hypoxanthine to xanthine and then to uric acid occurs at the molybdenum center,
with electrons being transferred intramolecularly via the Fe–S cluster to the FAD
cofactor. The oxidized enzyme form is then regenerated when O 2 accepts electrons
at the flavin center [34]. Another feature of this oxidase is that the mammalian
sourced enzyme can exist in two reversible, interconvertible forms: xanthine
dehydrogenase and XOD. The former prefers NAD
+ as the final electron acceptor,
while XOD uses O 2 exclusively. The oxidation of specific sulfhydryl residues in the
dehydrogenase form converts it reversibly into XOD, while the irreversible conversion is achieved via proteolysis [33, 35]. Key applications of XOD (and its
dehydrogenase form) include metabolizing nitrogenous heterocyclic organic compounds (e.g., caffeine and hypoxanthine/xanthine), elimination of by-products in
the synthesis of nucleoside analogs, clinical detection of xanthine/hypoxanthine
content in physiological fluids, and biological remediation [36].
Flavin-dependent oxidases have been widely used in the construction of
amperometric/voltammetric biosensors, being intimately related to the emergence
of the field. As mentioned in Sect. 1, Leland C. Clark Jr developed the first
biosensor ever proposed, a glucose biosensor based on GOx [37]. The enzyme was
entrapped between semipermeable membranes fixed on the surface of an O 2 electrode. By coupling the catalytic oxidation of glucose with the localized depletion of
O 2 levels, the indirect measurement of glucose concentration was achieved through
a bioelectrochemical strategy. This analytical tool was the steppingstone for a
myriad of biosensing devices that would soon follow [2, 38, 39]. In the late 80s and
early 90s, a number of hand-held devices for glucose self-monitoring were made
available to the public, which had an enormous impact on a clinical diagnosis of
diabetes mellitus [2, 13, 39]. In present days, apart from glucose monitoring, which
dominates the biosensor’s market [3, 40], other clinically and industrially relevant
analytes, such as lactate, cholesterol, and ethanol, drive the research focus towards
novel, wearable, noninvasive, and/or smart tools based on flavin-dependent oxidases [41–45]. Table 2 summarizes some examples of amperometric/voltammetric
bioanalytical tools designed for the detection of the mentioned analytes featuring
such oxidases. For more detailed reviews, the reader is directed to references [14,
21, 23, 28, 46–48].
The analyte’s detection by following the current associated with either the
decrease in O 2 tension or the formation of H 2 O 2 is a common protocol in
amperometric/voltammetric sensors based on oxidases [13, 14, 21, 46, 49]. Such
devices are classified as being first generation biosensors, where the communication
between the redox enzyme and the electrode is done via a freely-diffusing,
redox-active co-substrate (O 2 in this case) or co-product (H 2 O 2 ) [2, 13, 50] (Fig. 2).
However, this approach presents some disadvantages. The dependence on O 2 as the
detection principle can lead to incorrect measurements since its concentration varies
between samples. Given its restricted solubility in biological fluids, O 2 concentration
may also be insufficient to guarantee the full oxidation of the analyte, requiring a
sample dilution step in the detection protocol [2, 13, 51]. Finally, the oxidation of
H 2 O 2 requires quite positive overpotentials, which makes the analytical procedure
prone to electrochemical interference from other redox-active species commonly
Selective Enzymes at the Core of Advanced Electroanalytical …
313
with electrons being transferred intramolecularly via the Fe–S cluster to the FAD
cofactor. The oxidized enzyme form is then regenerated when O 2 accepts electrons
at the flavin center [34]. Another feature of this oxidase is that the mammalian
sourced enzyme can exist in two reversible, interconvertible forms: xanthine
dehydrogenase and XOD. The former prefers NAD
+ as the final electron acceptor,
while XOD uses O 2 exclusively. The oxidation of specific sulfhydryl residues in the
dehydrogenase form converts it reversibly into XOD, while the irreversible conversion is achieved via proteolysis [33, 35]. Key applications of XOD (and its
dehydrogenase form) include metabolizing nitrogenous heterocyclic organic compounds (e.g., caffeine and hypoxanthine/xanthine), elimination of by-products in
the synthesis of nucleoside analogs, clinical detection of xanthine/hypoxanthine
content in physiological fluids, and biological remediation [36].
Flavin-dependent oxidases have been widely used in the construction of
amperometric/voltammetric biosensors, being intimately related to the emergence
of the field. As mentioned in Sect. 1, Leland C. Clark Jr developed the first
biosensor ever proposed, a glucose biosensor based on GOx [37]. The enzyme was
entrapped between semipermeable membranes fixed on the surface of an O 2 electrode. By coupling the catalytic oxidation of glucose with the localized depletion of
O 2 levels, the indirect measurement of glucose concentration was achieved through
a bioelectrochemical strategy. This analytical tool was the steppingstone for a
myriad of biosensing devices that would soon follow [2, 38, 39]. In the late 80s and
early 90s, a number of hand-held devices for glucose self-monitoring were made
available to the public, which had an enormous impact on a clinical diagnosis of
diabetes mellitus [2, 13, 39]. In present days, apart from glucose monitoring, which
dominates the biosensor’s market [3, 40], other clinically and industrially relevant
analytes, such as lactate, cholesterol, and ethanol, drive the research focus towards
novel, wearable, noninvasive, and/or smart tools based on flavin-dependent oxidases [41–45]. Table 2 summarizes some examples of amperometric/voltammetric
bioanalytical tools designed for the detection of the mentioned analytes featuring
such oxidases. For more detailed reviews, the reader is directed to references [14,
21, 23, 28, 46–48].
The analyte’s detection by following the current associated with either the
decrease in O 2 tension or the formation of H 2 O 2 is a common protocol in
amperometric/voltammetric sensors based on oxidases [13, 14, 21, 46, 49]. Such
devices are classified as being first generation biosensors, where the communication
between the redox enzyme and the electrode is done via a freely-diffusing,
redox-active co-substrate (O 2 in this case) or co-product (H 2 O 2 ) [2, 13, 50] (Fig. 2).
However, this approach presents some disadvantages. The dependence on O 2 as the
detection principle can lead to incorrect measurements since its concentration varies
between samples. Given its restricted solubility in biological fluids, O 2 concentration
may also be insufficient to guarantee the full oxidation of the analyte, requiring a
sample dilution step in the detection protocol [2, 13, 51]. Finally, the oxidation of
H 2 O 2 requires quite positive overpotentials, which makes the analytical procedure
prone to electrochemical interference from other redox-active species commonly
Selective Enzymes at the Core of Advanced Electroanalytical …
313