samples, although efforts have been made to circumvent this by improving the
immobilization of the enzyme, creating a more effective microenvironment [124].
The plant AOx is a homodimeric enzyme, with one catalytic site per monomer,
which catalyzes the oxidation of ascorbate to dehydroascorbate via a disproportionation of the semidehydroascorbate radical [105, 107]. It has been obtained from
several sources (plants, fungi, and eubacteria). The possibility of direct ET has been
confirmed on gold (T1 center) and carbon-based electrodes (T1 and T2/T3 centers)
[66, 115, 125]. The enzyme has been applied in the construction of biosensors for
the detection of ascorbic acid [114, 117, 126–128] (ascorbate anion at physiological
pH [129]), as well as organophosphorus pesticides monitoring [130]. Determination
of ascorbic acid in the clinical and food industry is very important since its deficiency is known to cause several medical conditions (e.g., anemia, scurvy, muscle
degeneration, atherosclerotic plaques, and capillary hemorrhaging, neurotic disturbances) [128], while in excess it can lead to gastric irritation, renal problems,
inhibition of natural processes occurring in food, and can contribute to taste/aroma
deterioration [131]. In these devices, the analyte is detected by monitoring the
localized consumption of O 2 using a Clark electrode [126, 127, 130], or by
observing the biocatalytic oxidation of ascorbate [114, 115, 128].
As for BOD, it is a monomeric enzyme that has been identified in various fungi
and bacteria, and it catalyzes the oxidation of bilirubin to biliverdin, as well as other
tetrapyrroles, phenols, and aryl diamines [105, 132–135]. The enzyme has the
ability to produce mediated bioelectrocatalytic currents for the reduction of O 2 to
water near-neutral pH [109–111, 136]. Although direct ET between the electrode
and the T1 and T2/3 centers is possible [137–140], the use of mediators facilitates
the electrochemical communication between the active site and the surface of the
electrode [109–111, 136]. Owing to its high thermal stability, low sensitivity to
chloride ions, and high activity at neutral pH 7, BOD is considered an excellent
candidate for the elaboration of efficient biofuel cells and biosensors operating at
physiological conditions [107, 135]. BOD has been applied to the development of
electrochemical monitoring tools for a diverse group of analytes, such as glucose
[117], DNA sequences [122], O 2 [123], and bilirubin [116, 141, 142]. The former is
a bile pigment that results from the heme degradation of erythrocytes by reticuloendothelial cells and exists in the serum in free form, unconjugated
(albumin-bound) and conjugated (with glucuronic acid), which is excreted in the
bile [143, 144]. This form is a biomarker of liver function and is used in the
diagnosis of jaundice and hyperbilirubinemia, while the unconjugated form is
specifically used in newborns where the liver is not mature enough to convert the
unconjugated bilirubin to conjugated bilirubin resulting in jaundice [143, 144].
Regarding the detection principle of the analyte, many BOD-based bioelectrode
papers claim that it was performed indirectly by monitoring the increase in H 2 O 2
concentration that resulted from the enzymatic reaction [135, 145, 146]. However,
this goes against the established concept that BOD catalyzes the complete reduction
of O 2 to water, without the release of intermediary reactive species [136, 147]. For
this reason, such devices were not considered in this text. For the remaining works,
the detection of the analyte has been carried out by monitoring the non-mediated
320
T. Monteiro et al.
immobilization of the enzyme, creating a more effective microenvironment [124].
The plant AOx is a homodimeric enzyme, with one catalytic site per monomer,
which catalyzes the oxidation of ascorbate to dehydroascorbate via a disproportionation of the semidehydroascorbate radical [105, 107]. It has been obtained from
several sources (plants, fungi, and eubacteria). The possibility of direct ET has been
confirmed on gold (T1 center) and carbon-based electrodes (T1 and T2/T3 centers)
[66, 115, 125]. The enzyme has been applied in the construction of biosensors for
the detection of ascorbic acid [114, 117, 126–128] (ascorbate anion at physiological
pH [129]), as well as organophosphorus pesticides monitoring [130]. Determination
of ascorbic acid in the clinical and food industry is very important since its deficiency is known to cause several medical conditions (e.g., anemia, scurvy, muscle
degeneration, atherosclerotic plaques, and capillary hemorrhaging, neurotic disturbances) [128], while in excess it can lead to gastric irritation, renal problems,
inhibition of natural processes occurring in food, and can contribute to taste/aroma
deterioration [131]. In these devices, the analyte is detected by monitoring the
localized consumption of O 2 using a Clark electrode [126, 127, 130], or by
observing the biocatalytic oxidation of ascorbate [114, 115, 128].
As for BOD, it is a monomeric enzyme that has been identified in various fungi
and bacteria, and it catalyzes the oxidation of bilirubin to biliverdin, as well as other
tetrapyrroles, phenols, and aryl diamines [105, 132–135]. The enzyme has the
ability to produce mediated bioelectrocatalytic currents for the reduction of O 2 to
water near-neutral pH [109–111, 136]. Although direct ET between the electrode
and the T1 and T2/3 centers is possible [137–140], the use of mediators facilitates
the electrochemical communication between the active site and the surface of the
electrode [109–111, 136]. Owing to its high thermal stability, low sensitivity to
chloride ions, and high activity at neutral pH 7, BOD is considered an excellent
candidate for the elaboration of efficient biofuel cells and biosensors operating at
physiological conditions [107, 135]. BOD has been applied to the development of
electrochemical monitoring tools for a diverse group of analytes, such as glucose
[117], DNA sequences [122], O 2 [123], and bilirubin [116, 141, 142]. The former is
a bile pigment that results from the heme degradation of erythrocytes by reticuloendothelial cells and exists in the serum in free form, unconjugated
(albumin-bound) and conjugated (with glucuronic acid), which is excreted in the
bile [143, 144]. This form is a biomarker of liver function and is used in the
diagnosis of jaundice and hyperbilirubinemia, while the unconjugated form is
specifically used in newborns where the liver is not mature enough to convert the
unconjugated bilirubin to conjugated bilirubin resulting in jaundice [143, 144].
Regarding the detection principle of the analyte, many BOD-based bioelectrode
papers claim that it was performed indirectly by monitoring the increase in H 2 O 2
concentration that resulted from the enzymatic reaction [135, 145, 146]. However,
this goes against the established concept that BOD catalyzes the complete reduction
of O 2 to water, without the release of intermediary reactive species [136, 147]. For
this reason, such devices were not considered in this text. For the remaining works,
the detection of the analyte has been carried out by monitoring the non-mediated
320
T. Monteiro et al.