Lactate recycling with enzyme electrodes

An enzyme electrode based on coimmobilized cytochrome b2 and laccase (Scheller et al., 1987b) allows an explanation of the principle of substrate recycling in enzyme electrodes in greater detail (Fig. 100). The advantage of this system is that the cosubstrate, oxygen, as well as the analytes, hydroquinone and benzoquinone, are electrochemically active. This permits one to study different parts of the recycling process. Recycling of the analyte in the presence of the substrate of cytochrome b2, lactate, results in an increase in the sensitivity by a factor of 500 as compared with lactate-free operation. Under conditions that are optimal for laccase the analyte is almost completely in the oxidized state, i.e. it... 

Blaedel and Jenkins (1976) evaluated two LDH—NAD combinations for a reagentless lactate sensor (Fig. 55). NAD was either coimmobilized with LDH to cellulose or an NAD+-agarose complex was constrained together with LDH to a region near the electrode surface. By bringing the bound NAD into intimate physical contact with the electrode, the immobilized cofactor was recycled electrochemically and reused by the enzyme. This eliminated the need to supply NAD as a reagent. 

Shu H-C, HSkanson H and Mattiasson B 1993 D-lactic acid in pork as a freshness indicator monitored by immobilized D-lactate dehydrogenase using sequential injection analysis Anal. Chim. Acta 283 727-37 Schubert F, Kirstein D, Schrader K L and Scheller F 1985 Enzyme electrodes with substrate and coenzyme amplification. Anal. Chim. Acta 169 391-6 Scheller F, Siegbahn N, Danielsson B and Mosbach K 1985 High sensitivity enzyme thermistor determination of L-lactate by substrate recycling Anal. Chem. 57 1740-3... 

Figure I4-2 Schematic view of an enzyme electrode using a double recycling system for the determination of ATP or ADP. PK = pyruvate kinase HK = herokinase LOD = lactate oxidase LDH = lactate dehydrogenase PEP = phosphoenolpyruvat Reproduced from [327] with permission from Marcel Dekket, Inc.

In another recycling system we co-immobolized cytochrome bz and laccase in a gelatin layer in front of an oxygen electrode. In the presence of the cytochrome bz substrate, lactate, BQ is reduced to H2Q. Therefore, this enzyme substitutes the cathode of the previous cycling system (Figure 6). However, the substrate recyling is not restricted to the phase boundary (as with electrochemical recyling) but it proceeds in the total volume of the enzyme layer. At the optimum pH of cytochrome b2 (pH 6.5) and lactate saturation a maximum amplification of 500 has been obtained. 

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