NADH-sensor

Colorimetric and fluorimetric NH3 sensors contain mixtures of pH indicators having suitable dissociation constants at the tip of the fiber bundle. The measuring solution is separated from this indicator layer by an NH3 gas-permeable membrane covered by an immobilized de-aminating enzyme, e.g. urease (Wolfbeis, 1987 Arnold, 1987). The fluorimetric indication of NADH has been used in optical biosensors for lactate, pyruvate, and ethanol, where the respective dehydrogenase is immobilized at the tip of an optical NADH sensor (Arnold, 1987 Wangsa and Arnold, 1988). 

Ihble 14-9 shows the enzyme sensors developed so far using biocatalytic analyte recycling for signal amplification. The Hrst of such sensors have been studied for measurement of pyridine nucleotides, glucose, and lactate [321, 324]. The NAD /NADH sensor involves the oxidation-reduction of the analyte by horseradish peroxidase and glucose dehydrogenase ... 

The pH of the contacting solution has a profound effect on the reaction rate of eqn.(3). Under otherwise constant conditions, kobs will decrease with an increase in pH. This effect seems to be common to all phenoxazine mediators." For Meldola Blue, kobs reached almost a value of 10 M s" at pH 6.0. A virtually mass controlled current could thus be obtained at this pH with this CME working as an NADH sensor in a FIA-setup. ... 

An increased value of E of the adsorbed phenoxazine is expected to increase the reaction rate with NADH". If it is large enough kobs > 10 M s at all pHs) it should be possible to produce a virtually pH insensitive NADH sensor. A number of other phenoxazines (e.g. Brilliant Cresyl Blue, Fig.2) and other coupling reagents (Fig.4) are therefore under study at present. 

The fluorimetric indication of NADH has also been used in optical biosensors for lactate, pyruvate, and ethanol, where the respective dehydrogenase was immobilized at the tip of an optical NADH sensor. 

Ido Y, Chang K, Woolsey TA, Wdhamson JR. NADH sensor of blood flow need in brain, muscle, and other tissues. FASEB J 2001 15 1419-1421. 

NADH, 121, 122, 180 Nafion coating, 118, 123, 124, 126 Nanometer electrodes, 116, 128 Nernst equation, 3, 15, 80 Nernstian behavior, 143 Nernst Planck equation, 5 Neuronal sensors, 188 Neurotransmitters, 40, 116, 124 Neutral carrier electrodes, 154 Nickel, 123... 

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. 

Blum L. J., Gautier S.M., Coulet P.R., Continuous flow bioluminescent assay of NADH using a fibre-optic sensor, Anal. Chim. Acta 1989 226 331-336. 

Reliable measurements of L-lactate are of great interest in clinical chemistry, the dairy and vine industry, biotechnology, or sport medicine. In particular, blood lactate levels are indicative of various pathological states, including shock, respiratory insufficiencies, and heart and liver diseases. Silica sol-gel encapsulation of the lactate dehydrogenase and its cofactor was employed as a disposable sensor for L-lactate51. The sensor utilized the changes in absorbance or fluorescence from reduced cofactor nicotinamide adenine dinucleotide (NADH) upon exposure to L-lactate. 

Another type of sensor was based on the utilization of glucose dehydrogenase enzyme coupling with /ra(2,2 -bipyridylruthenium(II) complex [31]. This sensor can be used in the 10-2500-pmol/L concentration range. Several interferences occur, like NADH, oxalate, proline, and tripropylamine. However, gluconic acid and NAD+ do not interfere. 

Similar to the work described by Spohn et al. [34], a trienzyme sensor was developed recently for the determination of branched-chain amino acids (L-valine, L-leucine, and L-isoleucine). Leucine dehydrogenase, NADH oxidase, and peroxidase were coimmobilized covalently on tresylate-hydrophylic vinyl polymer beads and packed into a transparent PILL tube (20 cm X 1.0 id), which was used as flow cell. The sensor was free of interferences from protein and NH4+ and it was stable for 2 weeks. The sensor system was applied to the determination of branched-chain amino acids in plasma with recoveries ranging from 98 to 100% [36],... 

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],... 

Although much less so than pyrrole polymers, indole polymers are beginning to be synthesized and studied as new materials. Electropolymerized films of indole-5-carboxylic acid are well-suited for the fabrication of micro pH sensors and they have been used to measure ascorbate and NADH levels. The three novel pyrroloindoles shown have been electrochemically polymerized, and the polymeric pyrrolocarbazole has similar physical properties to polyaniline. 

Many bioluminescence sensors were investigated using enzymes for H202, NADH, ethanol, glucose, and various amino acids. 

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.)...

The monitoring of viable cell mass even in technical media is possible with this technique. However, fluorescence medium components might drastically interfere with the NADH-dependent fluorescence and signal disturbances occur caused by air bubbles (lowering the sensor readings) or large amounts of scattering particles. 

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