NADH determination with optical sensors

Gautier S., Blum L.J., Coulet P.R., Alternate determination of ATP and NADH with a single bioluminescence-based fiber-optic sensor, Sensor Actuat B-Chem 1990 1 580. 

Figure 3.6 — (A) Fibre-optic biosensor system a septum b needle guide c thermostated reaction vessel d fibre bundle e enzyme membrane f screw cap g stirring bar h reaction medium i black PVC jacket j 0-ring. (B) Continuous-flow fibre-optic sensor system for the bioluminescence determination of NADH. (Reproduced from [41] with permission of Marcel Dekker, Inc.)...

Gautier, S. M., Blum, L. J., and Coulet, P. R., Multi-function fibre-optic sensor for the bioluminescent flow determination of ATP or NADH. Ana/. Chim. Acta 235,243-253 (1990). Gautier, S. M., Blum, L. J., and Coulet, P. R., Bioluminescence-based fibre-optic sensor with entrapped co-reactant An approach for designing a self-contained biosensor. Ana/. Chim. Acta 243, 149-156 (1991). 

Blum, L. J., Gautier, S. M., Coulet, P. R., Alternate Determination of ATP and NADH with a Single Fiber-Optic Sensor , Sens. Actuators B1 (1990) 580-584. 

The creation of an optical sensor that detects the cofactors on which many enzymes dqiend is a much massociated with many enzymes, particularly dehydrogenases. This cofactor has a maximal absorption at 340 nm and a maximal fluorescence emission at 400 nm, which is easily detectable with a photomultiplier. NADH also has the advantage that it can be immobilized on the same support as the enzyme (see 3.3.1.e). nber-optic biosensors based on the fluorimetric detection of NADH have been constructed for the determination of lactate and pyruvate [208]. These sensors use inunobilized lactate dehydrogenase AD) to catalyse the following equilibrium reaction ... 

Pyruvate is determined by setting the pH to a more acidic value (pH 7.4) to favor production of lactate. The NADH is then consumed, and the fluorescence diminishes with the pyruvate concentration, which can be measured from 0.2 to 1 pM. The presence of the equilibrium means that the response of the optical sensor is non-linear for both lactate and pyruvate. 

Alcohol oxidase was used to generate H202 followed by its reaction with luminol in the presence of K3[Fe(CN)6] as a catalyst [53], The luminescence was transmitted from the flow cell to the detector via optical fibers. Ethanol can be determined in the 3-750-pmol/L concentration range, with a detection limit of 3 pmol/L. Also, using an immobilized alcohol dehydrogenase reactor in glass beads, a FIA sensor for a reduced form of NADH was constructed by the ECL using the above-mentioned ruthenium tris(2,2 -biryridine) complex. The sensor was satisfactorily applied to the determination of ethanol concentration [54],... 

Optodes provided with non-fluorescent esters of fluorophores have been used for the determination of external enzyme activities. The fluorophores are liberated by the enzymes and then seen by the optical Ober [214], As ecamples of p(02)-modulated optical biosensors, a glucose probe [213] and an ethanol probe [216] can be mentioned sensors based on glucose, alcohol, and other oxidases were reviewed by Opitz and Lttbbers [217]. The advantages of these 02-dependent optical biosensors are that, unlike corresponding amperometric sensors, they do not consume O2 and that they are strictly diffusion limited in their response. Fiber-optical devices are also available for the determination of substrates of dehydrogenases the NADH fluorescence produced by the immobilized enzyme is measured as a function of time [218, 219]. 

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