most commonly used ET mediators in second generation NiR-based biosensors
[206]. Aside from these chemical species, physiological redox mediators have also
been employed, namely pseudoazurine coupled with the CuNiR from Alcaligenes
faecalis S-6 and cytochrome c 552 from Marinobacter hydrocarbonoclausticus,
co-immobilized with the cd 1 NiR from the same organism (Table 5) [213, 242]. The
interaction between the NiRs and their natural ET partner proteins is thought to be
advantageous relative to biosensors based on artificial mediators, given that it
reproduces the ET processes occurring in vivo and improves detection selectivity
[243].
The ability of ccNiRs to directly exchange electrons with carbon electrodes has
been exploited for the construction of non-mediated, third generation, nitrite
biosensors [214, 223, 224, 244]. In these devices, the electrocatalytic currents arise
from the direct regeneration of enzyme cofactors at the electrode surface, following
substrate reduction, as shown in Fig. 11b. Since they are operated at potentials
directly related to the reduction potential of the enzyme, the selectivity is expected
to increase, and the interfering reactions minimized. The use of carbon nanomaterials for electrode modification was shown to improve the wiring of the enzyme
and to contribute to greatly enhanced enzyme catalysis [214, 245]. In effect, the
highest current density response to nitrite so far has been obtained with a bioelectrode based on Desulfovibrio desulfuricans ccNiR adsorbed on multilayered
single-walled CNTs (SWCNTs) deposits (sensitivity 2.4 AÁM
−1
Ácm
−2 ) [214].
Nitric oxide reductases (NORs) are involved in the third step of bacterial denitrification pathway, being responsible for the conversion of NO to nitrous oxide
[240, 241]. Recently the direct electrochemical response of M. hydrocarbonoclasticus NOR and the electrocatalytic activity towards NO have been studied at
carbon electrode interfaces [83, 215, 246]. The heterodimeric enzyme belongs to
the cNOR family of nitric oxide reductases and contains heme c and heme b ET
groups, and a catalytic di-iron center formed by a heme b 3 and a non-heme Fe B ,
bridged by a l-oxo/hydroxo group (Fig. 10c) [240, 241]. Direct ET of NOR was
observed upon adsorption to bare [215] and CNT composite modified pyrolytic
graphite (PG) electrodes [83, 246]. The resulting bioelectrodes were employed as
third generation biosensors for quantification of NO (Fig. 11b). The
nanocomposite-based electrodes provided much higher sensitivities in comparison
to the bare PG. The authors showed that the SWCNTs-lipid bilayer films and
MWCNTs-ionic liquid composites constitute suitable microenvironments for the
immobilization of the membrane protein, and for the preservation and enhancement
of its activity [83, 246].
4.3 Other Reductase Enzymes
Aside NaRs, three other molybdenum dependent reductase enzymes have been used
in amperometric biosensors, namely the DMSO reductase from Rhodobacter
sphaeroides, the trimethylamine N-oxide (TMAO) reductase (TorA) from E. coli,
and the perchlorate reductase from Dechlorosoma sp. Like the prokaryotic NaRs,
Selective Enzymes at the Core of Advanced Electroanalytical …
339
[206]. Aside from these chemical species, physiological redox mediators have also
been employed, namely pseudoazurine coupled with the CuNiR from Alcaligenes
faecalis S-6 and cytochrome c 552 from Marinobacter hydrocarbonoclausticus,
co-immobilized with the cd 1 NiR from the same organism (Table 5) [213, 242]. The
interaction between the NiRs and their natural ET partner proteins is thought to be
advantageous relative to biosensors based on artificial mediators, given that it
reproduces the ET processes occurring in vivo and improves detection selectivity
[243].
The ability of ccNiRs to directly exchange electrons with carbon electrodes has
been exploited for the construction of non-mediated, third generation, nitrite
biosensors [214, 223, 224, 244]. In these devices, the electrocatalytic currents arise
from the direct regeneration of enzyme cofactors at the electrode surface, following
substrate reduction, as shown in Fig. 11b. Since they are operated at potentials
directly related to the reduction potential of the enzyme, the selectivity is expected
to increase, and the interfering reactions minimized. The use of carbon nanomaterials for electrode modification was shown to improve the wiring of the enzyme
and to contribute to greatly enhanced enzyme catalysis [214, 245]. In effect, the
highest current density response to nitrite so far has been obtained with a bioelectrode based on Desulfovibrio desulfuricans ccNiR adsorbed on multilayered
single-walled CNTs (SWCNTs) deposits (sensitivity 2.4 AÁM
−1
Ácm
−2 ) [214].
Nitric oxide reductases (NORs) are involved in the third step of bacterial denitrification pathway, being responsible for the conversion of NO to nitrous oxide
[240, 241]. Recently the direct electrochemical response of M. hydrocarbonoclasticus NOR and the electrocatalytic activity towards NO have been studied at
carbon electrode interfaces [83, 215, 246]. The heterodimeric enzyme belongs to
the cNOR family of nitric oxide reductases and contains heme c and heme b ET
groups, and a catalytic di-iron center formed by a heme b 3 and a non-heme Fe B ,
bridged by a l-oxo/hydroxo group (Fig. 10c) [240, 241]. Direct ET of NOR was
observed upon adsorption to bare [215] and CNT composite modified pyrolytic
graphite (PG) electrodes [83, 246]. The resulting bioelectrodes were employed as
third generation biosensors for quantification of NO (Fig. 11b). The
nanocomposite-based electrodes provided much higher sensitivities in comparison
to the bare PG. The authors showed that the SWCNTs-lipid bilayer films and
MWCNTs-ionic liquid composites constitute suitable microenvironments for the
immobilization of the membrane protein, and for the preservation and enhancement
of its activity [83, 246].
4.3 Other Reductase Enzymes
Aside NaRs, three other molybdenum dependent reductase enzymes have been used
in amperometric biosensors, namely the DMSO reductase from Rhodobacter
sphaeroides, the trimethylamine N-oxide (TMAO) reductase (TorA) from E. coli,
and the perchlorate reductase from Dechlorosoma sp. Like the prokaryotic NaRs,
Selective Enzymes at the Core of Advanced Electroanalytical …
339