Fluorescent probes quenching effects

The conversion of squaraine 19a to the rotaxane 18 D 19a causes a modest red-shift only in both absorption (10 nm) and emission (7 nm) but an approximately threefold decrease in quantum yield. The addition of two triazole rings (dye 19b) did not significantly alter the quantum yield of 17b (Table 4). A macrocycle-induced quenching effect was verified by fluorescence titration experiments adding aliquots of 18 to a solution of squaraine 17b in methylene chloride [58]. Treatment of the 18 d 17b psuedorotaxane system with the tetrabutylammonium salts of chloride, acetate, or benzoate leads to the displacement of squaraine 17b from the macrocyclic cavity and the nearly complete restoration of its fluorescence intensity. The 18-induced quenching of 17b does not support the utility of this system as a bioimaging probe however, the pseudorotaxane system 18 Z> 17b acts as an effective and selective anion sensor with NIR fluorescence. 

It is important to notice that a change in lifetime is not a necessary result of a change in fluorescence intensity. For instance, the Ca2+ probe Fluo-3 displays a large increase in intensity on binding Ca2+, but there is no change in lifetime. This is because the Ca-free form of the probe is effectively nonfluorescent, and its emission does not contribute to the lifetime measurement. In order to obtain a change in lifetime, the probe must display detectable emission from both the free and cation-bound forms. Then the lifetime reflects the fraction of the probe complexed with cations. Of course, this consideration does not apply to collisional quenching, when the intensity decay of the entire ensemble of fluorophores is decreased by diffusive encounters with the quencher. 

Fluorescent compounds are sensitive to changes in their chemical environment. Alterations in media pH, buffer components, solvent polarity, or dissolved oxygen can affect and quench the quantum yield of a fluorescent probe (Bright, 1988). The presence of absorbing components in solution that absorb light at or near the excitation wavelength of the fluorophore will have the effect of decreasing luminescence. In addition, noncovalent interactions of the probe with other components in solution can inhibit rotational freedom and quench fluorescence. 

Many other fluorophores are temperature-sensitive only when they are bound to macromolecules. Figure 10.16 shows the effect of temperature on the fluorescence intensity of native and guanidine unfolded AEDANS-RNase. Increasing the temperature from 10 to 30°C induces a decrease in fluorescence intensity for both protein states. The intensity decrease in native protein is more affected by temperature than the guanidine-unfolded protein. This thermal quenching is the consequence of rapid movements of the protein structure around the fluorescent probe. These movements occur during the lifetime of the excited state, and their rate is temperature-dependent. 

As an example, the assay commonly used to measure the direct generation of ROS by nanoparticles is based on the conversion by ROS of the 2,7-dichlorodihydrofluorescein dye into a fluorescent product, 2,7-dichlorofluorescein. There is also a range of fluorescent probes that measure NM-induced ROS production inside the cells, in different intracellular compartments (e.g., dihydrorhoda-mine-1,2,3 in the mitochondria, 2,7-dichlorodihyydrofluorescein diacetate in the cytoplasm, dihydroethidium bromide in the nucleus) [59]. They are all relatively easy to use for quantification in a fluorimeter, multiwall plate reader, or by flow cytometry, but a potential drawback of all these assays is the background caused by particles as well as the fluorescence quenching effects that need to be controlled and taken into account to reliably measure the free radical production [59, 60]. 

---