Juan Yguerabide

Resonance light scattering particles as ultrasensitive labels for detection of analytes in a wide range of applications

We have developed a new detection technology that uses resonance light scattering (RLS) particles as labels for analyte detection in a wide range of formats including immuno and DNA probe type of assays in solution, solid phase, cells, and tissues. When a suspension of nano sized gold or silver particles is illuminated with a fine beam of white light, the scattered light has a clear (not cloudy) color that depends on composition and particle size. This scattered light can be used as the signal for ultrasensitive analyte detection. The advantages of gold particles as detection labels are that (a) their light producing power is equivalent to more than 500,000 fluorescein molecules, (b) they can be detected at concentrations as low as 10−15 M in suspension by eye and a simple illuminator, (c) they do not photobleach, (d) individual particles can be seen in a simple student microscope with dark field illumination, (e) color of scattered light can be changed by changing particle size or composition for multicolor multiplexing, and (f) they can be conjugated with antibodies, DNA probes, ligands, and protein receptors for specific analyte detection. These advantages allow for ultra‐senstive analyte detection with easiness of use and simple and relatively inexpensive instrumentation. We have shown that our RLS technology can indeed be used for ultra‐sensitive detection in a wide range of applications including immuno and DNA probe assays in solution and solid phases, detection of cell surface components and in situ hybridization in cells and tissues. Most of the assay formats described in this article can be adapted for drug fast throughput screening. J. Cell. Biochem. Suppl. 37: 71–81, 2001.

STEADY‐STATE FLUORESCENCE METHOD FOR EVALUATING EXCITED STATE PROTON REACTIONS: APPLICATION TO FLUORESCEIN

Abstract Fluorescein is a complex fluorophore in the sense that it displays four prototropic forms (cation, neutral, monoanion and dianion) in the pH range 1–9. In experiments with fluorescein‐labeled proteins we have sometimes observed complex nanosecond emission kinetics, which could be due to conversion of the excited monoanion into the excited dianion through an excited state proton exchange with a proton acceptor in the labeled protein. However, the literature is ambiguous on whether this possible excited state proton reaction of fluorescein does occur in practice. In this article we describe a general steady‐state fluorescence method for evaluating excited state proton reactions of simple as well as complex pH‐sensitive fluorophores and apply it to evaluate excited state proton reactions of fluorescein. The method depends on finding a buffer that can serve as an excited state proton donor‐acceptor but does not significantly perturb ground state proton equilibrium and especially does not form ground (or excited state complexes) with the fluorophore. Our results show that the excited monoanion‐dianion proton reaction of fluorescein does occur in the presence of phosphate buffer, which serves as a proton donor‐acceptor that does not significantly perturb ground state proton equilibria. The reaction becomes detectable at phosphate buffer concentrations greater than 20 mM and the reaction efficiency increases with increase in phosphate buffer concentrations. The reaction is most clearly demonstrated by adding phosphate buffer to a solution of fluorescein at constant pH 5.9 with preferential excitation of the monoanion. Under these conditions, the excited monoanion converts to the dianion during its lifetime. The conversion is detected experimentally as an increase in dianion and decrease in monoanion fluorescence intensities with increase in phosphate buffer concentration. The absorption spectrum is not significantly perturbed by the increase in phosphate buffer concentration. To quantitate the reaction, we have recorded titration graphs of fluorescence intensity versus pH for fluorescein solutions at low (5 mM) and high buffer (1 M) concentrations with preferential excitation of the monoanion and preferential detection of the dianion emission. We have also developed theoretical expressions that relate fluorescence intensity to pH in terms of the concentration of the four prototrophic forms of fluorescein, extinction coefficients, fluorescence efficiencies and ground and excited state pKa. The theoretical expressions give very good fits to the experimental data and allow evaluation of fundamental parameters such as pKa and fluorescence efficiencies. The analysis of the experimental data shows that the excited monoanion‐dianion reaction does not significantly occur at 5 mM phosphate buffer concentration. However, at 1 M buffer concentration the reaction is sufficiently fast that it practically achieves equilibrium during the lifetimes of the excited fluorescein monoanion and dianion. The pKa* of the excited monoanion‐dianion proton reaction is around 6.3. The results and methods presented here should be useful in the development and testing of pH‐sensitive labeling fluorophores and fluorescent indicators.

Partition of a Fluorescent Molecule between Liquid- Crystalline and Crystalline Regions of Membranes

SummaryWe have determined the partition coefficient of the fluorescent molecule perylene between liquid crystalline and crystalline regions of vesicle membranes formed from binary mixtures of several lipids. We measured the fluorescence intensity of perylene in these vesicles as a function of temperature and used the intensity profiles, together with a theory developed in a previous paper, to determine the partition coefficient defined as the ratio of the concentration of perylene in the liquid-crystalline (fluid) regions of the membrane to the concentration in the crystalline (solid) phase. In vesicles composed of dipalmitoyl phosphatidylcholine/distearoyl phosphatidylcholine (dppc/dspc) mixtures and of dipalmitoyl phosphatidylcholine/dipalmitoyl phosphatidylethanolamine (dppc/dppe) mixtures, the partition coefficient is close to unity. Its value is 1.04±0.18 for dppc/dsp mixtures and 1.10±0.26 for dppc/dppe mixtures. In vesicles composed of dimyristoyl phosphatidylcholine/distearoyl phosphatidylcholine mixtures, the partition coefficient was more difficult to determine and its value ranged from 0.3 to 7.

The effect of local anesthetics on the lateral mobility of lymphocyte membrane proteins

Abstract In this study, we have measured by fluorescence photobleach recovery (FPR) the relative mobilities of two rat lymphocyte membrane proteins, surface immunoglobulin (SIg) and AgB (histocompatability antigens). The results of the FPR experiments showed that SIg was relatively restricted in its mobility as compared with AgB. Treatment of cells with local anesthetics (LA), agents which are assumed to act, in part, by severing the linkage between the cytoskeleton and integral membrane proteins increased the mobility of SIg to a mobility similar to that of AgB. This finding suggests that SIg is relatively restricted in its mobility due to a tethering by cytoskeletal elements. LA also had a small, but significant effect on the mobility of AgB.

Rotational dynamics of immunoglobulin G antibodies anchored in protein a soluble complexes

The rotational dynamics of rabbit IgG anti-dansyl antibodies anchored in staphylococcal protein A (SpA) soluble complexes were studied by both steady-state and nanosecond fluorescence spectroscopy. To aid in the interpretation of the anisotropy data, the results of recently reported hydrodynamic and electron microscopic studies of IgG-SpA complexes were used to calculate global tumbling times of the various complexes and to estimate the steric hindrance of the antibody Fab segments. The anisotropy decays, fitted to the sum of two exponentials, indicated that the Fab arms of antibodies bound to SpA by their Fc regions exhibit considerable flexibility. For the different IgG-SpA mixtures examined, changes in the IgG preexponential anisotropy weighting factors, fS and fL, and the short rotational correlation time, phi S, were relatively small. On the other hand, the long rotational correlation time, phi L, increased systematically when the percentage of larger IgG-SpA complexes in a mixture was increased. The greatest restriction of Fab flexibility was observed for antibodies anchored in the exceptionally compact IgG4-SpA2 complexes. Available electron microscopic data suggest that increases in phi L correlate with increased steric hindrance of the antibody segments. Both native and hinge-disulfide-cleaved IgG experienced similar percentage increases in phi L when bound in SpA complexes. In agreement with our earlier interpretation, the results of this study provide rather striking evidence that phi L mainly represents flexible motions of the Fab segments and not global tumbling: the phi L-values of IgG bound in the various SpA complexes ranged from 101 to 162 nsec, whereas the calculated global tumbling times of the different complexes ranged from about 300 to 3000 nsec.

Theory of lipid bilayer phase transitions as detected by fluorescent probes

SummaryWe present a quantitative theory that relates the fluorescence intensityvs. temperature (I vs. T) profile of a fluorescent-labeled two-component lipid bilayer to the phase diagram of the bilayer and the partition coefficientK of the fluorophore between fluid and solid phases of the bilayer. We show how the theory can be used to evaluateK from experimentalI vs. T profiles and the appropriate phase diagrams as well as to understand the different shapes ofI vs. T profiles obtained with particular fluorophores and phase diagrams. Using calculatedI vs. T graphs, we discuss the meaning of parameters, such as midpoint of the phase transition and onset and termination of a transition, which are often used to characterize phase transitions on the basis of fluorescence intensityvs. temperature profiles.

Microviscosity of mucosal cellular membranes in toad urinary bladder: Relation to antidiuretic hormone action on water permeability

SummaryThe microviscosity of cellular membranes (or membrane fluidity) was measured in suspensions of single mucosal cells isolated from the urinary bladder of the toad,Bufo marinus, by the technique of polarized fluorescence emission spectroscopy utilizing the hydrophobic fluorescent probe, perylene. At 23°C, 5mm dibutyryl cyclic 3′,5′-AMP decreased the apparent microviscosity of the cell membranes from 3.31 to 3.07 P, a minimum decrease of 7.3% (P<0.001) with a physiological time course. Direct visualization of the cell suspension indicated that 98% of the cells were viable, as indicated by Trypan Blue dye exclusion. The fluorescent perylene could be seen only in plasma membranes, suggesting that the measured viscosity was that of plasma membrane with little contribution from the membranes of cellular organelles. Addition of antidiuretic hormone to intact hemibladders stained with perylene produced changes in fluorescence consistent with a similar 7% decrease in apparent microviscosity with a physiological time course. However, finite interpretation of the findings in intact tissue cannot be made because the location and the fluorescent lifetime of the probe could only be conducted on the isolated cells. Comparison with previously determined relationships between water permeability and microviscosity in artificial bilayers suggests that the 7% (a lower limit) decrease in microviscosity would produce only a 6.5% increase in water permeability.

Resonance light-scattering particles for ultra-sensitive detection of nucleic acids on microarrays

Resonance light-scattering particles for ultra-sensitive detection of nucleic acids on microarrays

Role of Membrane Fluidity in the Expression of Biological Functions

Biological membranes are composed of proteins, lipids, and carbohydrates. It is generally agreed that proteins are the components most directly responsible for the great diversity of functions displayed by natural membranes while the lipids, arranged in a bimolecular leaflet, provide a highly impermeable and supportive matrix for the proteins. Some of the membrane proteins, the so-called integral membrane proteins, extend into the hydrophobic regions of the lipid bilayer and are exposed on at least one of the membrane surfaces or may span the lipid bilayer and be exposed on both surfaces. Other membrane proteins, the peripheral proteins, are noncovalently attached to the membrane surface and do not extend significantly into hydrophobic regions of the membrane. The carbohydrates reside on the membrane surface, covalently attached to proteins or lipids. The lipid bilayer is not a static structure but can exist in different dynamic states in which the lipid molecules exhibit different degrees of rotational, segmental, and lateral mobilities. When mobility is high, the membrane is said to be in a high fluid state and free membrane proteins can readily translate and rotate in the lipid bilayer.

Fluorescence spectroscopy in biological and medical research

Abstract Fluorescence spectroscopy is one of the most sensitive and versatile tools in medical, biological and biochemical research. Here we discuss the use of (1) polarized fluorescence spectroscopy to study the conformational dynamics of proteins, (2) fluorescence recovery after photobleaching to study lateral mobility of proteins and lipids in biological cell membranes and (3) excitation energy transfer to measure distances between interesting sites in macromolecules and biological membranes.