Nicotinamide adenine dinucleotide fluorescence

Some proteins contain other native fluorophores in addition to fluorescent amino acids. These include cofactors such as nicotinamide adenine dinucleotide (fluorescent in its reduced, NADH state) and flavin adenine dinucleotide (FAD). NADH is weakly fluorescent in water, but its fluorescence yield increases markedly on binding to a protein-binding site with an emission peak around 470 nm (3). FAD and flavin mononucleotide (FMN) are also fluorescent with an emission maximum around 520 nm, but fluorescence is quenched on binding to many flavoproteins (4). 

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. 

Naphthalene-2,3-dicarboxaldehyde Nicotinamide adenine dinucleotide N-Acetylneuraminic acid 4-Fluoro-7-nitrobenzoxadiazole Naphthalene-2,3-dicarboxaldehyde Nondestructive readout Near infrared Near infrared fluorescence Nuclear magnetic resonance 2-Nitrophenyl oxalate 1,1 -Oxalyldiimidazole Polycyclic aromatic hydrocarbon Principal component analysis Photosensitized chemiluminescence Pentachlorophenyl oxalate Polymerase chain reaction... 

A final optical application deals with the measurement of intracellular nicotinamide adenine dinucleotide (NADH) by fluorescence [77], giving information about the physiological status of wastewater treatment plant biomass. This indirect method could be envisaged for toxicity estimation. 

A. Gafni and L. Brand, Fluorescence decay studies of reduced nicotinamide adenine dinucleotide in solution and bound to liver alcohol dehydrogenase, Biochemistry 15, 3165-3171 (1976). 

Autofluorescence of cells often complicates the studies with fluorescence microscopy (especially the application of green fluorescent substances). There are different reasons for the occurrence of this phenomenon (157) (i) the fluorescent pigment lipofuscin, which settles with rising age in the cytoplasm of cells (ii) cell culture medium, which often contains phenol red that increases autofluorescence (iii) endogen substances such as flavin coenzymes [flavin-adenine dinucleotide (FDA), flavin mononucleotide (FMN) absorp-tion/emission 450/515nm], pyridine nucleotides [reduced nicotinamide adenine dinucleotide (NADH) absorption/emission 340/460nm] or porphyrine (iv) substances taken up by cells (as mentioned above filipin) and (v) preparation of the cells fixation with glutaraldehyde increases autofluorescence. 

There are some methods that are specific to HCHO. For example, the Hantzsch reaction of HCHO, collected with a diffusion scrubber, with ammonium acetate, acetic acid, and acetylacetone to form diacetyldihydrolutidine, which is measured using its fluorescence at 470 nm, has been applied to air measurements (Dasgupta et al., 1988, 1990 Kleindienst et al., 1988a,b Lawson et al., 1990 Khare et al., 1997). Reaction with 1,3-cyclohexanedione and ammonium acetate to form a dihydropyridine derivative that is measured by fluorescence has been used in conjunction with a diffusion scrubber (Fan and Dasgupta, 1994). Enzymatic methods have been used in which formaldehyde dehydrogenase catalyzes the oxidation of HCHO to HCOOH in the presence of -nicotinamide adenine dinucleotide, NAD+, which is reduced to NADH. The latter is measured by fluorescence at 450 nm (Lazrus et al., 1988 Ho and Richards, 1990). 

An enzyme reactor with immobilized 3 -hydroxysteroid dehydrogenase has been successfully used for the analysis of residues of 17 -methyltestosterone in trout by high-performance liquid chromatography (HPLC) (269). Following their separation by reversed-phase chromatography, the major tissue metabolites of 17 -methyltestosterone, namely 5 -androstane-17 -methyl-3, 17 -diol, and 5 -androstane-17 -methyl-3, 17 -diol, were enzymatically modified in the presence of a coreactant, nicotinamide-adenine dinucleotide (NAD), to the corresponding ketone. The position at 3 was enzymatically oxidized, and NADH, the reduced form of NAD, was produced as a coproduct and subjected to fluorescence detection. Reoxidation of NADH to NAD provides the possibility for electrochemical detection. 

Tissue also contains some endogenous species that exhibit fluorescence, such as aromatic amino acids present in proteins (phenylalanine, tyrosine, and tryptophan), pyridine nucleotide enzyme cofactors (e.g., oxidized nicotinamide adenine dinucleotide, NADH pyridoxal phosphate flavin adenine dinucleotide, FAD), and cross-links between the collagen and the elastin in extracellular matrix.100 These typically possess excitation maxima in the ultraviolet, short natural lifetimes, and low quantum yields (see Table 10.1 for examples), but their characteristics strongly depend on whether they are bound to proteins. Excitation of these molecules would elicit background emission that would contaminate the emission due to implanted sensors, resulting in baseline offsets or even major spectral shifts in extreme cases therefore, it is necessary to carefully select fluorophores for implants. It is also noteworthy that the lifetimes are fairly short, such that use of longer lifetime emitters in sensors would allow lifetime-resolved measurements to extract sensor emission from overriding tissue fluorescence. 

Kong, K., Bers, M. W., and Tromberg, B. J (1997). Time-resolved and steady-state fluorescence measurements of beta-nicotinamide adenine dinucleotide-alcohol dehydrogenase complex during UVA exposure. Photochem. Photobio. B Biol. 37 91-95. 

Enzymatic cofactors, such as nicotinamide adenine dinucleotide (NADH), nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (EAD), flavin mononucleotide (EMN), and pyridoxal phosphate, are fluorescent and commonly found associated with various proteins where they are responsible for electron transport (see Fig. lb and Table 1). NADH and NADPH in the oxidized form are nonfluorescent, whereas conversely the flavins, FAD and EMN, are fluorescent only in the oxidized form. Both NADH and FAD fluorescence is quenched by the adenine found within their cofactor structures, whereas NADH-based cofactors generally remain fluorescent when interacting with protein structures. The fluorescence of these cofactors is often used to study the cofactors interaction with proteins as well as with related enzymatic kinetics (1, 9-12). However, their complex fluorescent characteristics have not led to widespread applications beyond their own intrinsic function. 

Fluorescence detection is also inherently more selective than absorbance detection, since both the excitation and emission wavelengths may be chosen to suit a particular reaction product. For example, assays employing dehydrogenase enzymes may monitor NAD+ or nicotinamide adenine dinucleotide phosphate (NADP+) absorbance at 340 nm with reasonable sensitivity and selectivity. However, if excited at 340 nm, the nicotinamide coenzymes fluoresce at 460 nm. Not only do the fluorescence measurements inherently have lower detection limits, but they also provide selectivity against potential interferents that may also absorb at 340 nm but do not emit at 460 nm. 

The reduced form of nicotinamide adenine dinucleotide (NADH) is an important and highly fluorescent coenzyme. It has an absorption maximum of 340 nm and an emission maximum at 465 nm. Standard solutions of NADH gave the following fluorescence intensities ... 

Figure 7.70 Structures of resazurin and resorufin. The transformation from blue resazurin to red fluorescing resorufin by reduction is catalysed by the redox reaction of nicotinamide adenine dinucleotide (NAD+, coenzyme 1) to NAD dehydrogenase.

TR-XRF totally reflecting X-ray fluorescence spectrometry (see TXRF) tryptophan essential amino acid, which participates in the biosynthesis of NAD (nicotinamide adenine dinucleotide) and is the initial precursor of the neurotransmitter serotonin... 

Rex, A., Hentschke, M.-P, and Fink, H., Bioavailability of reduced nicotinamide-adenin-dinucleotide (NADH) in the central nervous system of the anaesthetized rat measured by laser-induced fluorescence spectroscopy, P/jaraaco/. Toxicol, 90,2002. 

Most applications of native fluorescence detection involve the direct detection of a fluorescent analyte of interest. However, native fluorescence can also be used indirectly to detect the presence of nonfluorescent species. For example, the presence of a nonfluorescent enzyme can be detected based on its conversion of substrate into natively fluorescent products. This is often used in conjunction with electrophoretically mediated microanalysis, in which differences in buffer mobilities enable on-column mixing and reaction of enzymes with substrates. One such study exploited the intrinsic fluorescence of nicotinamide adenine dinucleotide (NADH) to investigate differences in the reactivity of individual molecules of lactate dehydrogenase. 

In addition to the development of absorbance-based fiber-optic biosensors, we have recently demonstrated the feasibility of fluorescence-based biosensors (9, 10). Our initial work in this area has focused on the development of fiber-optic biosensors based on the fluorometric detection of reduced nicotinamide adenine dinucleotide (NADH). Here, a dehydrogenase enzyme supplies the biocatalytic activity, and either the generation or consumption of NADH is oionitored. 

The detection of DNA hybridization using electrochemical readout is particularly attractive for the development of clinical diagnostics. The use of nanostructured materials in electrical detection for biomolecular sensing offers unique opportunities for electrochemical transduction of DNA sensing events. Tian and co-workers [175] have reported that PANI/Gold nanoparticle multilayer films electrocatalyze the oxidation of nicotinamide adenine dinucleotide (NADH) and detect DNA hybridization by both an electrochemical method and by surface plasmon enhanced fluorescence... 

Dimethylamino)ethanethiol DMAET-capped CdTe NPs were employed in the construction of aptamer-based biosensing system for proteins [109]. Highly efficient and sensitive determination of alkaloids and amino acids using CE in conjunction with sequential light-emitting diode-induced fluorescence (LEDIF) and ECL method was carried out. In the CE-LEDIF-ECL system, the ECL detector was located in the outlet of the capillary, while the LEDIF detector was positioned 12 cm from the outlet [110]. Ru(bpy)3 in MWCNTs/Nafion composite membrane was immobilized for an ECL method for reduced nicotinamide adenine dinucleotide (NADH) with a detection limit of 8.2 x 10 M [111]. 

The sensors for lactate and penicilline in Table 8.5 make use of fluorescence itself rather than fluorescence quenching. The lactate sensors stand for a group of optodes where the inherent fluorescence of NAD (nicotinamide-adenine-dinucleotide, see Chap. 7, Sect. 7.4.2) is evaluated. Reaction (8.8) can be influenced by establishing a specific pH so that it runs either from left to right or right to left. In this way, lactate as well pyruvate sensors can be manufactured. 

Fluorescence applications are ranging from in vivo and in vitro diagnostics to experimental biology. In the first case mainly intrinsic fluorescence is used, which arises from proteins (in particular the amino acid tryptophan [4,5]), extracellular fibres [6,7] or specific coenzymes, e.g. nicotinamide adenine dinucleotide (NADH). So far, fluorescence intensity of NADH has been correlated with metabolic functions [8,9] and was proposed to be an appropriate parameter for detection of ischemic [10,11] or neoplastic [12-14] tissues. The application of fluorescent probes for diagnostic purposes in humans has so far been limited to fluorescein or indocyanine green used... 

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