Laser-induced Fluorescence Polarization Research Articles

Off-DNA DNA-Encoded Library Affinity Screening.

DNA-encoded library (DEL) technology is emerging as a key element of the small molecule discovery toolbox. Conventional DEL screens (i.e., on-DNA screening) interrogate large combinatorial libraries via affinity selection of DNA-tagged library members that are ligands of a purified and immobilized protein target. In these selections, the DNA tags can materially and undesirably influence target binding and, therefore, the experiment outcome. Here, we use a solid-phase DEL and droplet-based microfluidic screening to separate the DEL member from its DNA tag (i.e., off-DNA screening), for subsequent in-droplet laser-induced fluorescence polarization (FP) detection of target binding, obviating DNA tag interference. Using the receptor tyrosine kinase (RTK) discoidin domain receptor 1 (DDR1) as a proof-of-concept target in a droplet-scale competition-binding assay, we screened a 67 100-member solid-phase DEL of drug-like small molecules for competitive ligands of DDR1 and identified several known RTK inhibitor pharmacophores, including azaindole- and quinazolinone-containing monomers. Off-DNA DEL affinity screening with FP detection is potentially amenable to a wide array of target classes, including nucleic acid binding proteins, proteins that are difficult to overexpress and purify, or targets with no known activity assay.

POSTMORTEM INTERVAL ESTIMATION BY THE AZIMUTH OF LASER-INDUCED FLUORESCENCE OF POLYCRYSTALLINE CEREBROSPINAL FLUID FILMS IMAGES

Abstract. There are many new optical polarimetric methods of biological tissue structure assessment, which ones are the most perspective in PMI estimation. The purpose of the work is to develop and to test the method of two-dimensional Stokes-polarimetric mapping of biological layers autofluorescence for PMI estimation by statistical analysis of postmortem changes dynamics of the coordinate distributions values of polycrystalline films of liquor (PFL) images laser-induced fluorescence polarization azimuth (LIFPA). Materials and methods. Objects of investigation are PFL, taken in 78 corpses with accurately known time of death and 20 healthy volunteers. Coordinate distributions of LIFPA image values were determined for each sample of PFL in the optical arrangement of the Stokes polarimeter in short-wave spectral bands of optical emission. Results and conclusions. Two-dimensional Stokes-polarimetric mapping distributions LIFPA of PFL images may be used in PMI estimation. Statistical points of the 3 rd and 4 th orders are the most sensitive PFL optical values to evaluate postmortem changes by short-range fluorescence. Optimal interval in 24 hours and the accuracy of PMI estimation 25 min for proteins and NADH were found.

Post-mortem interval estimation by laser-induced fluorescence of polycrystalline cerebro-spinal fluid films images

There are many new optical diagnostic methods (photometric, polarization and correlation techniques) of biological tissue structure assessment, which are the most perspective in post-mortem interval determination.
 The purpose of the work. Development and testing of the two-dimensional Stokes-polarimetric mapping of biological layers own fluorescence to evaluate accuracy of the post-mortem interval (PMI) assessment using statistical analysis of postmortem changes dynamics of the coordinate distributions values of polycrystalline films of liquor (PFL) images laser-induced fluorescence polarization intensity (LIFPI).
 Materials and methods. Objects of investigation are PFL, taken in 72 corpses with accurately known time of death and 20 healthy volunteers. The cause of the death was cardiovascular accident. Coordinate distributions of LIFPI image values were determined for each sample of PFL in the optical arrangement of the Stokes polarimeter in short-range spectrum of optical radiation.
 The value of statistical points 1 - 4 th order was perfomed for each two-dimensional distribution of PFL images LIFPI values. Statistical processing of the calculated values of set of points that characterize the LIFPI distributions was carry out. The depending on the time change of the most sensitive points of statistical values were built.
 Results and conclusions. Two-dimensional Stokes-polarimetric mapping distributions LIFPI of PFL images may be used in determination of the PMI. Statistical points of the first and fourth order are the most sensitive PFL optical values to evaluate postmortem changes by short-range fluorescence.

Measurements of localized potential profiles by LIF polarization spectroscopy in an inertial-electrostatic confinement discharge

Spatial distributions of the electric field were measured in inertial-electrostatic confinement plasmas generated in a glided cylindrical hollow cathode in He gas as a function of the applied voltage using laser-induced fluorescence polarization spectroscopy to obtain profiles of the potential and the charge density. With increasing applied voltage, the potential difference between the center of the plasma and the cathode decreased, while a virtual anode due to converging positive ions was clearly appeared at the center of the cathode. These are probably caused by the increase in the number of electrons emitted from the cathode surface due to the increasing ion bombardment with increasing applied voltage.

Fluorescence Polarization Combined Capillary Electrophoresis Immunoassay for the Sensitive Detection of Genomic DNA Methylation

Genomic DNA hypomethylation is epigenetically associated with aberrant gene expression and chromosome instability. Here we describe a method for rapid and sensitive detection of genomic DNA methylation without the need for bisulfite conversion, enzymatic digestion, or PCR amplification. The methylated DNA is first specifically recognized by an anti-5-methylcytosine IgG1 antibody and noncovalently labeled by a monovalent, fluorescently labeled, Fc-specific anti-IgG1 Fab fragment (secondary antibody). The formed immuno-complex of methylated DNA can be efficiently focused and separated from the DNA unbound secondary antibody by capillary electrophoresis (CE). The free fluorescent dye comigrates with the immuno-complex. However, by taking advantage of online laser-induced fluorescence polarization detection (LIFP), the target immuno-complex can be distinguished and accurately measured from the overlapped dye without further separation. The developed method is highly sensitive with a LOD of 0.3 nM and is highly specific for the detection of methylated DNA. Moreover, the CE-LIFP immunoassay is rapid (1.2 min for one analysis) and only consumes less than 0.1 ng of genomic DNA. The method was validated by examining human cell lines treated by methyltransferase inhibitor 5-aza-2'-deoxycytidine at low doses (10 nM-10 microM). Because of its sensitivity and speed, our method will be applicable for rapid epigenetic evaluation and for the study of tumorigenesis and chemical-cell interactions.

Spectroscopic observations, spin-orbit functions, and coupled-channel deperturbation analysis of data on theA Σ1u+andb Π3ustates ofRb2

We present experimentally derived potential curves and spin-orbit interaction functions for the strongly perturbed $A\text{ }{^{1}\ensuremath{\Sigma}}_{u}^{+}$ and $b\text{ }{^{3}\ensuremath{\Pi}}_{u}$ states of the rubidium dimer. The results are based on laser-induced fluorescence and optical-optical double-resonance polarization spectroscopy measurements combined with earlier laser-induced fluorescence data. We used an analytic potential (Hannover form) incorporated in a discrete variable representation of the Hamiltonian matrix for numerical energy-level calculation. A previous vibrational assignment of the $A$ levels is confirmed, and very probable vibrational assignment for the $b$ levels is also obtained. Currently, the rms residual of our fit is $0.053\text{ }{\text{cm}}^{\ensuremath{-}1}$ as compared to the typical experimental uncertainties that are estimated to be $0.005\text{ }{\text{cm}}^{\ensuremath{-}1}$. Fitted diagonal and off-diagonal spin-orbit functions are obtained and compared with ab initio calculations by all electrons and effective core pseudopotential methods. Analysis of the computational results yields an explanation for the ubiquitous single minima in these spin-orbit functions, which can be represented approximately by Morse-type functions.

DNA wrapping is required for DNA damage recognition in the Escherichia coli DNA nucleotide excision repair pathway

Localized DNA melting may provide a general strategy for recognition of the wide array of chemically and structurally diverse DNA lesions repaired by the nucleotide excision repair (NER) pathway. However, it is not clear what causes such DNA melting and how it is driven. Here, we show a DNA wrapping-melting model supported by results from dynamic monitoring of the key DNA-protein and protein-protein interactions involved in the early stages of the Escherichia coli NER process. Using an analytical technique involving capillary electrophoresis coupled with laser-induced fluorescence polarization, which combines a mobility shift assay with conformational analysis, we demonstrate that DNA wrapping around UvrB, mediated by UvrA, is an early event in the damage-recognition process during E. coli NER. DNA wrapping of UvrB was confirmed by Förster resonance energy transfer and fluorescence lifetime measurements. This wrapping did not occur with readily denaturable damaged DNA substrates ("bubble" DNA), suggesting that DNA wrapping of UvrB plays an important role in the induction of DNA melting around the damage site. Analysis of DNA wrapping of mutant UvrB Y96A further suggests that a cooperative interaction between DNA wrapping of UvrA(2)B and contact of the beta-hairpin of UvrB with the bulky damage moiety may be involved in the local DNA melting at the damage site.

Intensity and polarization of laser-induced fluorescence due to forbidden excitation of He atoms immersed in an electric field in plasmas

Laser-induced fluorescence (LIF) processes through the Stark and electric-quadrupole moment (QDP) transitions of HeI (2(1)S-->n1D-->2(1)P,n=3,4) have been investigated for reliable electric-field measurements in plasmas. A linear-polarization model is formulated for various configurations of electric fields, magnetic fields, and laser polarization. To extend the model to higher-particle-density plasmas we develop a rate-equation model involving a collisional disalignment term. Disalignment rates of n1D states, Rda, due to a collision with He gas were measured. Spatial distributions in intensity and polarization of LIF were observed in a discharge plasma. For n=3 with small Rda, the same electric-field distribution in the sheath was obtained from either of the intensity ratios of the Stark to QDP component and the polarization, and the sheath potential agreed well with that by an electric probe. For n=4 with large Rda, the distribution was also correctly obtained from partially depolarized LIF wave forms by using the extended model. These results show that our extended model provides an accurate measurement of the electric field. The minimum detectable field strength was 80 V/cm for n=4. Application and limitations of the methods are discussed.

Organic Osmolyte Mediated Kinetic Capillary Electrophoresis for Study of Protein−DNA Interactions

An organic osmolyte mediated kinetic capillary electrophoresis is developed and applied to study the binding of Escherichia coli RecA protein to single-stranded (ss) DNA. The complex of RecA and ssDNA can be significantly enhanced and stabilized by 20% glycerol over 5.6 times. Kinetic capillary electrophoresis study reveals that glycerol may function in the preservation of RecA native conformation, enhancement of specific binding affinity (20 times), and decrease in dissociation rate constant (1 time). The combined laser-induced fluorescence polarization (LIFP) study further indicates a tightened assembly of RecA on ssDNA induced by glycerol. The binding of RecA to ssDNA was also enhanced by two additional protecting organic osmolytes, sucrose and PEG 400. In contrast, a denaturing organic osmolyte, urea, even at a concentration of 110 mM can prevent the formation of the RecA-ssDNA complexes. The detection of the binding of E. coli single-stranded binding protein (SSBP) to ssDNA was also improved by the same method. By combining the protection of organic osmolytes with low-temperature storage, a method for preservation of protein-DNA complex up to 43 h is further demonstrated. By use of a long capillary and protecting glycerol together an enhanced kinetic CE analysis was also developed, providing a better separation and analysis of the undissociated complex, the dissociated complex, and the unbound probe. The results demonstrate the wide applicability of the method to the study of various protein-DNA binding.

Effect of Disalignment due to Electron Collision on Laser-Induced Fluorescence Polarization Spectroscopy for Measuring Electric Field in Plasmas

The effect of disalignment due to electron collisions on the determination of electric fields in laser-induced fluorescence (LIF) polarization spectroscopy was studied. The aligned He I n1D (n=3,4) atoms were produced by laser excitation of 21S state through electric quadrupole transition in Penning discharge plasma. The polarization components of the subsequent LIF (n1D→21P) were observed as a function of electron density in the range from 2×1010 to 2×1012 cm-3. From the dependences of disalignment rates for 31D and 41D states on electron density, their rate coefficients were estimated to be (1.5±0.3)×10-5 and (4.7±1.2)×10-5 cm3/s, respectively. Critical disalignment rates, where the polarization of LIF disappeared, were also estimated using a rate-equation model taking into account the disalignment effect. On the basis of experimental rate coefficients and critical disalignment rates, we deduced the limitation of measurable electric field for electron density in LIF polarization spectroscopy.

Simultaneous laser-induced fluorescence and sub-Doppler polarization spectroscopy of the CH radical

The first application of polarization spectroscopy (PS) to the CH radical is demonstrated. In particular, we report on the simultaneous application of laser-induced fluorescence (LIF) and sub-Doppler PS to CH. The conventional experimental setup for PS was supplemented with a second detection system in order to collect the LIF emission. At the same time a Fabry–Perot etalon and molecular iodine were utilized to obtain a precise relative and absolute frequency scale, respectively. CH was investigated in a low pressure methane–oxygen flame. The R2(5) transition of the B–X (0,0) band corresponding to a wavelength around 387.3nm was scanned while fluorescence emission was collected in the spectral region around 431nm from the B–X (0,1), A–X (1,1) and A–X (0,0) bands. The saturation behavior of both techniques is investigated as well as line broadening effects due to the pump laser pulse energy or rather fluence. Saturation fluence for LIF was found to be more than one order of magnitude higher as compared to PS.

2002 W.A.E. McBryde Award Lecture — Affinity recognition, capillary electrophoresis, and laser-induced fluorescence polarization for ultrasensitive bioanalysis

The combination of affinity recognition, capillary electrophoresis (CE), laser-induced fluorescence (LIF), and fluorescence polarization for the ultrasensitive determination of compounds of biological interest is described. Competitive immunoassays using CE–LIF eliminate the need for fluorescently labeling trace analytes of interest and are particularly useful for determination of small molecules, such as cyclosporine, gentamicin, vancomycin, and digoxin. Fluorescence polarization allows for differentiation of the antibody-bound from the unbound small molecules. Noncompetitive affinity CE–LIF assays are shown to be highly effective in the determination of biomarkers for DNA damage and HIV-1 infection. An antibody (or aptamer) is used as a fluorescent probe to bind with a target DNA adduct (or the reverse transcriptase of the HIV-1 virus), with the fluorescent reaction products being separated by CE and detected by LIF. Aptamers are attractive affinity probes for protein analysis because of high affinity, high specificity, and the potential for a wide range of target proteins. Fluorescence polarization provides unique information for studying molecular interactions. Innovative integrations of these technologies will have broad applications ranging from cancer research, to biomedical diagnosis, to pharmaceutical and environmental analyses.Key words: capillary electrophoresis, laser-induced fluorescence, fluorescence polarization, immunoassay, affinity probes, antibodies, aptamers, DNA damage, toxins, therapeutic drugs.

Competitive immunoassay for vancomycin using capillary electrophoresis with laser-induced fluorescence detection.

A competitive immunoassay using capillary electrophoresis with laser-induced fluorescence was developed for vancomycin. Capillary electrophoresis using a Tris-glycine running buffer provided adequate separation of the antibody-bound from the unbound fluorescent probe (tracer) in less than 4 min. Laser-induced fluorescence polarization (LIFP) provided high sensitivity detection and simultaneous monitoring of fluorescence intensity and polarization. A fluorescence polarization value of 0.30 confirmed the formation of the antibody-tracer complex. Calibration curves showed a working linear range of 2-3 orders of magnitude with a minimum detectable concentration of 0.98 ng mL(-1) (or 1.1 fg vancomycin). Clinical samples obtained from patients undergoing vancomycin treatment were analyzed for vancomycin and the results correlated well with a standard immunoassay based on latex particle detection that was routinely used by a hospital laboratory. Only 1/10 of the reagents were needed as compared with the standard immunoassay.

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.

Polarization effects and depolarizing collisions in atomic vapour and molecular gas

Polarization effects in gases are investigated by two methods of coherent spectroscopy - stimulated photon echo in molecular gas and laser-induced fluorescence in atomic vapour. A well developed photon echo experimental technique permitted us not only to measure relaxation rates of population, orientation and alignment on degenerated resonant levels in molecular gas but also to study their dependence on particles' longitudinal velocity. The degree of polarization of laser-induced fluorescence in Yb vapour in a mixture with Xe proves a large contribution of depolarizing collisions. The complicated non-exponential fluorescence decay at higher Yb pressures is explained by the radiation trapping.

Studies of protein--DNA interactions by capillary electrophoresis/laser-induced fluorescence polarization.

Protein-DNA interactions were studied on the basis of capillary electrophoretic separation of bound from free fluorescent probe followed by on-line detection with laser-induced fluorescence polarization. Changes in electrophoretic mobility and fluorescence anisotropy upon complex formation were monitored for the determination of binding affinity and stoichiometry. The method was applied to study the interactions of single-stranded DNA binding protein (SSB) with synthetic oligonucleotides and single-stranded DNA. Increases in fluorescence anisotropy and decreases in electrophoretic mobility upon their binding to SSB were observed for the fluorescently labeled 11-mer and 37-mer oligonucleotide probes. Fluorescence anisotropy and electrophoretic mobility were used to determine the binding constants of the SSB with the 11-mer (5 x 10(6) M(-1)) and the 37-mer (23 x 10(6) M(-1)). Alternatively, a fluorescently labeled SSB was used as a probe, and the formation of multiple protein-DNA complexes that differ in stoichiometry was observed. The results demonstrate the applicability of the method to study complex interactions between protein and DNA.

Capillary electrophoretic immunoassays for digoxin and gentamicin with laser-induced fluorescence polarization detection

New immunoassays for therapeutic drugs digoxin and gentamicin have been described, which involved the separation of free and antibody-bound drug by capillary electrophoresis (CE) and the detection by laser-induced fluorescence polarization (LIFP). While the fluorescein-labeled digoxin and gentamicin (tracers) displayed negligible fluorescence polarization in solution, the complex formation between these small molecules and their antibodies resulted in substantial increases in fluorescence polarization due to the increase in molecular size. The LIFP detection, capable of measuring vertically and horizontally polarized fluorescence components simultaneously, provides enhanced capability for the identification of complex in capillary electrophoretic immunoassays. Proper adjustments of the running buffer pH and the ratio of antibody to tracer are essential for optimization of the performance of these assays. The digoxin–antibody complex remained stable during CE separation with running buffer pH ranging from 9.3 to 12. Calibration curves covering a concentration range of 0.05 to 0.5 ng/ml were obtained with a running buffer of pH 12. The concentration and mass detection limits were 0.02 ng/ml and 26 zmol, respectively. For gentamicin assay, the running buffer pH 10 was used to reduce the adsorption of the tracer while minimizing the dissociation of the antibody–tracer complex during the separation. The calibration curves covered a concentration range 0.05–1.0 μg/ml, with a concentration detection limit of 25 ng/ml and a mass detection limit of 52 amol of gentamicin.

Fluorescence polarization studies of affinity interactions in capillary electrophoresis.

Capillary electrophoresis (CE) combined with molecular recognition for ultrasensitive bioanalytical applications often requires the formation of stable complexes between an analyte and its binding partner. Previous studies of binding interactions using CE involve multiple-step titration experiments and are time-consuming. We describe a simple method based on laser-induced fluorescence polarization (LIFP) detection for CE separation, which allows for on-line monitoring of affinity complex formation. Because fluorescence polarization is sensitive to changes in the rotational diffusion arising from molecular association or dissociation, it is capable of providing information on the formation of affinity complexes prior to or during CE separation. Applications of the CE/LIFP method to three binding systems including vancomycin and its antibody, staphylococcal enterotoxin A and its antibody, and trp operator and trp repressor were demonstrated, representing peptide-protein, protein-protein, and DNA-protein interactions. The affinity complexes were readily distinguished from the unbound molecules on the basis of their fluorescence polarization. The relative increase in fluorescence polarization upon complex formation varied with the molecular size of the binding pairs.

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.

Polarized laser-induced fluorescence technique to measure localized electric field induced perpendicularly to magnetic field in the plasma-edge

To develop a direct measurement of the weak electric field induced in a magnetically confined edge plasma, the crossed magnetic- and electric-field effect was investigated on the linear polarization of laser-induced fluorescence. The fluorescence was induced by laser excitation of metastable He atoms 2 1S to 4 1D state due to Stark-induced electric dipole and electric quadrupole moments. In a model-type experiment carried out in a cylindrical hollow cathode plasma column, the radial distribution of the polarization of fluorescence was analyzed to determine the electric field dependence. The measured degree of polarization agreed with estimates from a theoretical model based on the selection rules of the spontaneous radiative transition. The minimum detectable electric field is estimated to be a few tens of V/cm in our scheme. We briefly discuss techniques to increase our measurement sensitivity by the interference between both the forbidden transitions in the laser excitation.