Fluoresceinated probes

Tian and co-workers [148] have reported the synthesis of two chlorinated fluoresceins probes 4,7,2,7 -tetrachloro-6-(5-carboxypentyl) fluorescein and 4,7,4,5 -tetrachloro-6-(5-carboxypentyl) fluorescein for labeling proteins. More recently, the same research group has described the synthesis of two novel chlorinated fluoresceins, namely 2,4, 5, 7 -tetrachloro-6-(5-carboxypentyl)-4,7-dichloro fluorescein succinimidyl ester 52 and 2, 4, 5, 7-tetrachloro-6-(3-carboxypropyl)-4,7-dichlorofluorescein succinimidyl ester 53 [149]. 

Fig. 25. Cucurbit[n]uril cavitand bearing sugar clusters and fluorescein probe. 

Two general forms of amine-reactive fluorescein probes are available. Both of them react under alkaline conditions with primary amines in proteins and other molecules to form stable, highly fluorescent derivatives. 

Figure 17.14 An expressed protein containing a thioester intein tag that was subsequently modified by native chemical ligation to contain an alkyne group then can be labeled using an azido-fluorescein probe by the click chemistry reaction in the presence of Cu1+.

Fig. 8.4. Biosensors may become interesting for probe detection. In this example (from Olson et al., 1991), a biotinylated and a fluoresceinated probe are hybridized in solution to the target (I). The primer sequences should be located outside this region for PCR-product detection. Avidin acts as a bridge between the biotinylated probe and the biotinylated surface of the biosensor (II). Fluorescein is detected with a urease-antibody conjugate. The rise in pH, after the addition of urea, is detected with the pH-sensitive biosensor. The total assay time is less than 2 h with a detectability of 30 amol and a coefficient of variation (standard deviation/mean) of less than 10%.

In order to observe encapsulated flavor droplets (emulsion) inside a spray-dried particle, ethyl-n-butyrate was used as a model flavor. Nile red was dissolved in ethyl-n-butyrate and used as a fluorescein probe of the oil phase (ethyl-n-butyrate emulsion) of the solution. The labeled flavor was added to the carrier solution and was emulsified. 

Quantify the oligonucleotide by absorbance at 260 nm. Extinction coefficients may be calculated using the Oligo 4.0 program by Rychlik (5) see Note 13). The resulting yield is usually between 1 x lO mol/pL for the biotinylated probes and 5 x 10 mol/pL for fluoresceinated probes. 

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