Selected examples of reductase-based biosensors are also summarized in Table 5,
including a description of their analytical performance. Owing to the nature of the
catalytic reactions, the majority of devices rely on amperometric/voltammetric
transducers; nonetheless, other detection modes have also been employed, such as
potentiometric, impedimetric, conductometric, optical/spectrophotometric, and
colorimetric biosensors [206, 209].
A common feature to almost all reports of reductase-based amperometric/
voltammetric biosensors is the need to work in O 2 free conditions. In fact, with a
few exceptions, the described sensing platforms operate in N 2 or argon purged
solutions. Therefore, we start this section with some considerations on the importance of O 2 removal in biosensing with reductases.
4.1 The O 2 Scavenging Challenge
Perhaps the biggest problems for the commercialization of reductase-based
biosensors are the maintenance of assay reducing conditions and the low potentials required for the catalytic reactions (Table 5). Interference from O 2 , in particular, is a major challenge as it may react with the enzyme, analyte, or electron donor
(e.g., mediators, reducing agents) involved in the biosensing reaction. Furthermore,
in the case of amperometric/voltammetric biosensors, O 2 reduction to water or
H 2 O 2 at the electrode surface generates i) high cathodic background current (below * −0.1 V vs. NHE, Fig. 9) at the potentials typically used for analyte
detection (Table 5) and ii) reactive oxygen species that can damage the enzymes.
Consequently, in the majority of works, the solutions (and samples) are purged with
inert gases (argon or N 2 ) to remove dissolved O 2 , a method that is not compatible
with on-site monitoring or POCT applications. Alternative strategies to overcome
this problem have been explored with some success. The subject was reviewed by
Plumeré [220]. The approaches span from i) the addition of chemical or enzymatic
O 2 scavengers to the assay solution to remove dissolved O 2 , such as sodium sulfite
or GOx/catalase (Cat) system, ii) to the shifting of the measurement potential to
more positive values, by using redox mediators or iii) by monitoring the reductase’s
activity through a coupled oxidase enzyme sharing a common co-substrate [220,
221]. The GOx/Cat O 2 scavenger system, in particular, has been proven very
efficient in reductase biosensor applications [85, 222–224]. It is based on the
concerted catalytic reactions of the two enzymes: glucose oxidation and concomitant O 2 reduction by GOx generates H 2 O 2 that is dismutated by Cat into water
and O 2 . In the net catalytic reaction, two glucose molecules are used for the
reduction of one O 2 molecule, which leads to a rapid decrease of O 2 concentration
in solution [220]. This system was successfully implemented in a recently reported
NiR biosensor. GOx and Cat were co-adsorbed with the nitrite reductase in disposable carbon screen-printed electrodes (CSPEs). The work revealed the good
performance of this O 2 scavenger system in the immobilized state and consequently
its potential for application in disposable biosensors free from O 2 interference
[224].
332
T. Monteiro et al.
including a description of their analytical performance. Owing to the nature of the
catalytic reactions, the majority of devices rely on amperometric/voltammetric
transducers; nonetheless, other detection modes have also been employed, such as
potentiometric, impedimetric, conductometric, optical/spectrophotometric, and
colorimetric biosensors [206, 209].
A common feature to almost all reports of reductase-based amperometric/
voltammetric biosensors is the need to work in O 2 free conditions. In fact, with a
few exceptions, the described sensing platforms operate in N 2 or argon purged
solutions. Therefore, we start this section with some considerations on the importance of O 2 removal in biosensing with reductases.
4.1 The O 2 Scavenging Challenge
Perhaps the biggest problems for the commercialization of reductase-based
biosensors are the maintenance of assay reducing conditions and the low potentials required for the catalytic reactions (Table 5). Interference from O 2 , in particular, is a major challenge as it may react with the enzyme, analyte, or electron donor
(e.g., mediators, reducing agents) involved in the biosensing reaction. Furthermore,
in the case of amperometric/voltammetric biosensors, O 2 reduction to water or
H 2 O 2 at the electrode surface generates i) high cathodic background current (below * −0.1 V vs. NHE, Fig. 9) at the potentials typically used for analyte
detection (Table 5) and ii) reactive oxygen species that can damage the enzymes.
Consequently, in the majority of works, the solutions (and samples) are purged with
inert gases (argon or N 2 ) to remove dissolved O 2 , a method that is not compatible
with on-site monitoring or POCT applications. Alternative strategies to overcome
this problem have been explored with some success. The subject was reviewed by
Plumeré [220]. The approaches span from i) the addition of chemical or enzymatic
O 2 scavengers to the assay solution to remove dissolved O 2 , such as sodium sulfite
or GOx/catalase (Cat) system, ii) to the shifting of the measurement potential to
more positive values, by using redox mediators or iii) by monitoring the reductase’s
activity through a coupled oxidase enzyme sharing a common co-substrate [220,
221]. The GOx/Cat O 2 scavenger system, in particular, has been proven very
efficient in reductase biosensor applications [85, 222–224]. It is based on the
concerted catalytic reactions of the two enzymes: glucose oxidation and concomitant O 2 reduction by GOx generates H 2 O 2 that is dismutated by Cat into water
and O 2 . In the net catalytic reaction, two glucose molecules are used for the
reduction of one O 2 molecule, which leads to a rapid decrease of O 2 concentration
in solution [220]. This system was successfully implemented in a recently reported
NiR biosensor. GOx and Cat were co-adsorbed with the nitrite reductase in disposable carbon screen-printed electrodes (CSPEs). The work revealed the good
performance of this O 2 scavenger system in the immobilized state and consequently
its potential for application in disposable biosensors free from O 2 interference
[224].
332
T. Monteiro et al.