oxidases (Sect. 2), dehydrogenases (Sect. 3), reductases (Sect. 4), and hydrolases
(Sect. 5), each one representing a characteristic type of reaction for which specific
transducing schemes have been developed. While hydrolases do not involve any
exchange of electrons between reagents, the other three families belong to the
oxidoreductases class. Oxidases and dehydrogenases, in particular, participate in
substrate oxidation reactions, whereas reductases operate in the opposite direction
(substrate reduction). In what concerns the transducing component, we will mainly
focus on electrochemical dynamic techniques, which are based on current measurements at a “probe” (working electrode) as a function of a differential voltage
that can either be time-dependent (voltammetric methods) or constant (amperometry). In the negative potential window (typically below −0.1 V vs. normal
hydrogen electrode, NHE), dissolved O 2 can markedly interfere in the results,
forcing solution deaeration before starting the assay. In order to avoid complex
purging systems, several (bio)chemical O 2 scavenging systems have been used,
including O 2 reducing enzymes, as described in Sect. 4.1.
2 Oxidases
2.1 Flavoenzymes
In this group of oxidases, the redox cofactor is a flavin (Table 1)—either flavin
adenine dinucleotide (FAD) or flavin mononucleotide (FMN)—, which acts as the
initial electron acceptor (Eq. 1) for the substrate’s oxidation (e.g., glucose, lactate,
alcohol). Afterward, the oxidized state of the cofactor is regenerated by reacting
with O 2 (the final electron acceptor), leading to the formation of H 2 O 2 (Eq. 2) [6–
8]. Examples of such enzymes are glucose oxidase (GOx), alcohol oxidase (AOD),
choline oxidase (ChOx), cholesterol oxidase (ChOD), glutamate oxidase (GluOx),
lactate oxidase (LOx), and xanthine oxidase (XOD).
Substrate þ Enzyme FAD=FMN ! Product þ Enzyme FADH 2 =FMNH 2
ð1Þ
Enzyme FADH 2 =FMNH 2 þ O 2 ! Enzyme FAD=FMN þ H 2 O 2
ð2Þ
GOx (Fig. 1a) is a FAD-dependent, homodimeric, glycosylated enzyme, with
one tightly and non-covalently bound cofactor per monomer, which catalyzes the
oxidation of b-D-glucose to D-1,5-gluconolactone, via a ping-pong mechanism [8,
9]. This oxidase belongs to the glucose-methanol-choline oxidoreductase superfamily (GMC), along with AOD, ChOx, and ChOD, with all members presenting
common structural features, such as a highly conserved N-terminal FAD-binding
domain and a less conserved C-terminal substrate-binding domain [10]. GOx has
been isolated from several sources (e.g., red algae, bacteria, fungi), with the variant
from Aspergillus niger being the most studied one [8, 9, 11]. This oxidase has been
a very popular enzyme in several applications (e.g., clinical chemistry, energy, and
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T. Monteiro et al.
(Sect. 5), each one representing a characteristic type of reaction for which specific
transducing schemes have been developed. While hydrolases do not involve any
exchange of electrons between reagents, the other three families belong to the
oxidoreductases class. Oxidases and dehydrogenases, in particular, participate in
substrate oxidation reactions, whereas reductases operate in the opposite direction
(substrate reduction). In what concerns the transducing component, we will mainly
focus on electrochemical dynamic techniques, which are based on current measurements at a “probe” (working electrode) as a function of a differential voltage
that can either be time-dependent (voltammetric methods) or constant (amperometry). In the negative potential window (typically below −0.1 V vs. normal
hydrogen electrode, NHE), dissolved O 2 can markedly interfere in the results,
forcing solution deaeration before starting the assay. In order to avoid complex
purging systems, several (bio)chemical O 2 scavenging systems have been used,
including O 2 reducing enzymes, as described in Sect. 4.1.
2 Oxidases
2.1 Flavoenzymes
In this group of oxidases, the redox cofactor is a flavin (Table 1)—either flavin
adenine dinucleotide (FAD) or flavin mononucleotide (FMN)—, which acts as the
initial electron acceptor (Eq. 1) for the substrate’s oxidation (e.g., glucose, lactate,
alcohol). Afterward, the oxidized state of the cofactor is regenerated by reacting
with O 2 (the final electron acceptor), leading to the formation of H 2 O 2 (Eq. 2) [6–
8]. Examples of such enzymes are glucose oxidase (GOx), alcohol oxidase (AOD),
choline oxidase (ChOx), cholesterol oxidase (ChOD), glutamate oxidase (GluOx),
lactate oxidase (LOx), and xanthine oxidase (XOD).
Substrate þ Enzyme FAD=FMN ! Product þ Enzyme FADH 2 =FMNH 2
ð1Þ
Enzyme FADH 2 =FMNH 2 þ O 2 ! Enzyme FAD=FMN þ H 2 O 2
ð2Þ
GOx (Fig. 1a) is a FAD-dependent, homodimeric, glycosylated enzyme, with
one tightly and non-covalently bound cofactor per monomer, which catalyzes the
oxidation of b-D-glucose to D-1,5-gluconolactone, via a ping-pong mechanism [8,
9]. This oxidase belongs to the glucose-methanol-choline oxidoreductase superfamily (GMC), along with AOD, ChOx, and ChOD, with all members presenting
common structural features, such as a highly conserved N-terminal FAD-binding
domain and a less conserved C-terminal substrate-binding domain [10]. GOx has
been isolated from several sources (e.g., red algae, bacteria, fungi), with the variant
from Aspergillus niger being the most studied one [8, 9, 11]. This oxidase has been
a very popular enzyme in several applications (e.g., clinical chemistry, energy, and
306
T. Monteiro et al.