In a recent work by Yuan and collaborators, a very promising method of NADH
regeneration without redox mediators in solution was developed. Herein, NADH
was bioelectrocatalytically regenerated by immobilizing diaphorase in a
cobaltocene-modified poly(allylamine) redox polymer; faradaic efficiencies
between 78 and 99% were achieved with high operational stability [168]. Electroactive polymers comprising redox moieties covalently bound to a polymeric
backbone are proving to be viable alternatives to conventional redox mediators, due
to an efficient ET through self-exchange based conduction.
Another possible solution for the electrochemical regeneration of NAD(P)
+
relies on the cofactor’s photochemical restoration by introducing a photosensitizer
in the matrix, such as N-methyl phenazonium methyl sulphate, porphyrins, Ru(II)tris-bipyridine with viologens as electron acceptors [153]. For example, a photoelectrochemical NAD(P) regeneration system composed of Ru(bipy)*
+ as a photosensitizer and methyl viologen (MV
2+ ) as a primary electron acceptor
(re-oxidized at the anode), was coupled to enzyme-catalyzed oxidations of alcohols
[169]. Research on photochemical NAD(P) generation has been limited and its
description is out of the scope of this chapter.
Despite the tremendous attention received by the scientific community over the
last decades and a few commercial achievements, challenges persist in the implementation of NAD(P)-dependent hydrogenases in electrochemical biosensors. The
need of adding the free-diffusing coenzyme, its complex electrochemistry, and the
lack of long-term stability are the main setbacks [170]. Therefore, at some point, the
trend shifted into the direction of other classes of dehydrogenases.
3.2 Other Dehydrogenases
In this section, we will address all other biosensors based on DHs that do not
depend on the soluble coenzymes NAD(P)H/NAD(P)
+ . Instead, these enzymes
harbor a FAD, FMN, PQQ, or molybdenum cofactor group that can be strongly
bound to the polypeptide chain, markedly improving the biosensors’ stability.
Another interesting feature of these cofactors is their formal reduction potentials
(Table 1), which are less negative than that assigned to the NAD(P)
+
/NAD(P)H
couple [149]. In particular, the flavin nucleotides, FMN and FAD, are two-electron,
two-proton redox centers that act as carriers of reducing equivalents. In a biological
context, the oxidized form can either accept one-electron (yielding the flavosemiquinone form) or two electrons (yielding FADH 2 or FMNH 2 ) by hydride transfer.
Unlike NAD(P)
+ , the reduction potential of flavin nucleotides depends on the
protein environment, as local interactions with amino acid side chains can distort
the electron orbitals in the flavin ring, affecting the stability of both oxidized and
reduced forms [148]. Representative values for the E°′ of protein-bound
FMN/FMNH 2 and FAD/FADH 2 are −0.30 V and −0.12 V (vs. NHE), respectively [171]. PQQ is also reduced by two electrons but at a remarkably higher
potential (+0.90 V). This cofactor is coordinately bound to the apoenzymes via Ca
2
328
T. Monteiro et al.
regeneration without redox mediators in solution was developed. Herein, NADH
was bioelectrocatalytically regenerated by immobilizing diaphorase in a
cobaltocene-modified poly(allylamine) redox polymer; faradaic efficiencies
between 78 and 99% were achieved with high operational stability [168]. Electroactive polymers comprising redox moieties covalently bound to a polymeric
backbone are proving to be viable alternatives to conventional redox mediators, due
to an efficient ET through self-exchange based conduction.
Another possible solution for the electrochemical regeneration of NAD(P)
+
relies on the cofactor’s photochemical restoration by introducing a photosensitizer
in the matrix, such as N-methyl phenazonium methyl sulphate, porphyrins, Ru(II)tris-bipyridine with viologens as electron acceptors [153]. For example, a photoelectrochemical NAD(P) regeneration system composed of Ru(bipy)*
+ as a photosensitizer and methyl viologen (MV
2+ ) as a primary electron acceptor
(re-oxidized at the anode), was coupled to enzyme-catalyzed oxidations of alcohols
[169]. Research on photochemical NAD(P) generation has been limited and its
description is out of the scope of this chapter.
Despite the tremendous attention received by the scientific community over the
last decades and a few commercial achievements, challenges persist in the implementation of NAD(P)-dependent hydrogenases in electrochemical biosensors. The
need of adding the free-diffusing coenzyme, its complex electrochemistry, and the
lack of long-term stability are the main setbacks [170]. Therefore, at some point, the
trend shifted into the direction of other classes of dehydrogenases.
3.2 Other Dehydrogenases
In this section, we will address all other biosensors based on DHs that do not
depend on the soluble coenzymes NAD(P)H/NAD(P)
+ . Instead, these enzymes
harbor a FAD, FMN, PQQ, or molybdenum cofactor group that can be strongly
bound to the polypeptide chain, markedly improving the biosensors’ stability.
Another interesting feature of these cofactors is their formal reduction potentials
(Table 1), which are less negative than that assigned to the NAD(P)
+
/NAD(P)H
couple [149]. In particular, the flavin nucleotides, FMN and FAD, are two-electron,
two-proton redox centers that act as carriers of reducing equivalents. In a biological
context, the oxidized form can either accept one-electron (yielding the flavosemiquinone form) or two electrons (yielding FADH 2 or FMNH 2 ) by hydride transfer.
Unlike NAD(P)
+ , the reduction potential of flavin nucleotides depends on the
protein environment, as local interactions with amino acid side chains can distort
the electron orbitals in the flavin ring, affecting the stability of both oxidized and
reduced forms [148]. Representative values for the E°′ of protein-bound
FMN/FMNH 2 and FAD/FADH 2 are −0.30 V and −0.12 V (vs. NHE), respectively [171]. PQQ is also reduced by two electrons but at a remarkably higher
potential (+0.90 V). This cofactor is coordinately bound to the apoenzymes via Ca
2
328
T. Monteiro et al.