Fluorescence polarization detection for affinity capillary electrophoresis

Affinity capillary electrophoresis (ACE) with laser-induced fluorescence polarization (LIFP) detection is described, with examples of affinity interaction studies. Because fluorescence polarization is sensitive to changes in the rotational motion arising from molecular association or dissociation, ACE-LIFP is capable of providing information on the formation of affinity complexes prior to or during CE separation. Unbound, small fluorescent probes generally have little fluorescence polarization because of rapid rotation of the molecule in solution. When the small fluorescent probe is bound to a larger affinity agent, such as an antibody, the fluorescence polarization (and anisotropy) increases due to slower motion of the much larger complex molecule in the solution. Fluorescence polarization results are obtained by simultaneously measuring fluorescence intensities of vertical and horizontal polarization planes. Applications of CE-LIFP to both strong and weak binding systems are discussed with antibody-antigen and DNA-protein binding as examples. For strong affinity binding, such as between cyclosporine and its antibody, complexes are formed prior to CE-LIFP analysis. For weaker binding, such as between single-stranded DNA and its binding protein, the single-stranded DNA binding protein is added to the CE separation buffer to enhance dynamic formation of affinity complexes. Both fluorescence polarization (and anisotropy) and mobility shift results are complementary and are useful for immunoassays and binding studies.     

Fluorescence polarization: past,present and future

Fluorescence polarization was first observed in 1920 and during the next few decades the theoretical foundations of the phenomenon were clearly established. In the last two decades of the 20(th) century, fluorescence polarization became one of the most prevalent methods used in clinical and biomedical sciences. In this article we review the history of fluorescence polarization, its theoretical foundations and some of the more important practical developments, which helped to popularize the method. We also discuss important, but often misunderstood, practical considerations including the wavelength dependence of the limiting polarization and the effect of energy transfer on polarization. The present state of fluorescence polarization, both in pure research as well as in the applied biosciences is also reviewed. Finally, we speculate on possible future developments in the field, such as the use of multi-photon techniques.     

Capillary electrophoresis coupled with laser-induced fluorescence polarization as a hybrid approach to ultrasensitive immunoassays.

Immunoassays using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) is a powerful approach to the determination of trace amounts of analytes in a complex biological matrix. However, its applicability is limited by the requirement that the free and bound tracer (fluorescently labeled compound) be resolved for their identification and quantitation. Here we show that replacing LIF with laser-induced fluorescence polarization (LIFP) permits ultrasensitive immunoassays to be performed with or without the separation of the free and bound tracer. A binding system involving cyclosporin A (CyA) and monoclonal antibody to CyA was chosen to demonstrate both homogeneous and heterogeneous immunoassay approaches. In the homogeneous scheme where the free and bound tracer were not separated, the fluorescence polarization of the mixture was a quantitative measure of the antibody-bound tracer. The concentration and mass detection limits for CyA using the homogeneous competitive assay were found to be 1 nM and 1 amol (10(-18) mol), respectively. The heterogeneous assay involved a nearly baseline separation of the free and bound tracer using CE with a phosphate running buffer of pH 7.0. The complex of the tracer with the antibody had a fluorescence polarization of approximately 0.24 whereas the free tracer had negligible polarization. The fluorescence polarization was independent of analyte concentration, and the fluorescence intensity of either the free or bound tracer was used for quantitation. Results from both assays suggest that the CE-LIFP approaches may have a wider application than the immunoassays based on either CE-LIF or fluorescence polarization alone.     

Über magnetische Beeinflussung der Polarisation der Resonanzfluoreszenz

1. Historical remarks concerning magnetic influence on the polarization of resonance fluorescence and its explanation as the Zeeman effect. 2. Rotation of the plane of polarization of the resonance fluorescence of Hg by weak magnetic fields and determination of the lifetime of the excited state. 3. Experiments concerning the excitation of the resonance fluorescence of Hg by light with different kinds and directions of polarization.     

Noninvasive optical characterization of hydrogels

This paper presents results based on concentration quenching of fluorescence polarization (cqfp), which demonstrate the feasibility of using cqfp to measure changes in hydrogel-membrane hydration. This is accomplished by binding fluorescent molecules to the hydrogel matrix, and showing that the fluorescence polarization is a monotonic function of fluorophore concentration. Films based on crosslinked 2-hydroxyethyl methacrylate containing lissamine side groups (10⁻⁴-10⁻⁷ mol/l) were mounted in a special cell which provided an aqueous environment, and in which polarized fluorescence could be measured. An argon-ion laser provided polarized excitation at 488nm, and the polarized fluorescent emission was detected. The correlation between the fluorescence polarization and the bound dye concentration was found to correspond to the theoretically expected behavior.     

Fluorescence polarization spectroscopy in protein analysis

Information about concentration, molecular structure, binding events, and motion can be obtained using fluorescence spectroscopy methods. Fluorescence polarization spectroscopy is one such method, which studies the relationship between the polarization of light that is used for excitation and light that is subsequently detected from fluorescence. The extent of change of polarization between excitation and emission can be used to study physical processes such as rotational diffusion, extent of denaturation, and orientation at surfaces. In this article Mann and Krull describe the underlying principles to the technique and show how fluorescence polarization spectroscopy has contributed to protein analysis.     

Novel peptide–protein assay for identification of antimicrobial peptides by fluorescence quenching

The specific interaction of peptides with proteins is often a key factor which determines biological activities. The determination of K(d) values of such interactions is commonly performed with fluorescence polarization. However, fluorescence polarization assays are prone to false-positive results due to the potential for non-specific interactions and only afford very low signal-to-background ratios. Here, we present as an alternative a fluorescence resonance energy transfer based quenching assay to measure peptide-protein interactions in solution. In a test setup where antimicrobial peptides were tested for their affinity towards the protein DnaK, the assay provided high specificity and good reproducibility and correlated with the results obtained by fluorescence polarization methods. Furthermore, we established a fast prescreening method which will allow a highly efficient screening of peptide libraries by reducing the amount of sample by 98% compared to conventional fluorescence polarization assays.     

The polarization of excimer fluorescence from a dinucleotide

The polarization of the excimer fluorescence from the dinucleotide CpC has been observed to be strikingly different from that of the monomer. The mixed polarization in the absorption reflects significant ground state interaction. The mixed polarization in the fluorescence is most likely due to a component which is polarized between the chromophores, a manifestation of charge resonance character in the excimer state.     

Molecular and quantitative aspects of the chemistry of fulvic acid and its interactions with metal ions and organic chemicals : Part II. The fluorescence spectroscopy approach

Information in the literature on the use of fluorescence spectroscopy for the study of the chemistry of fulvic acid and its interactions with metal ions and organic chemicals is discussed. Basic principles, instrumentation, procedures, methodology and limitations of the fluorescence technique and the fluorescence polarization method are briefly summarized. This is followed by an extended discussion on the direct information that fluorescence spectra can furnish on the properties and nature of fluorescing structures in fulvic acids. The effects of molecular parameters and environmental factors (molecular weight, concentration, pH, ionic strength, temperature and redox potential) on the fluorescence behaviour of fulvic acids are discussed. Particular attention is devoted to the indirect information that fluorescence quenching and fluorescence polarization studies provide on molecular and quantitative aspects of the chemistry of fulvic acid in solution, especially in relation to molecular conformation and binding with metal ions and organic chemicals. The potential advantage of this non-separative, non-destructive technique for the study of environmental samples such as fulvic acids is emphasized.     

ACTION SPECTRA OF CATION EFFECTS ON THE FLUORESCENCE POLARIZATION AND INTENSITY IN THYLAKOIDS AT ROOM TEMPERATURE

Abstract— The degree of polarization of chlorophyll- a (Chl- a ) fluorescence is known to monitor the extent of excitation migration and/or the orientation of the photosynthetic pigment molecules. We report here the effects of cations, at room temperature, on the degree of polarization of Chl- a fluorescence, and fluorescence intensity in thylakoids as a function of excitation wavelength. Observations of maxima at 650 and 675 nm in the cation-induced changes in the excitation spectrum for fluorescence at 730 and 762 nm, and, in the action spectra for the depolarization of fluorescence lead us to suggest that the regulation of the initial distribution of excitation to photosystem II involves the better coupling of Chl- b and- a in the light harvesting complex with Chl- a in the reaction center II complex.     

Ultrasensitive detection of potassium ions based on target induced DNA conformational switch enhanced fluorescence polarization

We have developed a simple, highly sensitive and selective fluorescence polarization assay for the detection of potassium ions based on target induced DNA conformational switch from hairpin to G-quadruplex enhanced fluorescence polarization. The assay was applied in the detection of low nM concentrations of potassium ions and was highly selective over other cations.     

Determination of Fluorescence Polarization and Absorption Anisotropy in Molecular Complexes Having Threefold Rotational Symmetry

Abstract— The current work concerns investigation of the polarization properties of complex molecular ensembles exhibiting threefold (C₃) rotational symmetry, particularly with regard to the interplay between their structure and dynamics of internal energy transfer. We assume that the molecules or chromophores in such complexes possess strongly overlapped spectra both for absorption and fluorescence. Such trimeric structures are widely found in biological preparations, as for example the trimer of C-phycocyanin (C-PC). Higher order aggregates, e.g. hex-amers and three-hexamer rods, are also investigated and compared with the trimer case. The theory addresses both steady-state and 8-pulse excitation and establishes some links between them. Monochromophoric, bichro-mophoric and trichromophoric molecular complexes are individually examined. For steady-state excitation, analytical formulas are reported for the degree of fluorescence polarization and absorption anisotropy. It is shown that the polarization is dependent on the chromophore inclination relative to the symmetry axis, the relative efficiencies of absorption and fluorescence by chromophores of different spectral types, and the rates of energy equilibration. To assess the validity of the theory, it has been applied to C-PC aggregates. Here it was found that different C-PC aggregates provide practically identical polarization response. For S-pulse excitation we give analytical formulas for determination of the fluorescence depolarization, and also the depolarization associated with absorption recovery, both for a monochromophoric trimer and some particular cases of bichromophoric trimer. More complicated systems are analyzed by computer modeling. Thus it transpires that the initial polarization anisotropy r(t = 0) takes the value 0.4 for all considered aggregates; the long-time limit r(t →∞) has about the same value as is associated with steady-state excitation. We also show that with steady-state excitation the degree of fluorescence polarization is practically equal for various C₃ aggregates of C-PC, and that the major factor determining the polarization is the chromophore orientation relative to the symmetry axis.     

Fluorescence study of polyelectrolyte complex formation III: Mobility of probes in microdomains

The mobility of fluorescent probe molecules in the microdomains formed by polyelectrolyte complexes (PEC) in aqueous solution was investigated by the fluorescence polarization method. The degree of fluorescence polarization (p) for the anthracene (Anth) chromophores attached to anionic polyelectrolytes increased with complexation, indicating the restricted mobility of probe molecules in the PEC microdomains. These p values depended on the properties of added polycations, which were not in parallel with the fluorescence intensities of the Anth chromophores.     

Dual‐Polarization Imaging of a Dual‐Fluorophore Ion Sensor: A Single‐Molecule Study

The fluorescence emission of the dual‐fluorophore Ca²⁺ ion sensor molecule, calcium‐green 2 (CG‐2), has been characterized using dual‐polarization imaging at the single‐molecule level. By comparing the fluorescence intensity of individual CG‐2 molecules in two mutually orthogonal polarization image channels, information about the relative orientation of the two constituent fluorophores in the molecule is obtained. Experimental results from polarization measurements are compared with those predicted from a geometric model based on coupled‐fluorophores that are randomly distributed in space. The results confirm previous optical spectroscopy‐based predictions of the orientation of CG‐2′s fluorophores, and the general applications of this dual‐polarization imaging approach for characterizing the optical properties of molecules containing multiple fluorophores is discussed.     

Fluorescence quenching and polarization studies of segmental motion in polystyrene

Fluorescence quenching has been used in conjunction with measurements of fluorescence polarization to study the segmental relaxation of polystyrene. There is confirmation of the apparent variation in the intrinsic polarization of the bound fluorescent probe with the nature of the macromolecule. The results agree with previous observations, using a different fluorescence approach, that the rotational times for groups at the chain ends are less than one fifth of those for the same groups in the chain interiors. The variation of intrinsic polarization with polymer structure is discussed.     

Fluorescence Detected Circular Dichroism for Highly Sensitive Observation of Exciton Coupled CD

The fluorescence detected circular dichroism (FDCD) appearance of some exciton coupling compounds is reviewed by a newly developed ellipsoidal mirror system (FDCD465). The FDCD465 achieves both the complete elimination of the polarization artifact and a dramatic enhancement of detection sensitivity expanding the applicability of exciton coupling fluorophores. A variety of chromophores that possess large extinction coefficients and high fluorescence quantum yields are available for exciton coupled FDCD, regardless of the degree of fluorescence polarization. Different types of FDCD devices can regulate FDCD polarization artifacts and the photoselection effect, enabling effective surveys of FDCD potential.     

Tict fluorescence in rigid matrices: α-delayed fluorescence

The observation of the dual fluorescence of 4-N,N-dimethylaminobenzonitrile (DMABN) in polyvinylalcohol has been extended to other matrices able to act as hydrogen-bond donors (nylon and polyurethane) and to related molecules. A model of α-delayed fluorescence is proposed to account for the properties of the luminescence in rigid matrices. This proposal is consistent with: the positive polarization of the anomalous fluorescence (room temperature) while the polarization of phosphorescence is negative (at 77 K); the quenching of the anomalous fluorescence by lowering the temperature; the very long lifetime (τ ≈ 1 s) of the anomalous fluorescence, in the same range as the phosphorescence lifetime.     

Fluorescence Detected Circular Dichroism for Highly Sensitive Observation of Exciton Coupled CD

Summary. The fluorescence detected circular dichroism (FDCD) appearance of some exciton coupling compounds is reviewed by a newly developed ellipsoidal mirror system (FDCD465). The FDCD465 achieves both the complete elimination of the polarization artifact and a dramatic enhancement of detection sensitivity expanding the applicability of exciton coupling fluorophores. A variety of chromophores that possess large extinction coefficients and high fluorescence quantum yields are available for exciton coupled FDCD, regardless of the degree of fluorescence polarization. Different types of FDCD devices can regulate FDCD polarization artifacts and the photoselection effect, enabling effective surveys of FDCD potential.     

POLARIZATION OF FLUORESCENCE FROM BACTERIOCHLOROPHYLL IN CASTOR OIL, IN CHROMATOPHORES AND AS P870 IN PHOTOSYNTHETIC REACTION CENTERS

Abstract— The polarization of fluorescence from isolated photosynthetic reaction centers and from light harvesting chlorophyll in photosynthetic units was measured over a wide range of exciting wavelengths. In addition, the fluorescence polarization of bacteriochlorophyll was measured. The simplest interpretation of the data is that in the bacterial reaction center, the three chlorophyll molecules closely associated with photochemical oxidation do not have their transition moments parallel to one another. Highly polarized fluorescence was also observed from the intact photosynthetic unit.