Dehydrogenase based

NADH. Immobilized redox mediators, such as the phenoxazine Meldola Blue or phenothiazine compoimds, have been particularly useful for this purpose (20) (see also Figure 4-12). Such mediation should be useful for many other dehydrogenase-based biosensors. High sensitivity and speed are indicated from the flow-injection response of Figure 3-21. The challenges of NADH detection and the development of dehydrogenase biosensors have been reviewed (21). Alcohol biosensing can also be accomplished in the presence of alcohol oxidase, based on measurements of the liberated peroxide product. 

Describe the major problems encountered in the detection of the NADH product of dehydrogenase-based amperometric biosensors. Discuss a common approach to circumvent these problems. 

M. Niculescu, R. Mieliauskiene, V. Laurinavicius and E. Csoregi, Simultaneous detection of ethanol, glucose and glycerol in wines using pyrroloquinoline quinone-dependent dehydrogenases based biosensors, Food Chem., 82(3) (2003) 481 89. 

M. Niculescu, T. Erichsen, V. Sukharev, Z. Kerenyi, E. Csoregi and W. Schuhmann, Quinohemoprotein alcohol dehydrogenase-based reagentless amperometric biosensor for ethanol monitoring during wine fermentation, Anal. Chim. Acta, 463(1) (2002) 39-51. 

Scheme 4.10 shows the equilibrium for the enzyme glutamate dehydrogenase. Based on the name of the enzyme alone, would you expect this enzyme to drive the reaction to the left or right Based on the Km values, does your answer change Note that the position of the equilibrium for this reaction is not determined by Km values but the free energy difference between the products and starting materials. 

Hassler, B.L., Kohli, N., Zeikus, J.G., Lee, I., Worden, R.M. (2007). Renewable dehydrogenase-based interfaces for bio-electronic applications. Langmuir 23 7127-33. 

Tapia and Eklund (1986) carried out a Monte Carlo simulation of the substrate channel of liver alcohol dehydrogenase, based on the X-ray diffraction structure for this enzyme. The addition of substrate and the associated conformation change induce an order—disorder transition for the solvent in the channel. A solvent network, connecting the active-site zinc ion and the protein surface, may provide the basis for a proton relay system. A molecular dynamics simulation of carbonic anhydrase showed two proton relay networks connecting the active-site zinc atom to the surrounding solvent (Vedani et ai, 1989). They remain intact when the substrate, HCOf, is bound. 

Gorton et al. reported carbon paste electrodes based on Toluidine Blue O (TBO)-methacrylate co-polymers or ethylenediamine polymer derivative and NAD" " with yeast alcohol dehydrogenase for the analysis of ethanol [152,153] and with D-lactate dehydrogenase for the analysis of D-lactic acid [154]. Use of electrodes prepared with dye-modified polymeric electron transfer systems and NAD+/NADH to detect vitamin K and pyruvic acid has also been reported by Okamoto et al. [153]. Although these sensors showed acceptable performances, insensitivity to ambient oxygen concentration, sensor stability and lifetime still need to be improved to obtain optimal dehydrogenase based enzyme biosensors. 

Chiodini, P. L., Bowers, K., Jorgensen, P., Barnwell, J. W., Grady, K. K., Luchavez, J., Moody, A. H., Cenizal, A., and Bell, D. (2007). The heat stability of Plasmodium lactate dehydrogenase-based and histidine-rich protein 2-based malaria rapid diagnostic tests. Trans. R. Soc. Trop. Med. Hyg. 101, 331-337. 

Table 5.2 Selected oxidase- or dehydrogenase-based electrodes. 

Figure 23 Proposed cataiytic mechanism for the Ciass iA dihydroorotate dehydrogenase based on initiai characterization of the enzyme isoiated from L lactis. This mechanism was proposed by Fagan etal. Reproduced from R. L. Fagan ...

Fig. 15. Representation of the structure of methylamine dehydrogenase based on the crystal structure 60, 159). The secondary structure elements, j8-sheets and a-heli-ces, are indicated as plates. The cofactor TTQ is presented in a ball-and-stick form. (Top) This representation shows clearly the small and large subunits, with the extended arm of the large subunit embracing the small subunit. (Bottom) The molecule is turned 90° with respect to the orientation in the top drawing, along the sevenfold symmetry present in the large subunit. A remarkable feature is that the cofactor is located on the extension of this sevenfold axis.

Okuda, J. Wakai, J. Igarashi, S. Sode, K. Engineered pqq glucose dehydrogenase-based enzyme sensor for continuous glucose monitoring. 

Describe the major problems eneountered in the deteetion of the NADH produet of dehydrogenase-based amperometrie biosensors. Diseuss a eommon approaeh to circiunvent these problems. 

The electrochemical sensing of NADH is of great interest in the development of a dehydrogenase-based amperometric biosensor owing to the ubiquitous use of NADH as a cofactor for over 300 enzymes and in the fine chemicals industry using NAD -dependent biocatalysts [106]. The oxidation of NADH at bare and modified electrodes has been well studied and the oxidation process is dependent on the nature of the electrode used. The direct electrochemical oxidation of NADH at the bare electrode, irrespective of its nature, requires a high overpotential, despite the formal potential of the NAD /NADH redox couple at pH 7, which is reported to be... 

Figure 4. Response curve for the lactate from the lactate dehydrogenase-based biosensor.

Saleh, F.S., Mao, L., Ohsaka, T. Development of a dehydrogenase-based glucose anode using a molecular assembly composed of nile blue and functionalized SWCNTs and its applications to a glucose sensor and glucose/02 biofuel cell Sens. Actuators B 152(1), 130-135 (2011). doi 10.1016/j.snb.2010.07.054... 

Becker, T., W. Schuhmann, R. Betken, H. L. Schmidt, M. Leible, and A. Albrecht. 1993. An automatic dehydrogenase-based flow-injection system Application for the continuous determination of glucose and lactate in mammalian cell-cultures. J. Chem. Technol. Biotechnol. 58 183-190. 

Takagi, K. and S. Shikata. 2004. Flow injection determination of histamine with a histamine dehydrogenase-based electrode. Anal. Chim. Acta 505 189-193. 

Wang, Z.J., Etienne, M., Poller, S., Schuhmann, W., Kohring, G.W., Mamane, V., and Walcarius, A. (2012) Dehydrogenase-based reagentless biosensors electrochemically assisted deposition of sol-gel thin films on functionalized carbon nanotubes. Electroanalysis, 24, 376-385. 

In addition to the previously described dehydrogenase-based CNT electrodes, electrochemical biosensors that employ other types of enzyme-modified CNTs have also been reported. Kowalewska and Kulesza applied CNTs with adsorbed redox mediator tetrathiafulvalene (TTF) for electrochemical detection of glucose." TTF-modified CNTs were found to facilitate electron transfer between GOx and the electrode surface for glucose detection. Jia et al. reported a similar strategy for the detection of lactate using MWCNTs modified with TTF and lactate oxidase. Since TTF does not cause skin irritation and the CNT/TTF platform also enables low-potential sensing of lactate, CNT/ TTF/lactate oxidase-based electrochemical biosensors conld be used to detect lactate in perspiration directly on human skin. This was accomplished by preparing temporary tattoos from CNT/ TTF/lactate oxidase-conductive carbon ink that was transferred onto a human subject s skin. ... 

F. Ricci, A. Amine, D. Moscone, G. Palleschi, A probe for NADH and H2O2 amperometric detection at low applied potential for oxidase and dehydrogenase based biosensor applications. Biosens. Bioelectron. 22 (2007) 854-862. 

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