Fluorophore isothiocyanates

The nondestructive introduction of a fluorescent label would provide the molecule with a nonradioactive fluorophore, yet would preserve the option for direct radiolabelling of the fluorescent moiety with 125Iodine. This approach was pioneered by Nagasawa et al. (5) who reacted native or /V-desulfated heparins with a fluorescein isothiocyanate (FITC). The resulting degree of labelling was low... 

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]. 

The N-terminal amino group or a Lys residue can easily be used to label the PNA. This can be carried out in solution after purification, or more conveniently, while the PNA is still attached to the resin. Fluorescein and rhodamine are the most common fluorophore labels, while biotin is the most common affinity tag. Fluorescein or rhodamine are usually coupled to the amino group as -OSu esters or isothiocyanates. 4,4 -Dimethoxytrityl-protected biotin and Piv-pro-tected fluorescein have also been coupled to the N-terminus of a PNA as their 1-phenylpyr-azolin-5-one carboxylate esters.147 In 1997 a new fluorescein-conjugated Lys monomer (21, Scheme 12) was described. 48 ... 

The most used FIA reagents conjugate a fluorophore such as fluorescein—isothiocyanate (FITC) or rhodamine—isothiocyanate to antibody (or antigen) free amino groups. Examples of other commonly used fluorophores for FIA and their spectral characteristics are presented in Table 3. FIA assays are available in sandwich and competitive formats similar to EIAs. Unlike EIA kits which can be used directly with visual color determination, FIAs require a fluorometer, and thus are primarily laboratory-based. 

The isothiocyanate derivative of the fluorophore, fluorescein or rhodamine, is coupled to the amino groups of IgG antibody in a one-step procedure and excess label is removed by gel filtration. 

This approach has been shown to work with a number of different fluorescent probes such as the short-wavelength fluorophores dansyl sul-fonyl chloride and coumarin chloride and the long-wavelength fluorophores tetramethylrhodamine-5-(and-6)-isothiocyanate [5(6)-TRITC], 5-(and-6)-carboxytetramethylrhodamine, succinimidyl ester [5(6)-TAMRA, succin-imidyl ester] and lissamine rhodamine B sulfonyl chloride (each in conjunction with different binding functionalities on the SAM surface. 

Targeted delivery has also been used to investigate the structure of carbohydrates needed to obtain discrimination of cellular uptake between liver and tumor cells [184], A study was performed by doubly modifying BSA with both a fluorophore (via Lys isothiocyanate derivatization) and different fucose derivatives (via Tyr diazotization). Similar studies have used EDC-mediated coupling to also attach therapeutic agents for delivery to human embryonal carcinoma cells [185]. 

Of the many known fluorophores, only a few are routinely employed for immunofluorescence. Spectral maxima for such fluorophores are provided in Table 1. Conjugation of the fluorophores to antibodies is typically achieved through reactive isothiocyanate, sulfonyl chloride, or succinimidyl ester groups, which provide bonding to protein amines. Since most investigators purchase fluorophores already conjugated to antibodies, the chemistry of these and other reactive groups will not be further addressed (see Chapter 6 for details). Additional fluorophore information is summarized below. 

To date, the most popular fluorescent labels for FIA have been those derived from the long-wavelength, strongly emitting xanthene dyes fluorescein isothiocyanate (FTTC) and lissamine rhodamine B (RB200).F The isothiocyanates or isocyanates of these fluorophores can be used to label primary and secondary aliphatic amines in aqueous solutions by simple procedures. 

If the investigated drug does not possess a suitable fluorophore, derivatization may be required [fluorescein isothiocyanate for compounds possessing a free amino group (e.g., amphetamines) may serve as a typical example an Ar-ion laser emitting at 488 nm was used for this purpose]. Generally, detection limits achieved with laser-induced fluorescence after derivatization can be around 3 mM in concentration terms (or 3 /rmol in terms of absolute mass detection). This is about three orders of magnitude less... 

Another related phenomenon that results in a lower quantum yield than expected is called concentration quenching. This can occur when a macromolecule, such as an antibody, is heavily labeled with a fluorophore, such as fluorescein isothiocyanate. When this compound is excited, the fluorescence labels are in such close proximity that radiationless energy transfer occurs. Thus, the resulting fluorescence is much lower than expected for the concentration of the label. This is a common problem in flow cytometry and laser-induced fluorescence when attempting to enhance detection sensitivity by increasing the density of the fluorescing label. 

Not many compounds fluoresce naturally. However, some compounds, when added to another compound, cause that compound to fluoresce. These compounds are called fluorophores. Dansyl chloride and fluorescamine are two that are used to react with primary amines. o-Phthalaldehyde is used as a postcolumn reactant to produce fluorescent compounds. Other compounds are naphthalenedialdehyde (NDA), fluorescein isothiocyanate (FITC), and phenylthiohydantoin (PTH). See Chapter 19, p. 203, for more detail. 

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