Fluorescence probe polarity-dependent

Fluorescence probes possessing the PyU base 46 selectively emit fluorescence only when the complementary base is adenine. In this case, the chromophore of is extruded to the outside of the duplex because of Watson-Crick base pair formation, and exposed to a highly polar aqueous phase. On the contrary, the duplex containing a PyU/N (N = G, C and T) mismatched base pair shows a structure in which the glycosyl bond of uridine is rotated to the syn conformation. In this conformation, the fluorophore is located at a hydrophobic site of the duplex. The control of base-specific fluorescence emission is based on the polarity change in the microenvironment where the fluorophore locates are dependent on the l>yU/A base-pair formation. 

The concept of polarity covers all types of solute-solvent interactions (including hydrogen bonding). Therefore, polarity cannot be characterized by a single parameter. Erroneous interpretation may arise from misunderstandings of basic phenomena. For example, a polarity-dependent probe does not unequivocally indicate a hydrophobic environment whenever a blue-shift of the fluorescence spectrum is observed. It should be emphasized again that solvent (or microenvironment) relaxation should be completed during the lifetime of the excited state for a correct interpretation of the shift in the fluorescence spectrum in terms of polarity. 

Time-resolved fluorescence spectroscopy of polar fluorescent probes that have a dipole moment that depends upon electronic state has recently been used extensively to study microscopic solvation dynamics of a broad range of solvents. Section II of this paper deals with the subject in detail. The basic concept is outlined in Figure 1, which shows the dependence of the nonequilibrium free energies (Fg and Fe) of solvated ground state and electronically excited probes, respecitvely, as a function of a generalized solvent coordinate. Optical excitation (vertical) of an equilibrated ground state probe produces a nonequilibrium configuration of the solvent about the excited state of the probe. Subsequent relaxation is accompanied by a time-dependent fluorescence spectral shift toward lower frequencies, which can be monitored and analyzed to quantify the dynamics of solvation via the empirical solvation dynamics function C(t), which is defined by Eq. (1). 

Solvatochromic fluorescent probe molecules have also been used to establish solvent polarity scales. The solvent-dependent fluorescence maximum of 4-amino-V-methylphthalimide was used by Zelinskii et al. to establish a universal scale for the effect of solvents on the electronic spectra of organic compounds [80, 213], More recently, a comprehensive Py scale of solvent polarity including 95 solvents has been proposed by Winnik et al. [222]. This is based on the relative band intensities of the vibronic bands I and III of the % - n emission spectrum of monomeric pyrene cf. Section 6.2.4. A significant enhancement is observed in the 0 0 vibronic band intensity h relative to the 0 2 vibronic band intensity /m with increasing solvent polarity. The ratio of emission intensities for bands I and III serves as an empirical measure of solvent polarity Py = /i/Zm [222]. However, there seems to be some difficulty in determining precise Py values, as shown by the varying Py values from different laboratories the reasons for these deviations have been investigated [223]. 

In FP, the use of polarized light to excite a solution of fluorescent molecules results in preferential excitation of the molecules that have their transition moments (dipoles) parallel to the direction of the polarized light of excitation. The polarization of their emitted light depends upon how fast the fluorescent probes rotate during the lifetime of its excitation state the faster the rotation, the smaller the polarized signal (P). 

The fluorescent probe 4-aminophthalimide (63) as well as its /V,/V-diethyl homomorph (89) were used by Samanta and coworkers148 149 to study the polarity and fluorescence dynamics in l-alkyl-3-methylimidazolium salts. The alkyl groups were variously ethyl, butyl and octyl and the anions were nitrate, tetrafluoroborate, hexafluorophosphate and bis(triflyl)imide. Similar conclusions were reached concerning the effects of alkyl chain length and anion as those obtained by Carmichael and Seddon142 using Nile Red. The solvation dynamics were found to depend on the viscosity of the media. Further use of 63 in l-butyl-3-methylimidazolium hexafluorophosphate was reported by Ingram and... 

The role of vibrational relaxation and solvation dynamics can be probed most effectively by fluorescence experiments, which are both time- and frequency-resolved,66-68 as indicated at the end of Sec. V. We have recently developed a theory for fluorescence of polar molecules in polar solvents.68 The solvaion dynamics is related to the solvent dielectric function e(co) by introducing a solvation coordinate. When (ai) has a Lorentzian dependence on frequency (the Debye model), the broadening is described by the stochastic model [Eqs. (113)], where the parameters A and A may be related to molecular... 

Another extremely useful method for cac determination, especially in the light of high sensitivity, is fluorescence emission spectroscopy [15]. Some aromatic water-insoluble dyes that are present in trace amounts in mixed polyelectrolyte-surfactant solutions have an ability to solubilize within the self-assembled surfactant aggregates and to change their photophysical properties because of the change of environmental polarity. Through this, they offer a very sensitive method for the determination of cac values. A typical and lately frequently used compound is pyrene, which is used as a fluorescence probe to assess various micellar properties. Pyrene exhibits a polarity dependent fluorescence spectrum with the ratio /,//3 (the ratio of the intensity... 

Figure 33 shows the fluorescence rate (a) and the lifetime (b) of individual R-6G molecules plotted as a function of the lateral displacement of the aperture with respect to the molecule [108], In the upper panel, molecule a has a distinct double peak, the others have a maximum emission rate when at the center of the aperture. This finding can be explained by a dipole probing the electric field components of the aperture with an effective diameter of about 200 nm, in accordance with the polarization dependent single-molecule data by Betzig and Chichester [83] in Section V.A. In the lower panel, the emission lifetime has a maximum at the center of the aperture and shortens significantly toward the side as expected. Quantitatively, the same results were found by Xie and Dunn [109]. However, the opposite effect was also found (Fig. 34) with a shorter lifetime at the center... 

Pyrene derivatives are the widest used probes for qualitative solubilization [365] by virtue of the solvatochromic shifts of the absorption bands [255], the excimer formation [145,186], the polarity dependent quantum yields [197] and fluorescence life-times [185-187, 196, 197, 202, 215, 292], and the pyrene fluorescence fine structure [65, 74,78,103,112,167, 224, 363, 371] the intensity ratio of the fluorescence bands I at 372 nm and III at 383 nm is a convenient measure for the polarity of the environment of the pyrene label ( py -scale I/III values increase with polarity, cf. Fig. 27). As, however, the fluorescence of pyrene is very sensitive to the experimental set-up [372], absolute I/III values reported by different groups are difficult to compare. 

Time-dependent blue shifts of the transient triplet charge-transfer (CT) absorption of p-aminonitroterphenyl (p-ANTP) were used to probe association of ions with a photoinduced charge-separated species (6). Thanks to the long lifetime of the triplet excited state of p-ANTP (t > 3 (Jis), the study of ionpairing dynamics can be extended to much lower electrolyte concentrations, and consequently to much less polar media, than is possible with the shortlived fluorescent probes (t 3 ns) (3, 4). The ability to probe weakly polar and nonpolar solvents is important because the electrostatic interactions are much larger in these media than in strongly polar solvents. 

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