Fluorophores rhodamine derivatives

The sensing arrays were prepared by parallel synthesis of aminoterminated SAMs on glass, functionalised by different fluorophores, rhodamine derivatives (Fig. 9). 

Direct labeling of a biomolecule involves the introduction of a covalently linked fluorophore in the nucleic acid sequence or in the amino acid sequence of a protein or antibody. Fluorescein, rhodamine derivatives, the Alexa, and BODIPY dyes (Molecular Probes [92]) as well as the cyanine dyes (Amersham Biosciences [134]) are widely used labels. These probe families show different absorption and emission wavelengths and span the whole visible spectrum (e.g., Alexa Fluor dyes show UV excitation at 350 nm to far red excitation at 633 nm). Furthermore, for differential expression analysis, probe families with similar chemical structures but different spectroscopic properties are desirable, for example the cyanine dyes Cy3 and Cy5 (excitation at 548 and 646 nm, respectively). The design of fluorescent labels is still an active area of research, and various new dyes have been reported that differ in terms of decay times, wavelength, conjugatibility, and quantum yields before and after conjugation [135]. New ruthenium markers have been reported as well [136]. 

Fig. 6.21. Principle of detection of lipopolysaccharide (LPS) with the CD14-derived probe. It relies on the formation of a ground state complex between fluorescein and rhodamine in aqueous solution with quenching of donor and acceptor fluorescence. Spectrum A shows hypothetical fluorescence emission spectra of this complex. After LPS binding, the peptide sequence gets straightened prohibiting the close contact between the two fluorophores and leading to the recovery of red fluorescence (Spectra B).

The intense Texas Red fluorophore has a QY that is inherently higher than the tetrameth-ylrhodamine or Lissamine rhodamine B derivatives. Texas Red s luminescence is shifted maximally into the red region of the spectrum, and its emission peak only minimally overlaps with that of fluorescein. This makes Texas Red derivatives among the best choices of labels for use in double-staining techniques. 

DNA modified with a diamine compound to contain terminal primary amines may be coupled with amine-reactive fluorescent labels. The most common fluorophores used for oligonucleotide labeling are the cyanine dyes and derivatives of fluorescein and rhodamine (Chapter 9). However, any of the amine-reactive labels discussed throughout Chapter 9 are valid candidates for DNA applications. 

In this respect, Johansson 1 reported that ro is the same (within experimental accuracy) for fluorophores belonging to the same family, e.g. perylene and per-ylenyl compounds (0.369 + 0.002), or xanthene derivatives such as rhodamine... 

The use of antibodies labelled with a fluorescent derivative obtained from fluorescein, rhodamine or other fluorophore is well suited for the measurement of human or animal immunoglobulins. However, the same principle is not yet used for simple organic molecules. Discrimination between the labelled and non-labelled species at the isolation stage can occur for small organic molecules. However, there are a few examples of assays in which isolation is followed by fluorescence measurement. 

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. 

In addition to proflavin and rhodamine, the photobleaching-resistant Cy3 and Cy5 fluorophores are also frequently used in single-molecule experiments and have been incorporated in the form of hydrazide derivatives into tRNAs via D residues (Pan et al., 2009) (Fig. 4.2). However, quantitative uptake of these hydrazide dyes requires modification of three reaction parameters higher concentrations of the hydrazide dyes (40 mM) than that required for proflavin or rhodamine (22 mM), pH 3.7 rather than pH 3.0, and 2 h reaction time instead of 45—90 min. The requirement of higher concentration is to promote formation of hydrazide adduct, while the slighdy elevated pH prevents hydrolysis of the adduct, which is acid labile. Thus, while the labeling method can be adapted to incorporate new fluorophores besides proflavin and rhodamine, it is prudent to systematically evaluate for the fluorophores under consideration for coupling efficiency as a function of dye concentration, pH, and reaction time. 

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. 

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