Poly NADH oxidation

Simon et al. [92] investigated a biocatalytic anode based on lactate oxidation by lactate dehydrogenase (LDH). The anodic current is generated by the oxidation of NADH (produced by NAD+ and substrate) while LDH catalyzes the electro-oxidation of lactate into pyruvate. As previously mentioned, the oxidation of NADH at bare electrodes requires a large overpotential, so these authors used poly(aniline) films doped with polyanions to catalyze NADH oxidation. Subsequent research by this group focused on targeting mutants of LDH that are amenable to immobilization on the polyaniline surface [93],... 

Use of electrocatalyst conducting poly- o-phenylenediamine) film, NAD 1 oxidation products (can act as electrocatalysts for NADH oxidation) [22]... 

Gorton and coworkers have been particularly active in this field and produced an excellent review of the methods and approaches used for the successful chemical modification of electrodes for NADH oxidation [33]. They concentrated mainly on the adsorption onto electrode surfaces of mediators which are known to oxidise NADH in solution. The resulting systems were based on phenazines [34], phenoxazines [35, 36] and pheno-thiazines [32]. To date, this approach has produced some of the most successful electrodes for NADH oxidation. However, attempts to use similar mediators attached to poly(siloxane) films at electrode surfaces have proved less successful. Kinetic analysis of the results indicates that this is because of the slow charge transfer between the redox centres within the film so that the catalytic oxidation of NADH is restricted to a thin layer nearest the electrode surface [37, 38]. This illustrates the importance of a charge transfer between mediator groups in polymer modified electrodes. 

Having shown that poly(aniline) films can mediate NADH oxidation, studies of the effect of altering the applied potential and the rotation rate of the electrode were undertaken. Preliminary results from these studies showed that the maximum current response was obtained when the applied potential was 0.2 V vs. SCE and that the currents were two orders of magnitude higher for poly(aniline) modified electrodes when compared to a bare electrode indicating that poly(aniline) is a good catalytic surface for the oxidation of NADH. However, studies of the effect of rotation speed carried out at pH 5 show a decline in current with time (see Fig. 2.12). 

Catalysis of NADH oxidation Figure 2.15 shows the first cycle response of two different poly(aniline)/ poly(vinylsulfonate) composite films in 0.1 mol dm-3 pH 7 citrate/phos-phate buffer with and without added NADH. 

The excellent stability and reproducibility of these modified poly(aniline) electrodes for NADH oxidation allows detailed kinetic studies of the system to be completed. To determine the mechanism of NADH oxidation the effects of film thickness, electrode potential, rotation rate and NAD concentration have been investigated. 

Having ascertained where the reaction occurs, the effect of altering the potential applied to the poly(aniline) film on the amperometric response to NADH was studied. The resistance characteristics of the poly(aniline)/ poly(vinylsulfonate) at this pH show that within the range -0.10 to +0.15 V vs. SCE, the polymer is sufficiently conducting to ensure that the system has reasonable response times. Figure 2.19(A) shows the response of a poly(aniline)/poly(vinylsulfonate) film to NADH at seven different potentials, within this chosen range. It was found that increasing the applied potential increases the current for NADH oxidation—the linear... 

NADH oxidation to function equally well for the oxidation of NADPH. Figure 2.22 shows a direct comparison of the responses of a poly(aniline)-coated electrode to NADH and NADPH. At low concentrations, the currents are identical within experimental error but at higher concentration (in this case above 0.6 mmol dm-3), the currents for NADPH fall below those for NADH. These results clearly show some saturation of the current at high NADPH concentrations. 

The results presented above show that the poly(aniline)/poly(vinylsul-fonate) composite is an electrocatalytic surface for NADH oxidation at... 

Best-fit parameters from the analysis of the data in Fig. 2.23 for the currents for NADH oxidation in the absence of added NAD+ at poly(aniline)/poly(vinylsulfonate)-modified electrodes, in each case, the data were fitted to equation (2.20) for the I/II boundary (n is the number of data points used)... 

Fig. 2.28. Case diagram for NADH oxidation at a glassy carbon electrode coated with a poly(aniline)/poly(vinylsulfonate) film. The points are the experimental data. The surface and case boundaries have been determined from the inhibited fit parameters given in Table 2.8. The residuals are shown as a function of the concentration of NADH below the plot.

Best-lit parameters from the analysis of the currents for all NADH oxidation data fitted at once using both the uninhibited and inhibited fits at poly(aniline)/poly(vinylsulfonate)-modified electrodes. The values were obtained by non-linear least squares fits of the experimental data to equations (2.4)-(2.8) for the uninhibited fit, with the addition of equations (2.15), (2.17) and (2.18) for the inhibited fit. In each case n is the total number of data points used in the fit... 

Modified electrodes for this analytical purpose have mostly been formed by electrode adsorption of the mediator systems on the electrode surface or by electropolymerization [24,116]. Recently, for example, NAD(P)H oxidations have been performed on platinum or gold electrodes modified with a monolayer of pyrroloquinoline quinone (PQQ) [117] or on poly(methylene blue)-modified electrodes with different dehydrogenases entrapped in a Nafion film for the amperometric detection of glucose, lactate, malate, or ethanol [118]. In another approach, carbon paste electrodes doped with methylene green or meldola blue together with diaphorase were used for the NADH oxidation [119]. A poly(3-methylthio-phene) conducting polymer electrode was efficient for the oxidation of NADH [120]. By electropolymerization of poly(aniline) in the presence of poly(vinylsulfonate) counterions. 

In the study of NADH oxidation at poly(pyrrole), Schuhmann and colleagues used poly(pyrrole) and modified poly(pyrrole) films. For poly(pyrrole) itself and poly(pyrrole) containing electrostatically bound ferricyanide, vanadate, or molybdate anions, they observed no NADH oxidation, although these electroactive anions are known to oxidize the NADH in homogeneous solution. By using pyrrole modified by covalent attachment of chloranil or 2,3-dichloro-1,4-naphthoquinone (Fig. 9.19) structures, they were able to obtain catalytic oxidation of NADH and reasonable stability. It this case quinone functionalities are presumed to act as catalytic sites for oxidation, since they are known to be efficient homogeneous oxidants for NADH. ... 

Polymeric films active in the mediated oxidation of NADH have been prepared and studied. Films of the electropolymerized conducting polymer, poly(3-methylthionine) (P3MT) on platinum electrodes displayed a catalytic activity toward NADH oxidation and yielded extremely stable films with high resistance toward surface passivation. Hajizadeh et immobilized the electron transfer... 

Different lactate oxidising enzymes use different co-substrates and, therefore, a variety of electrochemical indicator reactions in biosensors can be utilised. Most of the lactate biosensors are based on enzymes like lactate oxidase (LOD) and lactate dehydrogenase (LDH). A needle-type lactate biosensor has been recently developed by Yang and coworkers who fabricated poly (1,3-phenylenediamine) electrodes immobilised with LOD for continuous intravascular lactate monitoring [185]. In the enzyme electrodes based on LDH, the biochemical reaction has been coupled to the electrode via NADH oxidation, either directly [119,123,163], or by using mediators [186] or additional enzymes [119]. This may lead to a shift of the unfavourable reaction equilibrium by partial trapping of the reduced cofactor. Direct oxidation of NADH requires potentials of more than 0.4 V ... 

Arechederra MN, Addo PK, Minteer SD (2010) Poly (neutral red) as a NAD reduction catalyst and a NADH oxidation catalyst towards the development of a rechargeable biobattery. Electrochim Acta 56(3) 1585-1590... 

Figure 4.6 shows the electrochemical activity of deposited poly(MG) onto RVC, and in each case, a nonlinear dependence that resembles Michaelis-Menten-type kinetics is observed. This observation agrees with the model proposed in 1985 by Gorton et al. [51] for mediator-modified electrodes for NADH oxidation, and it agrees with similar studies in 1990 and 2001 [53,105]. This model postulates the formation of a charge transfer (CT) complex in the reaction sequence between NADH and the mediator, because the observed reaction rate starts to decrease with the increase in NADH concentration, analogous to the Michaelis-Menten kinetics of enzymatic reactions. Catalytic activity of poly(MG) is inversely proportional to the thickness of the polymer, and the number of deposition cycles is consistent with observations in the literature for other NADH mediators [26,44,47,49]. This is attributed to the low partition coefficient of NADH and the diffusion coefficient of NADH within the... 

Karyakin AA, Karyakina EE, Schuhmann W, Schmidt HL, Varfolomeyev SD. New amperometric dehydrogenase electrodes based on electrocatalytic NADH-oxidation at poly (methylene blue)-modified electrodes. Electroanalysis 1994 6 821-829. 

Rincon RA, Artyushkova K, Atanassov P, Germain MN, Minteer SD, Lau C, Cooney MJ. Integrating poly-azine catalysts for NADH oxidation in biofuel cell aooAes.AbstrPapAm Chem Soc 2009 238. 

Polypyrrole shows catalytic activity for the oxidation of ascorbic acid,221,222 catechols,221 and the quinone-hydroquinone couple 223 Polyaniline is active for the quinone-hydroquinone and Fe3+/Fe2+ couples,224,225 oxidation of hydrazine226 and formic acid,227 and reduction of nitric acid228 Poly(p-phenylene) is active for the oxidation of reduced nicotinamide adenine dinucleotide (NADH), catechol, ascorbic acid, acetaminophen, and p-aminophenol.229 Poly(3-methylthiophene) catalyzes the electrochemistry of a large number of neurotransmitters.230... 

Assuming the P/O-quotient of NADH is 2 and NADPH can be used bioenerge-tically, about 0.5 acetate must be oxidized to neutralize the synthesis. This expenditure of substrate diminishes the product yield coefficient from 0.72 g poly(3HB) per g acetic acid (Table 3) to about 0.57 g per g. Since the experimentally obtained yield coefficient is lower (being, on average, about 0.33 g per g, Table 3), we may draw three conclusions. Firstly, the P/O-quotient is lower than 2. Secondly, the fate of acetate is not strictly determined, i. e., its utilization is not a one-way path and does not terminate in a dead end. Third, there is no doubt that some energy generated from acetate is necessary for homeostasis and turnover processes (maintenance) under conditions of poly(3HB) synthesis and accumulation (with acetic acid as an uncoupler). 

Depletion of ATP is caused by many toxic compounds, and this will result in a variety of biochemical changes. Although there are many ways for toxic compounds to cause a depletion of ATP in the cell, interference with mitochondrial oxidative phosphorylation is perhaps the most common. Thus, compounds, such as 2,4-dinitrophenol, which uncouple the production of ATP from the electron transport chain, will cause such an effect, but will also cause inhibition of electron transport or depletion of NADH. Excessive use of ATP or sequestration are other mechanisms, the latter being more fully described in relation to ethionine toxicity in chapter 7. Also, DNA damage, which causes the activation of poly(ADP-ribose) polymerase (PARP), may lead to ATP depletion (see below). A lack of ATP in the cell means that active transport into, out of, and within the cell is compromised or halted, with the result that the concentration of ions such as Na+, K+, and Ca2+ in particular compartments will change. Also, various synthetic biochemical processes such as protein synthesis, gluconeogenesis, and lipid synthesis will tend to be decreased. At the tissue level, this may mean that hepatocytes do not produce bile efficiently and proximal tubules do not actively reabsorb essential amino acids and glucose. 

In this chapter, we present an overview of the detailed kinetic analysis of the oxidation of NADH at a poly(aniline) modified electrode, both to demonstrate the basic approach which can be used, and to illustate the type of information which can be extracted. In many ways the design of... 

Fig. 2.7. Schematic representation of proposed reaction mechanism for oxidation of NADH by poly(aniline)/poly(vinylsulfonate) composite films (the symbols are defined in the text). Note There is no differentiation between the substrate/ site complex formation and the chemical reaction, kcm, that occur at the site.

Fig. 2.9. A postulate mechanism for the oxidation of NADH by poly(aniline).

Fig. 2.12. Plot of the current as a function of time for the oxidation of 4 mmol dm- 1 NADH at 0.2 V at a poly(aniline)-coated rotating disc electrode (area 0.38 cm2, deposition charge ISO mC) in 0.1 mol dm 1 citrate/phosphate buffer, pH 5. The rotation speed of the electrode was increased in the sequence I, 4, 9, 16, 25, 36 and 49Hz and reduced in sequence back to 1 Hz. The broken line connects segments of the curve corresponding to the different rotation speeds. Note The current decays more rapidly at the higher rotation speeds and responds rapidly to changes in rotation speed.

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