eukaryotic and prokaryotic NaRs, respectively. In addition, they contain other redox
centers that are involved in electron flow to the catalytic site, namely heme b,
flavins, and Fe-S clusters (Table 1 and Fig. 10a) [228, 229]. Although NaRs from
all types have been employed in nitrate biosensors, the most commonly used are the
commercially available NaRs from Aspergillus niger (fungal) and E. coli (Nar).
The majority of NaR biosensors are based on amperometric/voltammetric
transducers [209]. Less explored approaches include colorimetric, potentiometric,
and conductometric detection of the catalytic reaction [210, 211, 230–233]. As
mentioned earlier, this work focuses on electrochemical biosensors.
In what concerns NaR amperometric/voltammetric biosensors, very few studies
have explored direct ET for signal transduction [77, 234]. The majority of works
employ redox mediators to facilitate the electrical communication between NaR and
the electrode transducer. NAD(P)H and viologen derivatives are the most used
compounds, but several other electron carrier molecules have been employed, such
as azure A, safranins, hydroquinones, as well as, a heme-protein ET mediator,
microperoxidase-11 [209, 235, 236]. The reaction scheme for mediated NaR (and
other reductases) biosensors is depicted in Fig. 11a. In general, the redox mediators
are co-immobilized with NaR on the surface of the electrode, although in some
cases they are added to the assay solution. The immobilization methods used
include adsorption, cross-linking, entrapment, electropolymerization, and covalent
binding [208, 209]. As for the types of immobilization materials, they range from
conducting and non-conduction polymers (e.g., polypyrrole [PPy] and Nafion)
[212, 237] to self-assembled monolayers and nanomaterials (e.g., carbon nanotubes
[CNTs] and poly(3,4-ethylenedioxythiophene) [PEDOT] nanowires) [238, 239].
Apart from the detection modes, potentiometric and conductometric NaR biosensors follow very similar construction modes to the amperometric/voltammetric
counterparts. The enzymes are immobilized in polymeric matrices, such as Nafion
and PPy, and redox mediators (Azure A, NADH, methyl viologen [MV]) are used
to convert the enzyme to its reduced active state [210, 211, 232].
c
Fig. 10 Three-dimensional structures of reductase enzymes used in biosensors. a Catalytic
subunit of periplasmic nitrate reductase (Nap) from E. coli (2NYA); molybdenum cofactor active
site is depicted in green and Fe-S cluster in yellow. b Catalytic subunit of cytochrome c nitrite
reductase (ccNiR) from D. desulfuricans (1OAH); heme groups are shown in dark red. c Nitric
oxide reductase (NOR) from Pseudomonas aeruginosa (3O0R); non-heme iron (FeB) is
represented in blue and heme groups are depicted in orange. d DMSO reductase from R.
sphaeroides (1EU1); molybdenum cofactor active site is shown in green. e TMAO reductase from
Shewanella massilia (1TMO); molybdenum cofactor active site is depicted in cyan; f Perchlorate
reductase from Azospira oryzae PS (5CH7); molybdenum cofactor active site and Fe-S clusters are
represented in green and in yellow, respectively. g GSH reductase from Saccharomyces cerevisiae
(2HQM); Cys residues forming the disulfide bond at the catalytic site are represented in yellow and
FAD cofactors are shown in orange. h Nitroreductase from Enterobacter cloacae (1NEC); FMN
cofactors are shown in yellow. i Cytochrome c reductase (CcR) from Bos taurus (1BGY); hemes
are depicted in red and Rieske cluster in green. Structures were prepared using UCSF Chimera
software and the respective entries from RCSB Protein Data Bank (codes in brackets)
336
T. Monteiro et al.
centers that are involved in electron flow to the catalytic site, namely heme b,
flavins, and Fe-S clusters (Table 1 and Fig. 10a) [228, 229]. Although NaRs from
all types have been employed in nitrate biosensors, the most commonly used are the
commercially available NaRs from Aspergillus niger (fungal) and E. coli (Nar).
The majority of NaR biosensors are based on amperometric/voltammetric
transducers [209]. Less explored approaches include colorimetric, potentiometric,
and conductometric detection of the catalytic reaction [210, 211, 230–233]. As
mentioned earlier, this work focuses on electrochemical biosensors.
In what concerns NaR amperometric/voltammetric biosensors, very few studies
have explored direct ET for signal transduction [77, 234]. The majority of works
employ redox mediators to facilitate the electrical communication between NaR and
the electrode transducer. NAD(P)H and viologen derivatives are the most used
compounds, but several other electron carrier molecules have been employed, such
as azure A, safranins, hydroquinones, as well as, a heme-protein ET mediator,
microperoxidase-11 [209, 235, 236]. The reaction scheme for mediated NaR (and
other reductases) biosensors is depicted in Fig. 11a. In general, the redox mediators
are co-immobilized with NaR on the surface of the electrode, although in some
cases they are added to the assay solution. The immobilization methods used
include adsorption, cross-linking, entrapment, electropolymerization, and covalent
binding [208, 209]. As for the types of immobilization materials, they range from
conducting and non-conduction polymers (e.g., polypyrrole [PPy] and Nafion)
[212, 237] to self-assembled monolayers and nanomaterials (e.g., carbon nanotubes
[CNTs] and poly(3,4-ethylenedioxythiophene) [PEDOT] nanowires) [238, 239].
Apart from the detection modes, potentiometric and conductometric NaR biosensors follow very similar construction modes to the amperometric/voltammetric
counterparts. The enzymes are immobilized in polymeric matrices, such as Nafion
and PPy, and redox mediators (Azure A, NADH, methyl viologen [MV]) are used
to convert the enzyme to its reduced active state [210, 211, 232].
c
Fig. 10 Three-dimensional structures of reductase enzymes used in biosensors. a Catalytic
subunit of periplasmic nitrate reductase (Nap) from E. coli (2NYA); molybdenum cofactor active
site is depicted in green and Fe-S cluster in yellow. b Catalytic subunit of cytochrome c nitrite
reductase (ccNiR) from D. desulfuricans (1OAH); heme groups are shown in dark red. c Nitric
oxide reductase (NOR) from Pseudomonas aeruginosa (3O0R); non-heme iron (FeB) is
represented in blue and heme groups are depicted in orange. d DMSO reductase from R.
sphaeroides (1EU1); molybdenum cofactor active site is shown in green. e TMAO reductase from
Shewanella massilia (1TMO); molybdenum cofactor active site is depicted in cyan; f Perchlorate
reductase from Azospira oryzae PS (5CH7); molybdenum cofactor active site and Fe-S clusters are
represented in green and in yellow, respectively. g GSH reductase from Saccharomyces cerevisiae
(2HQM); Cys residues forming the disulfide bond at the catalytic site are represented in yellow and
FAD cofactors are shown in orange. h Nitroreductase from Enterobacter cloacae (1NEC); FMN
cofactors are shown in yellow. i Cytochrome c reductase (CcR) from Bos taurus (1BGY); hemes
are depicted in red and Rieske cluster in green. Structures were prepared using UCSF Chimera
software and the respective entries from RCSB Protein Data Bank (codes in brackets)
336
T. Monteiro et al.