Label-free detection methods fluorescent dyes

Fluorescent receptor ligands can provide a sensitive means of identifying and localizing some of the most pivotal molecules in cell biology. Many types of fluorescently labeled and unlabeled ligands exist for various cellular receptors, ion channels and ion carriers. Many of these site-selective fluorescent probes may be used on live or fixed cells, as well as in cell-free extracts. Many new dyes provide extremely sensitive detection, which enables measurement of low-abundance receptors. Various methods for further amplifying detection of these receptors have been reported. 

Fluorometric HPLC analysis is currently the most sensitive and reliable method for quantitative detection of Sia in the pmol range. In this analysis, a free form of various Sia species is labeled with the fluorescent dye l,2-diamino-4,5-methylene-dioxybenzene (DMB), and fluorometrically analyzed on HPLC [62, 63]. DMB is a reagent which specifically reacts with a-keto acids, and detects not only Sia but also typical a-keto acids such as pyruvate and a-ketoglutarate. Huorometric HPLC analysis consists of the following steps step 1, acid hydrolysis of sialoglycoconjugates to release free Sia step 2, fluorescent labeling of released Sia with DMB step 3, separation and quantification of DMB-labeled Sia on HPLC. 

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