Fluorophore sensitivity

Quenching of the emissions of fluorophores sensitized by dioxetanes has also been used for chemical analysis [29, 30] but this is probably less specific and less sensitive than direct measurement of the sensitized emission of the analyte. Most analytical reactions of the dioxetanes require an organic solvent for optimal solubility and CL yields [31]. This has led to considerable interest in the development of water-soluble oxalate esters [32],... 

The product s d>F is called fluorophore sensitivity. More the sensitivity is high, more important will be the fluorescence intensity. 

As the result of high specificity and sensitivity, nucleic acid probes are in direct competition with immunoassay for the analytes of some types of clinical analytes, such as infectious disease testing. Assays are being developed, however, that combine both probe and immunoassay technology. In such hybrid probe—immunoassays, the immunoassay portion detects and amplifies the specific binding of the probe to a nucleic acid. Either the probe per se or probe labeled with a specific compound is detected by the antibody, which in turn is labeled with an enzyme or fluorophore that serves as the basis for detection. 

Nitroxides have the property of quenching fluorescence. Thus radical trapping with nitroxides containing fluorophores (e.g. 114) can be monitored by observing the appearance of fluorescence.511015 The method is highly sensitive and has been applied to quantitatively determine radical yields in PLP experiments (Section... 

By using phase sensitive detection, the detector phase angle can be adjusted to be exactly out of phase with the phase-delayed emission from any single fluorophore, suppressing its contribution to the total emission signal. Phase sensitive detection, coupled with... 

Applications of the oxalate-hydrogen peroxide chemiluminescence-based and fluorescence-based assays with NDA/CN derivatives to the analysis of amino acids and peptides are included. The sensitivity of the chemiluminescence and fluorescence methods is compared for several analytes. In general, peroxyoxalate chemiluminescence-based methods are 10 to 100 times more sensitive than their fluorescence-based counterparts. The chief limitation of chemiluminescence is that chemical excitation of the fluorophore apparently depends on its structure and oxidation potential. 

Though we and others (27-29) have demonstrated the utility and the improved sensitivity of the peroxyoxalate chemiluminescence method for analyte detection in RP-HPLC separations for appropriate substrates, a substantial area for Improvement and refinement of the technique remains. We have shown that the reactions of hydrogen peroxide and oxalate esters yield a very complex array of reactive intermediates, some of which activate the fluorophor to its fluorescent state. The mechanism for the ester reaction as well as the process for conversion of the chemical potential energy into electronic (excited state) energy remain to be detailed. Finally, the refinement of the technique for routine application of this sensitive method, including the optimization of the effi-ciencies for each of the contributing factors, is currently a major effort in the Center for Bioanalytical Research. 

Theory. If two or more fluorophores with different emission lifetimes contribute to the same broad, unresolved emission spectrum, their separate emission spectra often can be resolved by the technique of phase-resolved fluorometry. In this method the excitation light is modulated sinusoidally, usually in the radio-frequency range, and the emission is analyzed with a phase sensitive detector. The emission appears as a sinusoidally modulated signal, shifted in phase from the excitation modulation and partially demodulated by an amount dependent on the lifetime of the fluorophore excited state (5, Chapter 4). The detector phase can be adjusted to be exactly out-of-phase with the emission from any one fluorophore, so that the contribution to the total spectrum from that fluorophore is suppressed. For a sample with two fluorophores, suppressing the emission from one fluorophore leaves a spectrum caused only by the other, which then can be directly recorded. With more than two flurophores the problem is more complicated but a number of techniques for deconvoluting the complex emission curve have been developed making use of several modulation frequencies and measurement phase angles (79). 

If the signal decay is a single-exponential curve, equations 16 and 17 result in values for X that are in agreement with each other. Dissimilar values indicate multiexponential decay, which usually means that the sample contains more than one fluorophore. Multiexponential decay can be resolved by using a phase fluorometer with phase sensitive detection. A time-independent, direct-current signal is produced that is proportional to the cosine of the difference between the phase angle of the detector ( D) and the phase angle of the fluorescence ( ) ... 

An alternative approach is the use of pH-sensitive fluorophores (Lichtenberg and Barenholz, lOSS). These probes are located at the lipid-water interface and their fluorescence behavior reflects the local surface pH, which is a function of the surface potential at the interface. This indirect approach allows the use of vesicles independent of their particle size. Recently, techniques to measure the C potential of Liposome dispersions on the basis of dynamic light scattering became commercially available (Muller et al., 1986). 

Phenolic copolymers containing fluorophores (fluoroscein and calcein) were synthesized by SBP catalysis and used as array-based metal-ion sensor. Selectivity and sensitivity for metal ions could be controlled by changing the polymer components. Combinatorial approach was made for efficient screening of specific sensing of the metals. 

Direct and indirect competition formats, illustrated in Figure 1, are widely used for both qualitative and quantitative immunoassays. Direct competition immunoassays employ wells, tubes, beads, or membranes (supports) on to which antibodies have been coated and in which proteins such as bovine semm albumin, fish gelatin, or powdered milk have blocked nonspecific binding sites. Solutions containing analyte (test solution) and an analyte-enzyme conjugate are added, and the analyte and antibody are allowed to compete for the antibody binding sites. The system is washed, and enzyme substrates that are converted to a chromophore or fluorophore by the enzyme-tracer complex are added. Subsequent color or fluorescence development is inversely proportionate to the analyte concentration in the test solution. For this assay format, the proper orientation of the coated antibody is important, and anti-host IgG or protein A or protein G has been utilized to orient the antibody. Immunoassays developed for commercial purposes generally employ direct competition formats because of their simplicity and short assay times. The price for simplicity and short assay time is more complex development needed for a satisfactory incorporation of the label into the antibody or analyte without loss of sensitivity. 

The simplest fluorescence measurement is that of intensity of emission, and most on-line detectors are restricted to this capability. Fluorescence, however, has been used to measure a number of molecular properties. Shifts in the fluorescence spectrum may indicate changes in the hydrophobicity of the fluorophore environment. The lifetime of a fluorescent state is often related to the mobility of the fluorophore. If a polarized light source is used, the emitted light may retain some degree of polarization. If the molecular rotation is far faster than the lifetime of the excited state, all polarization will be lost. If rotation is slow, however, some polarization may be retained. The polarization can be related to the rate of macromolecular tumbling, which, in turn, is related to the molecular size. Time-resolved and polarized fluorescence detectors require special excitation systems and highly sensitive detection systems and have not been commonly adapted for on-line use. 

Vazquez ME, Blanco JB, Imperiali B (2005) Photophysics and biological applications of the environment-sensitive fluorophore 6-N, N-Dimethylamino-2, 3-naphthalimide. J Am Chem Soc 127 1300-1306... 

Pyrene fluorophores are also used as probes. Derivatives of pyrene show /.max/ Xem 340/376 nm, e 4.3 x 104 M 1 cm-1, and environmental sensitivity, this fluorophore can be used to report on RNA folding [102]. Pyrene also displays a long-lived excited state (x > 100 ns), which allows for an excited pyrene molecule to associate with a pyrene in the ground state. The resulting eximer exhibits a red-shift in fluorescence intensity (A,em 490 nm). This characteristic can be used to study important biomolecular processes, such as protein conformation [103]. 

Willets KA, Callis PR, Moemer WE (2004) Experimental and theoretical investigations of environmentally sensitive single-molecule fluorophores. J Phys Chem B 108(29) 10465-10473... 

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