Fluorescent-labelled amino acids

Single-cell CE experiments in neurochemistry were first performed in the late 1980s with initial cytoplasmic sampling from P. corneas completed in 1988 and whole cell injection of Helix aspersia in 1989 to separate fluorescently labeled amino acids. Since these pioneering studies, many different small molecule neurotransmitters and other related species have been detected in single cells by CE. 

Jorgenson reported the use of glass capillaries for free solution electrophoresis 25 years ago (Jorgenson andLukacs, 1981,1983). Aplug of analyte was introduced into a buffer-filled capillary and separated at high electric fields. Capillaries of 75 im inner diameter were employed, and detection of labeled amino acids and peptides was based on fluorescence. 

Ouverney and Fuhrman (1999) made another application of the defined DOM component approach in which the uptake of radiolabeled amino acids by a coastal bacterioplankton community was monitored with a combination of microautoradiography and fluorescent in situ hybridization (STARFISH), tracking the abundance and activity of bacterial cells from a-Proteobacteria and CFB lineages. The results indicate that 80% of the cells from each subgroup use the labeled amino acid mixture. When taken together these two... 

Chiral separation of FITC-labeled amino acid enantiomers was performed on a glass chip using fluorescent detection. Analysis time ranged from 75 s for the most basic amino acids to 160 s for the most acidic ones. y-CD was used as the chiral selector [627]. Chiral separation of amino acids in extraterrestrial samples or meteorites were also performed [610,628],... 

The LED has also been used as the excitation source for fluorescent detection. For instance, two LEDs (red and blue) were used to detect FTTC-labeled amino acids on a PMMA chip. The blue LED was used for excitation and the red LED for background noise compensation, leading to a four-fold S/N enhancement [681], Moreover, blue LEDs (470 nm) were used to detect various dyes, such as... 

Dual LIF and amperometric detection are used to detect a five-component mixture (see Figure 7.24). The labeled amino acids (i.e., NBD-Arg, NBD-Phe, and NBD-Glu) are fluorescently detected. The EOF marker (DA) and internal standard (CAT) are amperometric ally detected. The fluorescent peaks are normalized to the internal standard, CAT. As a result, the RSD of migration time was improved from 2.7% to 0.8% [670]. 

T. Ueda, F. Kitamura, R. Mitchell, T. Metcalf, T. Kuwana, and A. Nakamoto, Chiral separation of naph-thalene-2,3-dicarboxaldehyde-labeled amino-acid enantiomers by cyclodextrin-modified micellar electro-kinetic chromatography with laser-induced fluorescence detection, Chem. 63 2919 (1991). 

Unfortunately, the OPA condensation products decompose to some extent on the HPLC column (as shown by means of 14C-labelled amino acids - the positions of emerging samples shown by their fluorescence lag behind maximum radioactivity profiles), although users favouring these OPA derivatives have learned to practise strict protocols for their use, in order to get reliable results. The OPA-TV-acetyl-L-cysteine condensation product is a diastereoisomer mixture when formed with a... 

As mentioned above, fluorescent labels can be attached to specific amino acid residues in Mb. Mb possessing a label at a single site can be adsorbed to PDMS in the TIRF test cell and challenged by solutions of proteolytic enzymes. The rate of decay of the fluorescence signal will indicate the degree of exposure of the labeled amino acid residue, because as bonds near the label are cleaved by the enzyme, the peptide with the fluorescent label will be released and subsequently washed out of the test cell. Since the position of the label will be known the regions of the Mb molecule exposed to solution can be determined. 

Arriaga, E. A., Zhang, Y. N., and Dovichi, N. J., Use of 3-(P-carboxybenzoyl)quinoline-2-carboxaldehyde to label amino-acids for high-sensitivity fluorescence detection in capillary electrophoresis, Anfl/. Chim. Acta, 299, 319, 1995. 

Capillary zone electrophoresis with microchip-based SCCE has been used to separate fluorescent dye from degradation products and to separate fluorescein isothiocyanate (FITC) labeled amino acids. Capillary zone electrophoresis (CZE) in fused-silica capillaries has been used to separate the racemic mixtures of (a-hydroxybenzyl)methyltrimethylammonium and (2-hydroxy-l-phenyl)ethyltrimethylammonium with f)-cyclodextrin as the chiral pseudo-stationary phase. L-Phenylalanine and L-phenylalanine-ring-D5 (the hydrogens in the aromatic ring were substituted with deuterium) and another separation of the closely related amino acids phenylalanine and tyrosine have been resolved using CZE with capillary-based SCCE. ... 

Amino acids are derivatized in order to enable their detection by standard chromatographic detectors. Even today the perfect label has not been found. Such an ideal label should be easy to introduce in a defined manner (only one reaction product per amino acid) and should result in a stable and highly active (UV-absorbance, fluorescent activity) amino acid derivative, and it should be universal and react with all possible amino acids of interest. 

The amino and carboxyl residues can be elucidated chemically by labeling them. Two common methods modify the N-terminal residue by the chemical addition of fluoro-2,4-dinitrobenzene or dansyl chloride. The protein is then hydrolyzed to its component amino acids. The amino acid labeled with fluoro-2, 4-dinitrobenzene is colored yellow and that with dansyl chloride is fluorescent. This enables visualization of the labeled amino acid. Comparison of further... 

Dansyl chloride (11), which absorbs maximally at 330 nm and fluoresces maximally at 510 nm, can be used to label amino acids in fluorescence microscopy and FRET studies. Tabulated below is the variation of the fluorescence intensity of an aqueous solution of dansyl chloride with time after excitation by a short laser pulse (with / the initial fluorescence intensity) ... 

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