Fluorescence labelling agents

Fluorescein and its derivatives represent one of the most popular of all fluorescent labeling agents. Its fluorescent character is created by the presence of a multi-ring aromatic structure due to the planer nature of its upper, fused three-ring system (Figure 9.4). In its most elementary... 

Often, treatment of samples with fluorescence labeling agent reacts with primary and secondary amines to give a fluorescent compound. This is especially important for detecting amino acids in protein hydrolyzates. Fluorescence detectors may also be integrated into a high performance liquid chromatographic (HPLC) system. 

A more stable aryldiazoalkane, 1-pyranyldiazomethane (PDAM), has been prepared as a new fluorescent labeling agent for carboxylic acids by Nimura et al. (40). The PDAM readily reacts with carboxylic acids at room temperature without a catalyst to give an intensely fluorescent ester. 

An active area of pyrrole materials science research involves the development of novel borondipyrromethene (BODIPY) for novel analytical applications and a small subset of the recently published work will be mentioned here. BODIPY derivatives have been developed for use as pH probes <05JOC4152>, chiral fluorescent labeling agents <05BCSJ464>, zinc(II)... 

Riggs JL, Seiwald RJ, Burckhalter JH, Downs CM, Metcalf TG (1958) Isothiocyanate compounds as fluorescent labeling agents for immune semm. Amer J Path 34 1081. 

FMOC was introduced in 1983 as a fluorescent labeling agent, reacting rapidly with both primary and secondary AAs under mild conditions (borate buffer, pH 7.7-8.0) to give stable derivatives. The excess reagent is extracted by pentane. Recent derivatization studies have shown that, depending... 

Figure 3. Structure of used fluorescence labelling agents for plasma proteins...

Influence of the Type of Fluorescence Labelling Agent on Plasma Protein Adhesion... 

The synthesis and characterization of a somatostatin receptor-specific peptide H2N-(DPhe)-cyclo[Cys-Phe-(D-Trp)-Lys-Thr-Cys]-Thr-OH, labeled with an indo-dicarbo- and an indotricarbocyanine dye at the V-terminal amino group were described in [34], The ability of these fluorescent contrast agents to target the somatostatin receptor was demonstrated by flow cytometry in vitro, wherein the indotricarbocyanine conjugate led to elevated cell-associated fluorescence on somatostatin receptor-expressing tumor cells. The intracellular localization was visualized using NIR fluorescence microscopy. 

To reduce the hydrazone bonds to more stable linkages, cool the cell suspension to 0°C and add an equal volume of 30 mM sodium cyanoborohydride in PBS. Incubate for 40 minutes. Note If the presence of a reducing agent is detrimental to protein activity, eliminate the reduction step. In most cases, the hydrazone linkage is stable enough for fluorescent labeling experiments. 

Figure 16.6 The solid phase ICAT reagent provides a thiol-reactive iodoacetyl group to capture cysteine peptides, a spacer containing stable isotopic labels, and a photo-cleavable group that can release the captured peptides for mass spec analysis. The VICAT mass tag is a solution phase labeling agent that also has a photo-cleavable site to release isolated peptides from a (strept)avidin affinity resin. This compound adds a fluorescent group to better detect labeled peptides as they are being isolated from a sample.

Amines are another important group of analytes. Mellbin and Smith [72] compared three different fluorescent reagents, dansyl chloride, 4-chloro-7-nitrobenzo-1,2,5-oxadiazole, and o-phthaldialdehyde, for derivatization of alkylamines. The dansyl tag was found to be the most effective. Hamachi et al. [73] described the application of an HPLC-POCL method for determination of a fluorescent derivative of the synthetic peptide ebiratide. Another comparative study was done by Kwakman et al. [74], where naphthalene-2,3-dialdehyde and anthracene-2,3-dial-dehyde were evaluated as precolumn labeling agents for primary amines. The anthracene-2,3-dialdehyde derivatives were not stable, especially in the presence of hydrogen peroxide, and the POCL detection of these derivatives was therefore... 

Although tetracyclines possess the inherent ability to fluoresce, few methods exploiting this property have been reported (300, 306, 309). Instead, fluorometric methods based on the reaction of tetracyclines with suitable derivatizing agents have been developed. The use, for example, of zirconyl chloride as a fluorescence label in the postcolumn derivation of tetracyclines, has allowed highly selective and sensitive detection of these antibiotics in animal tissues (294, 295). 

Raman spectroscopy can offer a number of advantages over traditional cell or tissue analysis techniques used in the field of TE (Table 18.1). Commonly used analytical techniques in TE include the determination of a specific enzyme activity (e.g. lactate dehydrogenase, alkaline phosphatase), the expression of genes (e.g. real-time reverse transcriptase polymerase chain reaction) or proteins (e.g. immunohistochemistry, immunocytochemistry, flow cytometry) relevant to cell behaviour and tissue formation. These techniques require invasive processing steps (enzyme treatment, chemical fixation and/or the use of colorimetric or fluorescent labels) which consequently render these techniques unsuitable for studying live cell culture systems in vitro. Raman spectroscopy can, however, be performed directly on cells/tissue constructs without labels, contrast agents or other sample preparation techniques. 

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