Phospholipids fluorescence detection

N. L. Thompson, H. M. McConnell, and T. P. Burghardt, Order in supported phospholipid monolayers detected by dichroism of fluorescence excited with polarized evanescent illumination, Biophys. J. 46, 739-747 (1984). 

Fig. 3 Separation of standard and liver phospholipids by HPLC on a 5-yttm Nucleosil 5 NH2 stationary phase with an isocratic mobile phase consisting of acetonitrile, methanol, water, and methyl phosphonic acid and subsequent UV and fluorescence detection. The second column was cut off from the eluent stream by valve switching after about 30 min. (Reprinted from Ref. 43 with the kind permission of Analytical Biochemistry.)...

SL Abidi, TL Mounts, KA Rennick. Reversed-phase high performance liquid chromatography of phospholipids with fluorescence detection. J Chromatogr 639 175-184,1993. 

Numerous applications have been reported in the literature on the use of cell membrane receptors which require the use of ligand bilayer or phospholipid vesicles to maintain functionality, but other immobilization methods were also successfully used. The biosensing of e.g. acetylcholine and cholinergies has been reported with different transducers (ISFETs, interdigitated electrodes with measurements of capacity changes and optical fiber optode with fluorescence detection). 

T. Ohyashiki, M. Nunomura, and T. Katoh, Detection of superoxide anion radical in phospholipid liposomal membrane by fluorescence quenching method using 1,3-diphenylisobenzofuran. Biochim. Biophys. Acta. 1421, 131-139 (1999). 

T. Parassassi, F. Conti, M. Glaser, and E. Gratton, Detection of phospholipid phase separation. A multifrequency phase fluorimetry study of l,6-diphenyl-l,3,5-hexatriene fluorescence, J. Biol. Chem. 259, 14011-14017 (1984). 

Derivatization is never used in the analysis of the different phospholipid classes present, but is very widely used in the analysis of molecular species. The main reason for (mostly precolumn) derivatization in this case is to reduce significantly the detection limits. To this end, both UV-ab-sorbing and fluorescent derivatives are frequently used, as described in more detail in Secs. IV.B and V.3. 

Table 7 Relative Sensitivity of Standard Phospholipids for Fluorescence and Ultraviolet Detection...

W Bernhard, M Linck, H Creutzburg, AD Postle, A Anting, I Martin-Carrera, KF Sewing. High performance liquid chromatographic analysis of phospholipids from different sources with combined fluorescence and ultraviolet detection. Anal Biochem 220 172-180, 1994. 

The nanosized detection area Ar or volume created by STED also extends the power of fluorescence correlation spectroscopy (FCS) and the detection of molecular diffusion [74,95]. For example, STED microscopy has probed the diffusion and interaction of single lipid molecules on the nanoscale in the membrane of a living cell (Fig. 19.6). The up to 70 times smaller detection areas created by STED (as compared to confocal microscopy) revealed marked differences between the diffusion of sphingo- and phospholipids [74]. While phospholipids exhibited a comparatively free diffusion, sphingolipids showed a transient ( 10 ms) cholesterol-mediated trapping taking place in a < 20-nm diameter area, which disappeared after cholesterol depletion. Hence, in an unperturbed cell putative cholesterol-mediated lipid membrane rafts should be similarly short-lived and smaller. 

Nioi P, Perry BK, Wang EJ, et al. In vitro detection of drug-induced phospholipidosis using gene expression and fluorescent phospholipid based methodologies. Toxicol Sci. 2007 99(1) 162-173. 

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