Alexa Fluor

The number of new NIR fluorophores that can be used in biological systems has grown substantially in recent years as a consequence of extensive research efforts to improve the properties of available dyes. A brief overview of the various types of long-wavelength (above 600 nm) fluorophores including phycobiliproteins, BODIPY, and Alexa Fluor dyes (Life Technologies), Cy dyes (GE Healthcare),... 

The excitation and emission maxima of Alexa Fluor 647, 680, and 750 (the numbers indicate approximate absorption maxima in nanometers), and the molar absorptivities are in the same range as those for Cy5, Cy5.5, and Cy7, but Alexa Fluor dyes are reported to have major advantages in terms of their photostability,... 

Fig. 1 Glycan array analysis of codakine as measured by fluorescence intensity (glycan array v3.0 at Consortium for Functional Glycomics). Purified codakine from white clams was purified with Alexa Fluor 488 Protein Labeling Kit (Molecular Probes , Invitrogen) and tested on glycan array v3.0 at Consortium for Functional Glycomics.

Filter set for visualization of AMCA, Alexa Fluor 350 or nuclear dye DAPI. 

Dramatic advances in modem fluorophore technology have been achieved with the introduction of Alexa Fluor dyes by Molecular Probes (Alexa Fluor is a registered trademark of Molecular Probes). Alexa Fluor dyes are available in a broad range of fluorescence excitation and emission wavelength maxima, ranging from the ultraviolet and deep blue to the near-infrared regions. Because of the large... 

Berlier JE et al (2003) Quantitative comparison of long-wavelength Alexa Fluor dyes to Cy dyes fluorescence of the dyes and their bioconjugates. J Histochem Cytochem 51 1699-1712... 

Similarly to dyes, some fluorescent proteins can be incorporated into polymeric beads to be used as an alternative for ion sensing. For example, a reporter protein (composed of a phosphate-binding protein, a FRET donor (cyan fluorescent protein) and a FRET acceptor (yellow fluorescent protein)) was incorporated into polyacrylamide nanobeads by Sun et al. [46]. FRET was inhibited upon binding of phosphate. Kopelman and co-workers [47] used a similar approach to design a nanosensor for copper ions. They have found that fluorescence of red fluorescent protein DsRed (commonly used as a label) is reversibly quenched by Cu2+ and Cu+. Both DsRed and Alexa Fluor 488 (used as a reference) were entrapped into polyacrylamide nanobeads. Typically, up to 2 ppb of copper ions can be reliably measured. It should be mentioned, that in contrast to much more robust dyes, mild conditions upon polymerization and purification are very important for immobilization of the biomolecule to avoid degradation. 

Sumner JP, Kopelman R (2005) Alexa Fluor 488 as an iron sensing molecule and its application in PEBBLE nanosensors. Analyst 130 528-533... 

Figure 5 shows two typical core-shell structures (a) contains a metal core and a dye doped silica shell [30, 32, 33, 78-85] and (b) has a dye doped silica core and a metal shell [31, 34]. There is a spacer between the core and the shell to maintain the distance between the fluorophores and the metal to avoid fluorescence quenching [30, 32, 33, 78-80, 83]. Usually, the spacer is a silica layer in this type of nanostructures. Various Ag and Au nanomaterials in different shapes have been used for fluorescence enhancement. Occasionally, Pt and Au-Ag alloys are selected as the metal. A few fluorophores have been studied in these two core-shell structures including Cy3 [30], cascade yellow [78], carboxyfluorescein [78], Ru(bpy)32+ [31, 34], R6G [34], fluorescein isothiocyanate [79], Rhodamine 800 [32, 33], Alexa Fluor 647 [32], NIR 797 [82], dansylamide [84], oxazin 725 [85], and Eu3+ complexes [33, 83]. 

Figure 2 Principle of spectral bio-imaging HUVEC incubated with pH-sensitive DOPE CHEMS liposomes (loaded with fluorescein isothiocyanate-dextran), cholera toxin subunit B (Alexa Fluor 594 labeled), and diamidino-phenylindole-dihydrochloride.

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