET [149, 187].
Therefore, the electrode modification strategy has played a fundamental role in
the construction of CDHs based third generation biosensors. Gorton’s research
group has done extensive work in this field [170, 182]. In one of their first studies
[188], it was proved that gold electrodes modified with self-assembled monolayers
promote the correct protein orientation, so one could observe both the direct ET
with the heme cofactor and a good catalytic response in the presence of cellobiose.
The latter was dependent on pH, showing a close agreement with the fact that
internal ET between the two cofactors is switched off at high pH values [189, 190].
Similar catalytic behavior was found in many other heme-containing
PQQ-dehydrogenases where the substrate oxidation takes place at PQQ site and
electrons are subsequently transferred to the electrode via the heme domain. This is
the case of FDH, which has been largely used in the development of several third
generation fructose biosensors (see examples in Table 4), though in a less extensive
way than CDH, most likely due to the little relevance of fructose analysis [149, 191,
192]. Other examples are alcohol dehydrogenase [186, 193], pyruvate dehydrogenase [194] or lactate dehydrogenase [195].
4 Reductases
This section is focused on biosensors based on reductase enzymes. The emphasis
was put on enzymes alone, therefore, the reductase activities of ET proteins, such as
chromate reduction by cytochrome c 3 [204], or of O 2 binding proteins, e.g., nitrite
reduction by hemoglobin and myoglobin [205], are not discussed.
A small but diverse group of reductase enzymes has been explored, most of
which are metalloproteins containing iron or molybdenum in their active sites
(Table 1). The majority of works concerns nitrogen oxide reductases, namely
nitrate and nitrite reductases (NaRs and NiRs, respectively), or glutathione reductase [206–209]. The remaining cases include three molybdenum dependent
enzymes (dimethyl sulfoxide (DMSO) reductase, trimethylamine N-oxide (TMAO)
reductase (TorA) and perchlorate reductase), nitroreductase (NR), and cytochrome
c reductase (CcR), for which very few biosensor proposals have been described.
Herein, representative biosensing applications of these enzymes are discussed
alongside with transduction methods, immobilization strategies, and target samples.
Selective Enzymes at the Core of Advanced Electroanalytical …
331