Fluorescence sensitivity

In the analysis of complex PAH mixtures obtained from environmental samples, reversed-phase LC-FL typically provides reliable results for only 8-12 major PAHs (Wise et al. 1993a). To increase the number of PAHs determined by LC-FL, a multidimensional LC procedure is used to isolate and enrich specific isomeric PAHs that could not be measured easily in the total PAH fraction because of interferences, low concentrations, and/or low fluorescence sensitivity or selectivity. This multi-dimensional procedure, which has been described previously (Wise et al. 1977 May and Wise 1984 Wise et al. 1993a, 1993b), consists of a normal-phase LC separation of the PAHs based on the number of aromatic carbon atoms in the PAH, thereby providing fractions containing only isomeric PAHs and their alkyl-substituted isomers. These isomeric fractions are then analyzed by reversed-phase LC-FL to separate and quantify the various isomers. 

Milbemectin consists of two active ingredients, M.A3 and M.A4. Milbemectin is extracted from plant materials and soils with methanol-water (7 3, v/v). After centrifugation, the extracts obtained are diluted to volume with the extraction solvent in a volumetric flask. Aliquots of the extracts are transferred on to a previously conditioned Cl8 solid-phase extraction (SPE) column. Milbemectin is eluted with methanol after washing the column with aqueous methanol. The eluate is evaporated to dryness and the residual milbemectin is converted to fluorescent anhydride derivatives after treatment with trifluoroacetic anhydride in 0.5 M triethylamine in benzene solution. The anhydride derivatives of M.A3 and M.A4 possess fluorescent sensitivity. The derivatized samples are dissolved in methanol and injected into a high-performance liquid chromatography (HPLC) system equipped with a fluorescence detector for quantitative determination. 

Because FRET results in a decrease in donor fluorescence intensity and excited state lifetime with a corresponding increase in the acceptor fluorescence (sensitized emission), various methods for measuring FRET have been based on assessing one of the above photophysical consequences [22], The most commonly employed methods for measuring FRET in living systems (described elsewhere in more detail in this volume) are ... 

As an extension of the fluorescent sensitizer concept, Forrest et al. have applied this approach to phosphorescent OLEDs, in which the sensitizer is a phosphorescent molecule such as Ir(ppy)3 [342]. In their system, CBP was used as the host, the green phosphor Ir(ppy)3 as the sensitizer, and the red fluorescent dye DCM2 as the acceptor. Due to the triplet and the singlet state energy transfer processes, the efficiency of such devices is three times higher than that of fluorescent sensitizer-only doped device. The energy transfer processes are shown in Figure 3.21. 

In the field of molecular biology, the substitution of a polyphenylene den-drimer with fluorescent dyes and receptor units can be used for fluorescence labeling of biologically active compounds. The multiplicative effect of the number of dyes or receptor units can result in an increase in fluorescence sensitivity and an augmentation of the binding constant in the substrate receptor complex. Since we have shown that perylenemonoimide dyes at the periphery of our den-... 

Drug residues in foods that strongly fluoresce can be more efficiently detected by fluorescence detectors. Typically, fluorescence sensitivity is 10-1000 times higher than that offered by a UV detector for strong UV-absorbing materials (125). Using a fluorescence detector, it has been possible to detect the presence of even a single analyte molecule in an LC flow cell. This type of detection is very versatile because of its ability to measure the intensity of the fluorescent radiation emitted from analytes excited by UV. 

Add Fluorescence sensitivity (units/fig) Fluorescence sensitivity relative to UV response... 

This possibility has been explored by Pappayee and Mishra [130], It was found that in DMPC and DPPC liposomes, simultaneous presence of both P and DP form fluorescence is observed in a pH interval of 11-13. Within this pH interval, the maximal fluorescence sensitivity (largest changes in neutral to anionic form fluorescence ratio) was observed at pH 12. At this pH, CBZ partitions well into the liposome membrane with a large Kp value of 2 X 106 M 1 for DMPC and 3 X 106 M 1 for DPPC liposomes. Fluorescence quenching studies with the hy-... 

Cao H, Diaz DI, DiCesare N, Lakowicz JR, Heagy MD. Monoboronic acid sensor that displays anomalous fluorescence sensitivity to glucose. Organic Letters 2002, 4, 1503-1505. 

Dansyl-amino acid (fluorescence-sensitive detection)... 

The demonstrated ZnO NR-enabled fluorescence sensitivity is even more remarkable, when considering the fact that such fluorescence enhancement of ZnO NR platforms is achieved without the use of chemical and biological ampliflcation processes, major improvements of detection apparatus and analysis software, or application of specially designed fluorophores. Although telomerase and cytokine assays are discussed in this section as example cases, the versatility and general applicability of ZnO NR-based assays can be extended to other disease-marker or biomarker systems. 

Fig. 4.1 Fluorescent sensitive monolayer (SAM) on a glass surface. The sensitive fluorescent monolayer comprises a monolayer modified with fluorophores (F) and the binding molecules. In the presence of an analyte, the fluorescence emission of the SAM changes due to interaction of the analyte with the layer36 - Reproduced with permission of The Royal Society of Chemistry...

Fig. 4.2 General schematic cartoon (a), and chemical structures (b) of the fabrication procedure of a fluorescent sensitive monolayer on glass (i) Silanation of the glass slide with lV-[3-(trimethoxysilyl)propyl]ethylenediamine to form the amino terminated monolayer TPEDA, (ii) reaction with an activated fluoro-phore, and (iii) covalent attachment of a binding molecule...

Ion Reagent Wavelength, nm Absorption Fluorescence Sensitivity, p.g/mL Interference... 

Fluorescence Sensitization. The optical pumping efficiency and thereby the prospects for oscillation of several actinides may be improved by codoping with sensitizer ions using schemes similar to those employed for the lanthanides in Table IV. For example, the first excited J states of l)3+, Np3+, and Pu3+ could be sensitized by energy transfer from lanthanide codopants such as Ho3+, Er3+, Tm3+, and Yb3+ as in the "alphabet" Ho3+ scheme (31). Other schemes using actinides include Cf3+-sensitized Bk3+ ( 4- 6), l)3+-sensitized Np3+... 

The luminescent decomposition of certain oxalic acid esters (Fig. 9) in the presence of hydrogen peroxide and fluorescent sensitizers represents one of the most efficient nonbiological light-producing reactions (Rl, R2). The two most commonly used esters are shown in Fig. 45. As diagnostic agents, oxalate esters... 

Fig. 46. Possible mechanism for chemiluminescence of oxalate esters. Attack by peroxide ions leads to a peroxyoxalate intermediate which, in turn, breaks down to an unstable excited-state dio-xetaneone. In the presence of an appropriate fluorophore, energy transfer from the dioxetaneone leads to luminescence from the fluorescent sensitizer.

Fluorescence detectors are becoming more popular due to their selectivity and sensitivity. Selectivity frequently means that the compounds of interest can be readily detected when present in a complex mixture of compounds that do not exhibit fluorescence. Sensitivity can be measured in terms of parts per billion. Both fixed wavelength and scanning fluorescence units are available. 

When an acridonylalanine (acdAla) was incorporated at different positions of camel single-chain antibody against hen-lysozyme, the Tyrl06acdAla mutant sensitively responded to the binding of nanomolar concentration of the antigen, whereas the Trpl23acdAla mutant was insensitive to the binding (Fig. 5.1-16) [71]. When the same fluorescent amino acid was incorporated into streptavidin, some mutants responded to even a picomolar quantity of biotin [71]. The lower limit of the detectable concentration is determined not by the fluorescence sensitivity, but by the dissociation constants of the protein-small molecule interactions. 

Fluorescence sensitivity of calcofluor to the medium is common to many fluorophores such as TNS (McClure and Edelman, 1966), Trp residues (Burstein et al. 1973) and flavin (Weber, 1950). However, the fluorescence emission maxima of the above fluorophores are also viscosity dependent. Thus, the solvent polarity scale is insufficient to describe the spectral properties of a fluorophore in a protein (case of Trp residues) or bound to a protein (case of TNS). In fact, when the fluorophore is sun ounded by a rigid or viscous environment, or when it is bound tightly to a protein, its fluorescence emission will be located at short wavelengths. In this case, the emission occurs from a non-relaxed state, and the spectrum obtained will be identical to that obtained when the emission occurs from a hydrophobic environment such as isobutanol. Therefore, emission of calcofluor on HSA may be the result of an emission from a hydrophobic binding site and/or a highly rigid binding site. 

Fluorescent sensitivity is commonly enhanced by forming fluorescent derivatives with appropriate fluorescing reagents. This procedure can also be used in chiral chromatography providing the fluorescent derivatives are still enantiomeric and subject to the chiral selectivity of the stationary phase. There is, however, another method of using the fluorescence as a detection method and that is by employing a UV... 

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