of these GDHs is their insensitivity to O 2 , whereas their main weakness is their
lower substrate selectivity. The Abbott FreeStyle Optium and FreeStyle Optium
Neo blood glucose meters, for example, employ the NAD
+
-dependent GDH. Furthermore, by using the NAD
+
-dependent b-hydroxybutyrate DH, they also test for
b-ketone [152].
3.1 NAD(P)H Dependent Dehydrogenases
The prevalence of NAD(P)
+ DHs is easily explained by the fact that they are
ubiquitous in nature making this cofactor a universal electron acceptor [148]. As a
result, many of their substrates, including glucose, lactate, ethanol among others,
are quite relevant from the analytical standpoint.
In general, the pair NAD
+
/NADH is involved in catabolic pathways, whereas
NADP
+
/NADPH participates in anabolic reactions. Aside from the differences in
their biological functions, both forms have similar thermodynamic properties (E°
′ = −0.315 V vs. NHE, at pH 7) and reaction mechanisms [153]. Equations (3) and
(4) show the typical catalytic mechanism associated with NAD(P)
+ -linked dehydrogenases in a biological environment. The enzyme (E) removes two hydrogen
atoms from its substrate (S). While one hydrogen is transferred as a hydride ion
(H
− ; the equivalent of a proton and two electrons) to NAD
+
, the other is released as
a proton (H
+
), in a reversible process [148].
S þ E þ NADðPÞ
þ ! S Á E Á NADðPÞ
þ ! E þ P þ NADðPÞH þ H
þ
ð3Þ
NADðPÞH ! NADðPÞ
þ þ H
þ
þ 2e
À
ð4Þ
Since the enzyme’s substrate (analyte) is stoichiometrically equivalent to NAD
(P)
+ (1:1), the electrochemical monitoring of the cofactor would allow the quantification of the former. In broad terms, this is the strategy underlying most of the DHs
biosensors. However, the electroanalysis of the redox pairs NAD(P)H/NAD(P)
+ is
highly challenging due to the marked lack of reversibility and the high overvoltages
required to drive the electrochemical reaction. Unlike in nature, the electro-oxidation
of NAD(P)H at bare electrodes, especially when made of carbonaceous or metallic
materials, occurs through much complex high energy reaction routes, which require
large overpotentials that typically go up to 1 V vs. saturated calomel electrode (SCE).
This greatly increases the risks of interfering phenomena, including high background
currents, electrode fouling due to the formation of stable adducts between surface
species and radical intermediates as well as the adsorption of biologically inactive
NAD dimers. The occurrence of unwanted reactions with other easily oxidizable
compounds that are usually present in physiological samples such as, ascorbate,
urate, and acetaminophen, can also interfere in the analysis [152, 154, 155].
On the other hand, owing to the low formal reduction potential of NAD(P)
+
, its
electrocatalytic reduction requires the application of a voltage within a very negative window (below −0.315 V vs. NHE), where dissolved O 2 also responds,
Selective Enzymes at the Core of Advanced Electroanalytical …
323
lower substrate selectivity. The Abbott FreeStyle Optium and FreeStyle Optium
Neo blood glucose meters, for example, employ the NAD
+
-dependent GDH. Furthermore, by using the NAD
+
-dependent b-hydroxybutyrate DH, they also test for
b-ketone [152].
3.1 NAD(P)H Dependent Dehydrogenases
The prevalence of NAD(P)
+ DHs is easily explained by the fact that they are
ubiquitous in nature making this cofactor a universal electron acceptor [148]. As a
result, many of their substrates, including glucose, lactate, ethanol among others,
are quite relevant from the analytical standpoint.
In general, the pair NAD
+
/NADH is involved in catabolic pathways, whereas
NADP
+
/NADPH participates in anabolic reactions. Aside from the differences in
their biological functions, both forms have similar thermodynamic properties (E°
′ = −0.315 V vs. NHE, at pH 7) and reaction mechanisms [153]. Equations (3) and
(4) show the typical catalytic mechanism associated with NAD(P)
+ -linked dehydrogenases in a biological environment. The enzyme (E) removes two hydrogen
atoms from its substrate (S). While one hydrogen is transferred as a hydride ion
(H
− ; the equivalent of a proton and two electrons) to NAD
+
, the other is released as
a proton (H
+
), in a reversible process [148].
S þ E þ NADðPÞ
þ ! S Á E Á NADðPÞ
þ ! E þ P þ NADðPÞH þ H
þ
ð3Þ
NADðPÞH ! NADðPÞ
þ þ H
þ
þ 2e
À
ð4Þ
Since the enzyme’s substrate (analyte) is stoichiometrically equivalent to NAD
(P)
+ (1:1), the electrochemical monitoring of the cofactor would allow the quantification of the former. In broad terms, this is the strategy underlying most of the DHs
biosensors. However, the electroanalysis of the redox pairs NAD(P)H/NAD(P)
+ is
highly challenging due to the marked lack of reversibility and the high overvoltages
required to drive the electrochemical reaction. Unlike in nature, the electro-oxidation
of NAD(P)H at bare electrodes, especially when made of carbonaceous or metallic
materials, occurs through much complex high energy reaction routes, which require
large overpotentials that typically go up to 1 V vs. saturated calomel electrode (SCE).
This greatly increases the risks of interfering phenomena, including high background
currents, electrode fouling due to the formation of stable adducts between surface
species and radical intermediates as well as the adsorption of biologically inactive
NAD dimers. The occurrence of unwanted reactions with other easily oxidizable
compounds that are usually present in physiological samples such as, ascorbate,
urate, and acetaminophen, can also interfere in the analysis [152, 154, 155].
On the other hand, owing to the low formal reduction potential of NAD(P)
+
, its
electrocatalytic reduction requires the application of a voltage within a very negative window (below −0.315 V vs. NHE), where dissolved O 2 also responds,
Selective Enzymes at the Core of Advanced Electroanalytical …
323