Fluorescent sensors nucleotides

The design of fluorescent sensors is of major importance because of the high demand in analytical chemistry, clinical biochemistry, medicine, the environment, etc. Numerous chemical and biochemical analytes can be detected by fluorescence methods cations (H+, Li+, Na+, K+, Ca2+, Mg2+, Zn2+, Pb2+, Al3+, Cd2+, etc.), anions (halide ions, citrates, carboxylates, phosphates, ATP, etc.), neutral molecules (sugars, e.g. glucose, etc.) and gases (O2, CO2, NO, etc.). There is already a wide choice of fluorescent molecular sensors for particular applications and many of them are commercially available. However, there is still a need for sensors with improved selectivity and minimum perturbation of the microenvironment to be probed. Moreover, there is the potential for progress in the development of fluorescent sensors for biochemical analytes (amino acids, coenzymes, carbohydrates, nucleosides, nucleotides, etc.). 

Cywinski P (2006) Molecular and polymeric fluorescent pyrazolequinoline sensors for nucleotides. PhD Dissertation, Technical University of Lodz... 

Yamana et al. [67] used bis-pyrene-labeled DNA aptamer for detection of ATP. The pyrene excimer was incorporated into several nucleotide positions. Addition of ATP resulted in an increase in fluorescence only for aptamers labeled by fluorescence probe between residues that were responsible for ATP binding. Using this sensor it was possible to detect ATP with mmol/L sensitivity. 

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. 

In the initial screen, one compound (given the name GTP Green, Fig. 6-8) displayed fluorescence enhancement ( tum-on ) upon GTP binding and was favorably photostable compared to other candidates [76], Prior to the discovery of GTP Green, the only other GTP sensor (rationally designed) was a turn-off sensor [78], GTP Green displayed a red-shifted 80-fold fluorescence enhancement (Tern = 540 nm) in the presence of GTP and only < two-fold increases with the other nucleotide triphosphates ATP, TTP, and UTP. This compound was also found to be selective for GTP over the mono- and diphosphate homologs. 

There are 11 different PDE families (PDEl-11) with different substrate specificity, affinity, sensitivity to inhibitors, and tissue localization. In addition, there are exonucleases such as 5 -nucleotide phosphodiesterase (PDE 1) and 3 -nucleotide phosphodiesterase (PDE 11) that hydrolyze phosphodi-ester bounds. These enzymes have several known functions in nucleic acids. By using all these enzymes in a sensor surface and by measuring changes in fluorescence polarization it was concluded that YTX binds to cyclic nucleotide PDEl, with a calcium-dependent effect, PDE3, and PDE4, and shows high affinity by exonuclease PDE 1 [15,16]. 

See also Electrophoresis Two-Dimensional Gels Nucleic Acids. Enzymes Enzyme-Based Assays. Flow Injection Analysis Principles. Fluorescence Quantitative Analysis. Lab-on-a-Chip Technologies. Mass Spectrometry Matrix-Assisted Laser Desorption/loniza-tion Time-of-Flight. Microelectrodes. Microscopy Overview. pH. Process Analysis Overview Chromatography Electroanalytical Techniques Sensors Acoustic Emission Maintenance, Reliability, and Training. Proteins Overview. Proteomics. Purines, Pyrimidines, and Nucleotides. Sensors Oven/iew. Spectrophotometry Overview. 

Hasegawa, T. Hagihara, M. Fukuda, M. Morii, T. Stepwise functionalization of ribonucleopeptides Optimization of the response of fluorescent ribonucleopeptide sensors for ATP. Nucleosides, Nucleotides Nucleic Acids 2007, 26, 1277-1281. 

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